Agenda T November 9th - 11th, 2018 abstract book · Agenda & abstract book 27th Gail Cassell...

Agenda

&

abstract

book

27th Gail Cassell

Microbiology Research

Retreat

Lake

Guntersville

State Park

November 9th - 11th, 2018

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Lake Guntersville State Park Map

Table of Contents

Special Thanks 1

Welcome 2

Program Agenda 4

Oral Abstracts 9 - 30

Poster Abstracts 31 - 62

Saturday Night Buffet Menu 63

Notes 64

Please note, WIFI is open, but very limited.

For an electronic version of the abstract

booklet, please click here, or scan the

below QR code.

https://www.uab.edu/medicine/microbiology/images/Research_Retreat/2018_Micro_Research_Program.pd

Special Thanks

Distinguished Alumnus Speaker

Kelly McNagny

Committee Members

Chair

Peter Burrows

Members

Todd Green | Rodney King | Bea León-Ruiz

Hubert Tse | Janet Yother

Judges

Peter Burrows | Ashlesha Deshpande | Terje Dokland

Joel Glasgow | Michael Gray | Todd Green

Hui Hu | John Kearney | Rodney King | Frances Lund

Guangxiang Luo | Jan Novak | Carlos Orihuela

Troy Randall | Jessica Scoffield | Sunnie Thompson

Charles Turnbough, Jr. | Mark Walter | Allan Zajac

Retreat Coordinators

Andrea Davis | Beth Graf | Kristina Sinclair

Host

Our deepest gratitude and appreciation to

Frances Lund for hosting this event. 1

2

Welcome

Dear Faculty, Postdocs and Students,

Welcome to the annual Gail Cassell Microbiology Research Retreat! The Micro retreat has always been a wonderful opportunity for students, faculty and post docs to focus on science and to establish new collaborations away from the distractions of everyday life.

The poster session is scheduled for Friday afternoon with our keynote speaker Dr. Kelly McNagny, on Saturday evening. All student and post doc abstracts, either poster or oral presentations, will be considered for the best poster and oral presentation awards. Lucrative prizes will be awarded!

Thank you again for coming to the Micro Research Retreat 2018 –

may you learn something exciting and develop new collaborations

while you are here!

Best!

Frances E. Lund, Ph.D. Chair Peter D. Burrows, Ph.D. Charles H. McCauley Professor and Chair Professor of Microbiology and Genetics Department of Microbiology Department of Microbiology

Agenda

Friday, November 9th

12:30 pm Meeting Registration Concierge Desk

2:00 pm WELCOME Grandview Frances Lund

2:05 pm INTRODUCTORY REMARKS Peter Burrows

2:15 pm Benjamin HuntNI

Swords Laboratory Title: Experimental modeling of infectious exacerbations in a cigarette smoke-exposure induced ferret model of COPD.

2:30pm Melissa McDanielNI Swords Laboratory Title: Stenotrophomonas maltophilia colonization and virulence in mono-and polymicrobial infections.

2:45pm Ashleigh RieglerNI

Orihuela Laboratory Title: Bacterial-induced necrosis: A key immune response initiator to colonization by Streptococcus pneumoniae.

3:00pm Terry BrissacPD

Orihuela Laboratory Title: Polysaccharide capsule modulates barrier crossing by Streptococcus pneumoniae.

3:15pm SangSang ParkPD Briles Laboratory Title: Streptococcus pneumoniae binds to apoptotic cells via interaction of pneumococcal PspA with host GAPDH.

3:30pm Jason NeedhamNI Thompson Laboratory Title: BK polyomavirus requires a nonessential ribosomal protein eS25 for efficient S phase induction and productive infection.

3:45pm Richard MurphyGS Saad Laboratory Title: Structural basis for HIV-1 envelope incorporation into virus particles.

4:00pm Ashley ConnellyGS Hel Laboratory Title: Characterization of newly identified human neutrophil subsets in inflammation-induced pathogenesis.

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Agenda Continued

F

Friday, November 9th

4:15 - 5:00pm BREAK / POSTER SET-UP

5:00 - 7:00 pm POSTER SESSION Camellia Room

7:00 pm DINNER Pinecrest Dining Room Seafood Buffet (or order from the menu up to the price of the buffet)

9:00 pm AFTER PARTY Riverview Room 320

Saturday, November 10th

6:30 - 8:30am BREAKFAST Pinecrest Dining Room

8:30am Jessica KeppleNI Grandview Hunter Laboratory Title: Illuminating the novel roles of the Ldb1 transcriptional co-regulation in maintaining brown adipose function.

8:45am Jessie BarraNI Tse Laboratory Title: Encapsulation to enhance islet transplantation.

9:00am Samuel BlumNI Tse Laboratory Title: The duality of MDA5 in Coxsackievirus- accelerated autoimmune diabetes and protection.

9:15am Meagan JenkinsNI Ballesteros-Tato Laboratory Title: Trafficking of lung-migratory cDC2s into the spleen and their effects on CD8 T cell responses to influenza virus infection.

9:30am Jessica PeelGS Lund Laboratory Title: Germinal center organization mediated by T-bet dependent expression of CXCR3 and CCR6.

9:45am Ashley LanduytGS Maynard Laboratory Title: Synergy between T-dependent antibodies and IL-10 limits susceptibility to inflammatory bowel disease.

10:00am J. Stewart NewPD Kearney Laboratory Title: Identification of commensal bacteria that induce the B-1 differentiation of GlcNAc-reactive B cells in mice.

5

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Agenda

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10:15am Michael SchultzGS Lund Laboratory Title: Blockade of NAD salvage pathway as a novel antiparasitic therapy for schistosomiasis.

10:30am BREAK

10:45am Joseph GouldGS Green Laboratory Title: Structure-function characterization of a novel polymerase-cofactor interaction in vesicular stomatitis virus.

11:00am Sushma BoppanaGS Goepfert Laboratory Title: Vaccine-encoded adapted epitopes infrequently induce CD8 T Cell responses.

11:15am Claire Hui WuGS Britt Laboratory Title: HCMV envelope protein gpUL132 controls viral production through efficient viral AC formation.

11:30am Cathy SungGS Britt Laboratory Title: Dysregulation of cerebellar Granule Neuron Precursor Cell (GNPC) proliferation upon CMV infection in newborn mice.

11:45am Rhea DerkeGS Gray Laboratory Title: The HOCl-response protein RclA reduces copper (ll) during oxidative stress in E. coli.

12:00pm Avishek MitraPD Niederweis Laboratory Title: The Dpp transporter is essential for heme and hemoglobin utilization by Mycobacterium tuberculosis.

12:30pm GROUP PICTURE TBA

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Agenda Continued

F


12:45pm LUNCH Pinecrest Dining Room

1:30pm FREE TIME

THINGS TO DO

AL VS Mississippi State @ 3:30pm airing on CBS

Zip line

Hiking, Biking & Horse Trails

Golfing

Fishing

Geocaching Challenge

Cathedral Caverns

7

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https://www.alapark.com/hiking-biking-and-horse-trails

https://www.alapark.com/lake-guntersville-state-park-eagles-nest-golf-course-18-holes

https://www.alapark.com/fishing-2

https://www.alapark.com/geocaching-guntersville-albertville-alabama-state-park

https://www.alapark.com/cathedral-caverns-state-park

Agenda


6:00 - 6:30pm SOCIAL HOUR Grandview Lobby

6:30pm DINNER Grandview

7:00pm DISTINGUISHED Grandview ALUMNUS SPEAKER Kelly McNagny Title: Role of innate lymphoid cells in inflammation, fibrosis and tissue repair.

9:00pm AFTER PARTY Riverview Room 320 320

Sunday, November 11th

6:30 - 9:00am BREAKFAST Pinecrest Dining Room

9:00am Hui Hu Camillia Room Title: Tfh cell differentiation: where to start?

9:30am Jan Novak Title: Molecular basis of pathogenesis of IgA1 immune complexes in IgA nephropathy.

10:00am Jessica Scoffield Title: Insights into Pseudomonas aeruginosa antagonism by commensal bacteria.

10:30am AWARDS Peter Burrows

11:00am CLOSING REMARKS Frances Lund

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Oral Abstracts

GS - Graduate Student (Year 4 and up)

NI - Graduate Student (1 - 3 years)

PD - Postdoctoral Fellow or PhD equivalent

Experimental modeling of infectious exacerbations in a

cigarette smoke-exposure induced ferret model of COPD

Benjamin Hunt, Denise Stanford, Niroop Kaza, Stephen Byzek, Jennifer LaFontaine, Steven M. Rowe, S. Vamsee Raju and W. Edward Swords

Division of Pulmonary, Allergy, and Critical Care Medicine, The University of Alabama at Birmingham, Birmingham, AL.

Patients with chronic obstructive pulmonary disease (COPD) suffer

from persistent airway infections that are typically caused by naso-

pharyngeal pathobionts, such as nontypeable Haemophilus influenzae

and Streptococcus pneumoniae. The population dynamics of the mi-

crobial populations within the lung of COPD patients are dynamic

and subject to rapid change, and the introduction of newly colonizing

strains or species has been associated with onset of approximately half

of all exacerbations in patient studies. However, in the absence of a

physiologically relevant animal model has precluded experimentation

on the impact of infection in the clinical context of COPD exacerba-

tions. Therefore, we utilized a novel chronic cigarette smoke-induced

ferret model of COPD that is physiologically reminiscent of the

human condition, with animals with COPD displaying symptoms

including defective mucociliary transport, mucus obstruction and

bacterial respiratory infections with the same bacterial phylotypes

that are observed in human patients. To model exacerbations,

cigarette smoke-exposed ferrets and control animals were infected via

the intratracheal route with nontypeable Haemophilus influenzae, and

the presence, density and persistence of the bacterial populations

were longitudinally monitored for 14 days by viable plate count and

quantitative PCR of bronchoalveolar lavages obtained by broncho-

scopy. Experimentally instilled bacteria persisted within the lungs of

COPD ferrets at considerable levels for at least 7 days before bacterial

presence is cleared by day 14. In vivo µCT imaging, histopathologic

and cytokine analyses also allowed estimation of the host inflamma-

tory response following infection. We conclude that COPD ferrets due

to cigarette smoke exposure serve as a suitable model for prolonged

infections with H. influenzae, and potentially other COPD related

opportunists. Moreover, acute infection initiates a robust inflamma-

tory response that is a hallmark of exacerbation of COPD disease.

Overall, this data is encouraging that this ferret model can be used for

further studies attempting to elucidate the role of NTHi in COPD

pathogenesis and exacerbation events.

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Oral Abstracts

Stenotrophomonas maltophilia colonization and virulence

in mono- and polymicrobial infections

Melissa S. McDaniel1, Trenton Schoeb2 and W. Edward Swords1

1. Department of Medicine/Pulmonary Allergy and Critical Care Medicine, 2. Department of Genetics/Genomics, The University of

Alabama at Birmingham. Birmingham, AL.

Stenotrophomonas maltophilia is a Gram-negative bacillus known to colonize the cystic fibrosis (CF) airway. At present, it is unclear whether presence of this organism is a causative agent of exacerba-tions. Moreover, the effect of poly-microbial interactions with this organism on CF disease progression is also uncertain. To investi-gate this, we established infection models in WT and CFTR-/- mice, as well as a co-infection model with Pseudomonas aeruginosa, and measured bacterial persistence and virulence, quantified by viable colony counting, weight loss, immune cell counts from BALF, and lung histopathology. S. maltophilia strains persisted in the lung up to 72 hours post-infection, and confocal microscopy of lung sections revealed multicellular foci at 24 hours post-infection, con-sistent with tissue-associated infection. Infection with S. maltophilia led to significant weight loss and immune cell infiltration, accompanied by histopathological changes. While loss of CFTR did not confer an increase in bacterial persistence or virulence, we found that the presence of Pa, a signature of CF patients, permits a profound increase in S. maltophilia persistence at 24, 48, and 72 hours post-infection. Bacterial counts of S. maltophilia and P. aeruginosa are positively correlated, although presence of both organisms does not increase infection severity. Based on these results, we conclude that S. maltophilia initiates a robust inflammatory response consistent with clinical significance in the context of acute exacerbations, and can establish a cooperative infection in concert with P. aeruginosa.

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Oral Abstracts

Bacterial-induced necrosis: A key immune response

initiator to colonization by Streptococcus pneumoniae

Ashleigh N. Riegler, Terry Brissac and Carlos J. Orihuela

Department of Microbiology, The University of Alabama

at Birmingham, Birmingham, AL.

Necroptosis, a highly inflammatory programmed form of lytic cell

death, is initiated during many bacterial infections through ion

dysregulation and energy depletion initiated by bacterial pore-

forming toxins (PFTs). PFT-producing pathogens, such as the lead-

ing cause of community acquired pneumonia, Streptococcus pneu-

moniae (Spn), cause necroptosis of macrophages and epithelia

during pneumonia and other disseminated infections. This results

in damaging inflammation and decreased survival, which can be

reduced through necroptosis inhibition. Asymptomatic naso-

pharyngeal colonization is a prerequisite to the development of

disseminated Spn diseases like pneumonia, bacteremia, and men-

ingitis. Notably, Spn colonize the nasopharynx as a biofilm, the

phenotype which expresses and releases more of the PFT pneumo-

lysin (Ply). We examined whether PFT-mediated necroptosis oc-

curs within the nasopharynx during colonization and if the subse-

quent inflammation drastically alters the immune responses to

Spn. Using a mouse model of Spn colonization, we determined

that nasopharyngeal epithelial cells (nEC) died of Ply-dependent

necroptosis. Necroptosis deficient mice, i.e. MLKL KO, had less

sloughed nEC in nasal lavage fluid (NALF). Additionally, Spn colo-

nized MLKL KO had decreased levels of IL-1α, IL-6, and IL-17; yet

increased levels of CXCL2 and neutrophils in NALF compared to

wildtype mice. Recruitment of CD11c+ cells in Spn-associated sub-

mucosa was correlated with nEC necroptosis. Importantly, colo-

nized MLKL KO mice or wildtype mice colonized with Spn ex-

pressing non-lytic Ply had less antibody against common Spn pro-

tein antigens, delayed Spn clearance, and were more susceptible to

secondary Spn challenge. Thus, PFT-induced necroptosis is a criti-

cal initiator of the immune response to Spn colonization. Grant

acknowledgements NIH-5T32AI007051-38 and NIH-AI114800. 11

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Oral Abstracts

Polysaccharide capsule modulates barrier crossing by

Streptococcus pneumoniae

T. Brissac, K.L. Kruckow, K.M. Bradley, S.M. Beno,

E.J. Benos and C.J. Orihuela



The ability of Streptococcus pneumoniae (Spn) to cross cell barri-

ers is a critical aspect of its pathogenesis. This property allows for

disease progression from pneumonia to invasive pneumococcal

diseases (IPD). To identify new ways to prevent IPD and

disseminated organ infections it becomes critical to understand

how the bacteria interact with and crosses endo/epithelium

barriers. The main virulence factor of Spn is its polysaccharide

capsule, without which Spn is mostly avirulent. Capsule surrounds

and protects the bacteria from opsonophagocytosis and killing by

immune cells. Yet, capsule also interferes with Spn adhesion to

and invasion of host cells by masking bacterial adhesins.

Therefore, modulation of capsule on the bacterial surface is pivotal

for Spn pathogenesis. Herein, we explored a role for capsule

during barriers crossing once the bacteria reach the intracellular

space; a role which has not been studied before. By comparing Spn

serotype 4 strain TIGR4 to its non-encapsulated isogenic

derivative, or isogenic switches (different capsule types in the

same genetic background) we observed differences in ability to:

(i) cross mouse cardiovascular endothelial cells, (ii) survive within

cells, (iii) tolerate endogenous stresses (iv) trigger intracellular

signaling. This was function of the presence of capsule or the

capsule type expressed at the surface of the bacteria. These

findings reveal a completely new role for a well-established

pneumococcal virulence factor. Moreover, the differences between

isogenic switches may explain, among others, differences in

therapeutic outcomes observed among different serotypes of Spn.

This knowledge can potentially allow the development of

targeted therapeutics preventing IPDs. 12

Oral Abstracts

Streptococcus pneumoniae binds to apoptotic cells via

interaction of pneumococcal PspA with host GAPDH

Sang-Sang Park, Norberto Gonzalez-Juarbe,

Carlos J. Orihuela and David E. Briles



Pneumococcal surface protein A (PspA) is an important pneumo-

coccal surface virulence factor that is found in almost all

Streptococcus pneumoniae. PspA binds to and blocks bactericidal

peptides of lactoferrin and inhibits host complement deposition

on the surface of S. pneumoniae. In this study, we report a novel

mechanism by which PspA affects pneumococcal-host interaction

by binding to glyceraldehyde-3-phosphate dehydrogenase

(GAPDH). We found that PspA does not bind to bacterial or

healthy cell GAPDH, but binds with great affinity to GAPDH

exposed on the surface of apoptotic cells. Using pulldown and

surface plasmon resonance (SPR) assays, we observed that purified

PspA bound strongly to human GAPDH but did not bind to

bacterial GAPDH. In addition, we found that the binding site for

GAPDH was within the C-terminal 60 aa of the α-helical domain

of PspA. Flow cytometry demonstrated direct PspA-dependent

binding of pneumococci to FITCconjugated GAPDH. However,

ΔpspA pneumococci and three non-pneumococcal bacterial

pathogens did not bind to human GAPDH. Interestingly, by using

western blot and fluorescent microscopy, PspA was observed to

bind only apoptotic cells and not normal, pyroptotic, or

necroptotic cells. Synthesized GAPDH peptide with or without

PspA fragment inhibited binding of PspA on apoptotic cells and

reduces S. pneumoniae lung colonization. Finally, we also

observed that pneumococcal interaction with apoptotic cells

enhanced the production of the pore-forming toxin pneumolysin.

Our observations suggest that host apoptotic cell surface GAPDH

is a major binding target of pneumococcal PspA. This mechanism

may be important in host colonization/infection by the

pneumococcus. 13

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Oral Abstracts

BK polyomavirus requires a nonessential ribosomal protein

eS25 for efficient S phase induction and productive infection

Jason Needham and Sunnie Thompson



BK polyomavirus (BKPyV) is a small, double-stranded DNA virus

that chronically infects up to 90% of the adult population

worldwide. In healthy adults, primary infection and subsequent

reactivations are asymptomatic. In organ transplant and immuno-

compromised patients, however, reactivation of BKPyV often

results in graft rejection and the development of autoimmune

disorders. During infection, BKPyV induces the host into a

sustained S phase for required for viral replication. We found that

knocking down a nonessential ribosomal protein, eS25, decreased

S phase entry and subsequent cell cycle progression. Since eS25 is

required for cap-independent translation initiation, this suggests

that e25 may be required for the expression of a regulator of S

phase entry. Consistent with this hypothesis, eS25 knockdown

decreased both BKPyV viral titers and genome replication,

although early steps in the viral lifecycle such as the timing of the

early gene Large T-antigen was unaffected. Others have reported

that SV40, a related polyomavirus, uses a cap-independent

mechanism to translate a structural protein VP3, however BKPyV

VP3 levels were not specifically decreased relative to the other viral

proteins in eS25 knockdown cells. Instead all viral proteins were

similarly decreased although eS25 knockdown does not affect

global host translation. We also observed a decrease in S phase

entry of mock infected eS25 knockdown cells, which was not be

rescued by BKPyV at 2 days post infection but was at 3 days post

infection. These results suggest that eS25 may be important for

priming or promoting cell cycle progression.

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Oral Abstracts

Structural basis for HIV-1 envelope incorporation

into virus particles

R. Elliot Murphy, Alexandra B. Samal, Jiri Vlach and Jamil S. Saad



The surface envelope glycoprotein (Env) of human immunodefi-

ciency virus type 1 (HIV-1) mediates viral infection of host cells.

The mechanism by which Env is incorporated into budding virus

particles is poorly understood. However, there is an abundance of

evidence suggesting that this mechanism may be mediated by an

interaction between the cytoplasmic tail of Env (gp41CT) and

Matrix (MA), the membrane-binding domain of the Gag

polyprotein. We have recently determined the structure of gp41CT

and characterized its interactions with the membrane using

nuclear magnetic resonance (NMR) techniques. We now set our

sights on characterizing the interactions between gp41CT and MA

bound to a membrane memetic. Cryoelectron tomography studies

have provided details about the structure of the immature Gag

lattice. However, the structure of MA bound to membrane remains

largely unresolved. We have devised the proper conditions for the

formation of a MA-membrane complex. Structural

characterization of this complex will provide insights into the

organization of MA on the inner leaflet of the plasma membrane,

where it purportedly interacts with gp41CT. The complex we have

produced consists of three components: lipid nanodiscs, a protein

construct comprised of MA tethered to a trimerization domain

called Foldon, and an antigen binding fragment (fab) for improved

stability and increased mass. With this construct, we intend to

utilize single particle cryo-electron microscopy to obtain high-

resolution structural data on MA bound to membrane and

ultimately to gp41CT incorporated into membrane. Altogether, our

studies will provide insights into the mechanism of HIV-1 envelope

incorporation into virus particles. 15

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Oral Abstracts

Characterization of newly identified human neutrophil

subsets in inflammation-induced pathogenesis

Ashley Connelly1, Krystle Ong1, Marcus Davis1, Harish Pal1, Ashish

Dhyani1, Valeriya Kuznetsova1, E. Turner Overton2 and Zdenek Hel1.

1. Department of Pathology, 2. Department of Medicine,

The University of Alabama at Birmingham, Birmingham, AL.

Neutrophils, the most abundant circulating leukocyte population,

were traditionally considered as a homogenous population of ter-

minally differentiated cells with restricted effector function. It is

increasingly clear that circulating neutrophils represent a diverse

population of subsets with distinct roles in immune regulation and

disease pathogenesis. Progress in the field is hampered by an in-

herent instability of neutrophils ex vivo, their tendency to form

multiplets with other cell types, and high background staining due

to the release of cationic granular proteins following neutrophil

activation. We present novel and optimized methods for the char-

acterization of human neutrophils close to their in vivo state. We

show how commonly employed methods of lysis and fixation in-

duce nonspecific binding of antibodies and formation of multiplets

are difficult to discern based on scatter properties. Our optimized

protocol, based on a one-step fixation/lysis and specific multiplet

removal, allowed identification of distinct, previously uncharacter-

ized subsets that exert distinct functional properties consistent

with specific roles in inflammatory conditions. A subset of

CD16lowCD62Llow neutrophils expands in HIV-1-infected patients

and in older individuals following pneumococcal vaccination. Fol-

lowing stimulation, neutrophils release chromosomal DNA, form-

ing neutrophil extracellular traps (NETs) to trap pathogens. We

have developed novel methods for NET detection, based on flow

cytometry and time-lapse microscopy. We show that current

methods underestimate the frequency of neutrophils undergoing

NETosis because of washing that fragments fragile NET structures.

The newly developed methods of neutrophil characterization will

facilitate detailed monitoring of neutrophil subsets in diverse con-

ditions and increase our understanding of neutrophils in health

and disease.

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Oral Abstracts

Illuminating the novel roles of the Ldb1 transcriptional

co-regulation in maintaining brown adipose function

Jessica Kepple, Yanping Liu, Teayoun Kim, Glenn Rowe,

Kirk Habegger and Chad S. Hunter

Comprehensive Diabetes Center and Department of Medicine, Divi-

sion of Endocrinology, Diabetes and Metabolism, The University

of Alabama at Birmingham, Birmingham, AL.

Brown adipose tissue (BAT) is critical for thermogenesis and glu-

cose homeostasis. BAT utilizes fatty acids and glucose for heat pro-

duction via mitochondrial uncoupling and is thus an attractive

therapeutic target for combatting obesity. Exploiting the energy

uncoupling capacity of this tissue requires a greater understanding

of underlying BAT transcriptional mechanisms. We recently

reported on a transcriptional co-regulator, LIM domain binding

protein 1 (Ldb1), which appears to have novel roles in BAT biology.

Ldb1 acts as a dimerized scaffold allowing for the assembly of

transcriptional complexes and is important for the development

and function of many metabolic tissues. However, direct roles for

BAT-expressed Ldb1 have not been elucidated. We set out to test

the hypothesis that Ldb1 directly impacts BAT function. We

developed a mouse model in which Ldb1 was deleted in

thermogenic adipocytes using a Ucp1- driven Cre recombinase,

termed Ldb1ΔBAT. These knockout mice have reductions in BAT-

selective genes including Ucp1 and Elovl3, a result similarly

observed in cell lines lacking Ldb1. Ldb1ΔBAT mice were unable to

defend body temperature during a cold challenge, suggesting

thermogenic defects. We also observed glucose intolerance in

Ldb1ΔBAT mice via intraperitoneal glucose challenge. These data

suggest a direct role for Ldb1 in maintaining thermogenic and

metabolic function in BAT. Ldb1ΔBAT will be crossed with a

reporter mouse to allow greater selection of adipocytes for

transcriptional and protein analyses. Additional examination of

glucose/lipid homeostasis and transcriptional profiling will in-

terrogate the mechanisms underlying Ldb1 control of BAT

function, potentially leading to novel obesity and diabetes

therapeutic targets.

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Oral Abstracts

Antioxidant encapsulation to enhance islet transplantation

Jessie M. Barra1, Veronika Kozlovskaya2, Eugenia Kharlampieva2,3 ,

Gregory Korbutt4 and Hubert M. Tse1

1. Department of Microbiology, Comprehensive Diabetes Center,

2. Department of Chemistry, 3. Center of Nanoscale Materials and Bio-

integration, The University of Alabama at Birmingham, Birmingham,

AL. 4. Department of Surgery, University of Alberta, Alberta, Canada.

Type 1 diabetes (T1D) is characterized by pancreatic β-cell death. Islet

transplantation can restore glucose homeostasis, however, limited

islet availability, toxicity of immunosuppressants, and poor graft sur-

vival are hurdles for clinical application. We hypothesize that islet

encapsulation with a nanothin coatings consisting of tannic acid (TA),

an immunomodulatory antioxidant, and poly (N-vinylpyrrolidone)

(PVPON), will provide an immunoprotective barrier and maintain β-

cell function after transplantation. (PVPON/TA)-encapsulated synge-

neic NOD.Rag islets (n=13) significantly delayed graft failure without

immunosuppression after transplantation into streptozotocin-treated

NOD mice by more than 30 days compared to non-encapsulated

grafts (n= 13; p=0.0021). Encapsulated syngeneic islets maintained glu-

cose responsiveness after transplantation better than non-

encapsulated controls (n=9, p=0.0032). Gene expression of syngeneic

NOD islet grafts 5 days post-transplant displayed a significant increase

in Ins2 (p<0.05) and Arg1 (p<0.0001) with a decrease in Ccl5

(p=0.0006), Cxcl10 (p=0.043), and Grzmb (p=0.032) mRNA accumula-

tion in (PVPON/TA)-encapsulated islet grafts, suggesting a reduction

in autoimmune inflammation. To address limited islet availability, we

encapsulated differentiated neonatal porcine islets and demonstrate

glycemic control for more than 200 days after transplantation into

diabetic NOD.scid mice. Gene expression analysis of xenotransplant

grafts 5 days post-transplant demonstrated an increase in Ins2

(p<0.001), Gcg (p<0.0001), and Arg1 (p<0.0001) with a decrease in Ccl5

(p<0.001) mRNA accumulation in (PVPON/TA)-encapsulated porcine

grafts. Our results support the hypothesis that islet encapsulation

with (PVPON/TA) coatings may elicit immunoprotection following

islet transplantation. These results also open the possibility of utiliz-

ing (PVPON/TA) nano-materials for clinical islet transplantation

while potentially reducing requirements for immunosuppressive

drugs.

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Oral Abstracts

The duality of MDA5 in Coxsackievirus-accelerated

autoimmune diabetes and protection.

Samuel I. Blum1, Ashley R. Burg1, Yi-Guang Chen2

and Hubert M. Tse1

1. Department of Microbiology, Comprehensive Diabetes Center, The

University of Alabama at Birmingham, Birmingham, AL. 2. Depart-

ment of Pediatrics, Medical College of Wisconsin, Milwaukee, WI.

While microbial infections can trigger autoimmune diabetes,

ultimately, innate immune activation and the synthesis of free

radicals, proinflammatory cytokines, and Type I interferons

contribute to pancreatic β-cell destruction in Type 1 diabetes (T1D).

We previously demonstrated that Coxsackievirus B3 (CB3) infection of

Non-Obese Diabetic (NOD) mice can accelerate T1D, partly due to the

induction of oxidative stress and antiviral signaling pathways

including melanoma differentiation-associated protein 5 (MDA5).

Single-nucleotide polymorphisms within Ifih1, the gene encoding for

MDA5, is associated with multiple autoimmune diseases including

T1D, but the molecular mechanism contributing to innate immune

dysregulation is not known. Since T1D is a chronic proinflammatory

autoimmune disease, we hypothesized that attenuated MDA5

expression and signaling can protect from diabetogenic viral-

accelerated T1D. NOD mice mutated in Ifih1 that resulted in loss of

MDA5 expression (NOD.Ifih1-m1) or an in-frame truncation in the

helicase domain of MDA5 (NOD.Ifih1-m4) exhibited a delay in

spontaneous T1D. Interestingly, CB3-infected NOD and NOD.Ifih1-m1

mice displayed accelerated virusinduced T1D, but NOD.Ifih1-m4 mice

were significantly (p < 0.001) delayed. CB3-infected NOD.Ifih1-m4

mice were efficient in pancreatic viral clearance and exhibited

decreases in local Type I interferons. Infiltrating CD8 T cells, and

activated macrophages (F4/80+ , I-A g7+ ) were reduced by ~34% and

~32%, respectively in contrast to infected NOD mice. The protective

Ifih1- m4 mutation may establish an acute level of Type I interferons

and islet-infiltrating lymphocytes to sufficiently inhibit CB3

replication without mediating bystander activation of autoreactive T

cells following virus-accelerated autoimmune diabetes. 19

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Trafficking of lung-migratory cDC2s into the spleen and their

effects on CD8 T cell responses to influenza virus infection

Meagan Jenkins1, Holly Bachus1, Beatriz León-Ruiz2

and André Ballesteros-Tato1

1. Department of Medicine, Division of Clinical Immunology and

Rheumatology, 2. Department of Microbiology, The University

of Alabama at Birmingham, Birmingham, AL.

Trafficking of antigen-bearing, migratory conventional type 1

(cDC1) and type 2 (cDC2) dendritic cells from the lung into the

lung-draining mediastinal LN (mLN) is essential for priming T cell

responses to influenza virus. In the absence of cDC1s and cDC2s,

or when they are unable to traffic from the lung into the mLN, T

cell responses are compromised and virus is not efficiently cleared,

highlighting the importance of these DC subsets in the generation

of T-cell-dependent immunity to influenza. Importantly, it is

generally considered that cDCs die shortly after reaching the mLN

and therefore do not recirculate back into the blood or traffic

further than the mLN. However, DCs purified from the spleen of

mice intranasally infected with influenza efficiently prime CD8+ T

cell responses. Paradoxically, the lungs and spleen are not

connected by the lymphatic vasculature and intranasally-

administered antigens do not freely reach the blood circulation.

Therefore, it is unclear how DCs in the spleen acquire influenza

antigens following intranasal infection, or how this affects the

total CD8 T cell response to influenza virus. Our work shows that

a fraction of the lung-migratory cDC2s that reach the mLN

following influenza virus infection egress from the mLN, reenter

the blood circulation and subsequently migrate into the spleen to

prime CD8+ T cell Responses in vivo. Collectively, our results

demonstrate a new paradigm of DC migration, offer new insights

into how systemic T cell responses to influenza are initiated, and

will ultimately reveal new strategies to elicit systemic responses

to vaccination. 20

Oral Abstracts

Germinal center organization mediated by T-bet

dependent expression of CXCR3 and CCR6

Jessica Peel,1 Sara Stone,1 Chris D. Scharer2 and Frances E. Lund1

1. Department of Microbiology, The University of Alabama at Bir-

mingham, Birmingham, AL. 2. Department of Microbiology

and Immunology, Emory University, Atlanta, GA.

The germinal center (GC) is composed of two anatomically and

functionally distinct regions with proliferation and affinity

maturation occurring within the dark zone (DZ) and antigen-

mediated selection in light zone (LZ). Despite the important role

that GCs play in the development of enduring humoral immunity,

we know remarkably little about the dynamics of the GCB cell

response. We previously showed that T-bet, an IFN -inducible

transcription factor is expressed by GCB cells and is required for

the development of LL-PCs following influenza infection. Based on

these data, we hypothesized that T-bet may regulate the selection

of GCB cells into the LLPC lineage. We observed an overrepresen-

tation of Tbx21-/- GCB cells relative to WT GCB cells in the LZ.

Given that the LZ is the site of affinity-driven selection, we

hypothesized that the Tbx21-/- GCB cells may be better able to

bind the influenza antigens, NP and HA. Although preliminary,

our data indicated minimal differences in the capacity of WT and

Tbx21-/- GCB cells to bind influenza antigens. Therefore, we

hypothesized that efficient migration for GC B cells is dependent

on the expression of CXCR3, a known target of T-bet.

Interestingly, we saw that Cxcr3-/- GCB cells were enriched in the

LZ while the CXCR3 sufficient GCB cells were enriched in the DZ

of the GC. Furthermore, CCR6+, a repressive target of T-bet, GCB

cells are enriched in the LZ. Thus, our data suggests that T-bet-

mediated CXCR3 and CCR6 expression may regulate the migration

of GCB cells within the GC during influenza infection.

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Oral Abstracts

Synergy between T-dependent antibodies and IL-10 limits

susceptibility to inflammatory bowel disease

Landuyt, A.E.1, Klocke, B.J.1 and Maynard, C.L.1

Department of Pathology, The University of Alabama


Inflammatory bowel diseases (IBD) are chronic autoinflammatory

conditions in which the immune system loses tolerance to gastro-

intestinal microbes. The role of humoral immunity in IBD is

largely undefined. We examined the potential of T-dependent

antibodies to prevent intestinal inflammation using mouse models

of IBD. For this, we used mice without inducible T cell

costimulator ligand (ICOSL). Icosl-/- mice have fewer germinal

centers and significantly fewer T-dependent antibodies.

Additionally, Icosl-/- mice have greater frequencies of CD4 T cells

producing IL-10, an immunosuppressive cytokine, in the colon. As

such, we hypothesized that IL-10 and T-dependent antibodies

cooperatively promote intestinal homeostasis. We first tested this

using mice deficient in CD4 T cell-derived IL-10 (Il10cKO) and

ICOSL. This codeletion resulted in colitis before 28 days of age.

However, fostering Icosl-/- Il10cKO mice on wild-type mothers

significantly delayed this early-onset inflammation. These results

strongly suggest that the T-dependent antibodies present in breast

milk are protective against early-onset colitis. We sought to

further investigate the synergy between T-dependent antibodies

and IL-10 in adults. For this, we temporarily eliminated IL-10

producing cells from adult mice. Icosl-/-, but not wild type or IgA-/-

mice, rapidly developed colitis under these conditions. This colitis

could be prevented if mice were first depleted of gram-negative

bacterial flora. Further examination revealed that Icosl-/- mice had

no defect in IgA coating of colonic commensals, but had

significantly reduced IgG binding to mucosally-associated

microbes. Collectively, our results suggest that novel cooperation

between T-dependent IgG targeting mucosal commensals and

IL-10 maintains intestinal homeostasis throughout life. 22

Oral Abstracts

Identification of commensal bacteria that induce the B-1

differentiation of GlcNAc-reactive B cells in mice

J. Stewart New, R. Glenn King, Austin R. Lenox and John F. Kearney



Natural antibodies reactive with conserved carbohydrate epitopes

represent a therapeutic target for intervention and prevention of

autoimmune and allergic airway diseases. We showed previously

that colonization by the commensal microbiota is essential for the

establishment of Nacetylglucosamine (GlcNAc)-reactive B-1 B cells

and their natural IgM products in mice. Herein, we identify a

member of the bacteroides phylum, Bacteroides thetaiotaomicron

(B. theta), as a candidate in driving activation of GlcNAc-reactive B

cells at the intestinal mucosa. Monocolonization of germ-free mice

with B. theta induces the differentiation of plasma cells producing

IgA and IgM serum antibody reactive with GlcNAc. Moreover, B.

theta colonization drives peritoneal cavity expansion of GlcNAc-

reactive B cells bearing innate-like B cell phenotypes. B theta

boasts a genome specialized for carbohydrate degradation,

possessing numerous glycosyl hydrolases organized into discrete

polysaccharide utilization loci, which support its expression of

phase-variable capsular polysaccharide antigens. Our results

indicate that expression of GlcNAc-containing capsular antigens

by B theta is strain-dependent, and is modulated based on the

availability of dietary glycans and host carbohydrate substrates,

including gastric mucin. Thus, we propose that the activation of

GlcNAc-reactive B cells at the mucosal interface during

colonization by B theta is contingent on the milieu of

carbohydrate substrates metabolized by the bacteria, and the

incorporation of GlcNAc moieties into its capsular polysaccharide

as bacterial surface epitopes.

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Oral Abstracts

Blockade of NAD salvage pathway as a novel antiparasitic

therapy for schistosomiasis

Michael D. Schultz1, Tulin Dadali1, Helène Muller-Steffner2, Esther

Kellenberger3, Leonardo Sorci4, Davide Botta1 and Frances E. Lund1

1. Department of Microbiology, The University of Alabama at Birmingham,

Birmingham, AL. 2. Laboratoire des Systèmes Chimiques Fonctionnels, Uni-

versité de Strasbourg, Illkirch, France. 3. Laboratoire d’Innovation Théra-

peutique, Université de Strasbourg, Illkirch, France. 4. Department of Medi-

cine and Surgery, Università Politecnica delle Marche, Ancona, Italy.

Schistosomiasis is the third most common parasitic infection

worldwide and infects over 200 million people. Treatment of the

disease relies solely on praziquantal, which, despite its effective-

ness, has an unknown mechanism of action and its large-scale use

in endemic areas poses a high risk for drug resistance. Relying on

this single treatment is unsustainable and risky, and novel anti-

schistosomal drug targets and therapies are desperately needed. A

comparative genome analysis revealed that NAD biosynthesis in S.

mansoni occurs solely via the salvage pathway and therefore is

dependent on host-derived NAD precursors. We confirmed the

expression of NAD salvage-specific genes in adult parasites by PCR

and hypothesized that blockade of the NAD salvage pathway

would impact parasite survival. In vitro incubation of S. mansoni

with inhibitors of the NAD salvage pathway decreased NAD levels

in adult parasites, which correlated with reduced lactic acid levels,

egg production, survival and mobility. Histological analysis

revealed significant damage to the papillae and outer membrane

following in vitro treatment of adult male parasites. Importantly,

in vivo blockade of the NAD salvage pathway decreased egg and

worm burden in mice that were infected with S. mansoni.

Furthermore, S. mansoni-infected mice had lower degrees of

splenomegaly and hepatomegaly, and a reduced number of

granulomas in the liver. In conclusion, our data show that

inhibition of the NAD salvage pathway impairs S. mansoni

reproduction and survival both in vitro and in vivo and suggest

that targeting the NAD salvage pathway is a promising

therapeutic approach for the treatment of schistosomiasis. 24

Oral Abstracts

Structure-function characterization of a novel polymerase-

cofactor interaction in Vesicular stomatitis virus

Joseph R. Gould1, Shihong Qiu1, Qiao Shang1, Peter E. Prevelige1,

Chad M. Petit2 and Todd J. Green1

1. Department of Microbiology, 2. Department of Biochemistry and

Molecular Genetics, The University of Alabama


Vesicular stomatitis virus (VSV) is a critically important model

system for Mononegavirales, the order of negative sense RNA

viruses which includes important pathogens like Ebola and

Hendra. VSV has also seen an emerging role in human health,

both as a vaccine platform and as an oncolytic agent. The lifecycle

of VSV relies upon the interaction of the viral large (L) and

phospho- (P) proteins to form the RNA-dependent RNA

polymerase (RdRP) holoenzyme. Despite this interaction being

absolutely required for the generation of viral mRNA and genomic

ribonucleoproteins, the interface or interfaces at which L and P

interact have not to date been reported. Using a structure-based

approach, we have identified that the connector domain of VSV L,

which previously had no assigned function, accommodates part of

the P protein. Sequence alignments demonstrate the conservation

of a VxF/W motif in Vesiculovirus and Sprivivirus

phosphoproteins, which was found to be functionally significant in

a cell-based reporter system. Using our results to inform

computational modeling, we assert that a novel, conserved, and

functionally relevant L-P interaction has been identified in VSV,

and that a functional assignment can be made for the connector

domain of L.

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Oral Abstracts

Vaccine-encoded adapted epitopes infrequently

induce CD8 T cell responses

Sushma Boppana, Sarah Sterett, Anju Bansal and Paul Goepfert

Department of Medicine, The University of Alabama


Recently, adapted epitopes (AE) containing HLA-I associated

polymorphisms were shown to be less immunogenic than non-

adapted epitopes (NAE) in acute HIV-1 infection, and infection

with AE-encoding viruses correlated with poorer clinical

outcomes. As AE are encoded by all current HIV-1 vaccines in

testing, this study aims to determine the impact of vaccine-

encoded AE on recipients’ CD8 responses. Eighty-two HVTN502

vaccinees were screened by IFNγ ELISpot for responses to

vaccine encoded NAE and AE restricted to each one’s HLA-I al-

leles. Antigen sensitivity was quantified as an EC50, and

polyfunctionality was assessed using flow cytometry. Vaccine

recipients responded to a significantly higher proportion of NAE

than AE (p<0.0001). Twelve vaccinees (14.6%) had AE-specific

responses, which were restricted to 7 unique epitopes. Although

vaccine-encoded AE had significantly weaker predicted HLA-I

binding affinities than NAE, immunogenic NAE and AE had

similar HLA-I binding affinities. These immunogenic NAE and AE

responses were also found to have similar antigen sensitivities and

cross-reactivity. In a subset, we found that the polyfunctionality of

AE-specific CD8s was comparable to NAE-specific CD8s. Vaccine-

encoded AE were poorly immunogenic; however, immunogenic AE

responses were equal in magnitude, avidity, and polyfunctionality

compared to NAE ones. Collectively, these data indicate that many

of these AE encoded by vaccines are unlikely to elicit effective CD8

responses, limiting the benefits of including these variants in

vaccine inserts.

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Oral Abstracts

HCMV envelope protein gpUL132 controls viral production

through efficient viral AC formation

Wu, H.1, Mach, M.2 and Britt, W.J.1

1. Departments of Pediatrics and Microbiology, University of Ala-

bama at Birmingham, Birmingham, AL. 2. Institute of Clinical and

Molecular Virology, University of Erlangen, Erlangen, Germany.

The human cytomegalovirus UL132 open reading frame encodes a

270-amino acid type I envelope glycoprotein, gpUL132, that is

required for efficient virus production. Deletion of UL132 from the

HCMV genome resulted in a pronounced deficit in virus yield with

an approximately 2-3 log decreases in infectious titer. To

determine the role of gpUL132 in the virus life cycle, we first

studied the characteristics of the ∆UL132 HCMV mutant. Using

density gradient centrifugation in potassium-tartrate, we observed

that ∆UL132 extracellular particles banded at different densities

compared to wild type (wt) HCMV particles. Additional studies

indicated that the defects in the ∆UL132 mutant virus resulted

from the altered morphogenesis of the membranous viral assembly

compartment (AC), rather than deficits in virus entry, genome

replication, or cell-to-cell spread. Expression of gpUL132 in trans

rescued the defects in the AC in cells infected with the ∆UL132

mutant virus and infectious virus production. Importantly, we

utilized a fusion protein combining the ecto- and transmembrane

domains from an irrelevant protein, TrkB, with the cytosolic

domain from gpUL132 for these experiments, which demonstrates

that the cytosolic domain of gpUl132 is sufficient to rescue the

defects in AC formation in cells infected with the ∆UL132 mutant.

These findings argue that gpUL132 is essential for HCMV AC

formation and highlight its importance for viral production.

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Oral Abstracts

Dysregulation of cerebellar granule neuron precursor cell

(GNPC) proliferation upon CMV infection in newborn mice

Cathy Yea Won Sung1 and William Britt1,2,3

1. Department of Microbiology, 2. Department of Pediatrics Infec-

tious Disease, 3. Department of Neurobiology, The University of

Alabama at Birmingham, Birmingham, AL.

Human cytomegalovirus (HCMV) infection in the developing

central nervous system (CNS) results in long-term neurological

damage including motor disorders, microcephaly, and mental

retardation. Although the connection between CMV and neurode-

velopmental defects is widely recognized, the underlying

mechanisms are poorly understood. Utilizing a mouse model in

which newborn animals are infected intraperitoneally with murine

CMV (MCMV), we have previously showed that the virus spreads

hematogenously to the developing brain inducing robust

inflammatory response. Infection resulted in the disruption of

proper cerebellar development characterized by reduced cerebellar

size, but an increase in the number of cerebellar granule neuron

precursor (GNP) cells in the external granular layer (EGL) of the

cerebellar cortex. This increase was attributed to prolonged cell

cycle duration, premature cell cycle exit, and decreased GNP

migration from EGL to IGL for final maturation of cerebellar

GNPs. Specifically, MCMV infection led to prolonged G1 and S

phase of the GNP cell cycle. Furthermore, we have found that

sonic hedgehog (Shh), a GNP mitogen expressed in purkinje cells,

was reduced in MCMV-infected mice cerebella consistent with the

altered GNP cell cycle progression. Collectively, our results

demonstrate that compromised GNP proliferation is a major

determinant of increased cell cycle exit and differentiation

contributing to reduced cerebellar size in MCMV-infected mice.

These findings may lead to the foundation of a therapeutic

approach aimed to prevent delayed brain development of fetuses

exposed to the HCMV as a result of maternal infection. 28

Oral Abstracts

The HOCl-response protein RclA reduces copper (II)

during oxidative stress in E. coli.

Rhea Derke and Michael Gray



Commensal bacteria must resist oxidative stress to survive on host

epithelial surfaces. How bacteria survive treatment with

hypochlorous acid (HOCl), an oxidant produced by neutrophils

during inflammation, has not been well characterized. This project

focuses on identifying the function of a protein called RclA, which

is highly upregulated by HOCl-stressed E. coli, protective against

HOCl stress through an unknown mechanism, and is conserved

among many host-associated bacteria. Because RclA shares

sequence homology to disulfide and mercuric reductases, we

hypothesized that it reduces an unknown substrate using a

conserved cysteine motif. To test this hypothesis, a series of

experiments were done to identify the substrate of RclA. Thiol-

trapping experiments showed that RclA did not function to reduce

oxidized proteins. Surprisingly, in vivo ICP-MS experiments

revealed that E. coli ∆rclA cells exported more copper than wild-

type E. coli after HOCl stress. In vitro studies then showed that

RclA is a thermostable copper (II) reductase, with a melting

temperature (Tm) about 10 o C higher than the mean Tm of the E.

coli proteome. RclA is able to function in denaturing conditions,

including urea and HOCl treatment. These results indicate that

the HOCl response and copper regulation in E. coli are integrated

in a previously underappreciated way. Further studies are being

conducted to determine differences in HOCl sensitivity of E. coli in

the presence of copper and whether RclA is needed to mediate

such effects.

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Oral Abstracts

The Dpp transporter is essential for heme and hemoglobin

utilization by Mycobacterium tuberculosis

Avishek Mitra1, Ying-Hui Ko2, Gino Cingolani2,3

and Michael Niederweis1


mingham, Birmingham, AL. 2. Department of Biochemistry and

Molecular Biology, Thomas Jefferson University, Philadelphia, PA.

3. Institute of Biomembranes and Bioenergetics, National Research

Council, Bari, Italy.

Iron is essential for growth of Mycobacterium tuberculosis (Mtb).

More than 70% of the iron in the human body is stored in heme,

mostly sequestered within hemoglobin. In this study, we

demonstrated that the inner membrane Dpp importer of Mtb is

essential for both heme and hemoglobin utilization. The 1.27 Å

crystal structure of the periplasmic DppA protein associated with

the Dpp transporter revealed a tetrapeptide bound in the protein

core and a large solventexposed crevice for specific, yet reversible

heme binding. Mutation of arginine 179 at this position eliminated

both heme binding to DppA in vitro and growth of Mtb in heme or

hemoglobin media. The surface-accessible and membrane-

anchored PPE36 and PPE62 proteins are required for heme and

hemoglobin utilization indicating that these pathways converge at

the cell surface of Mtb. These results identified the sole heme

transporter of Mtb and revealed a novel mechanism of bacterial

heme uptake.

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Poster Abstracts

JR - Graduate Student (years 1-2) MID - Graduate Student (year 3-4) SR - Graduate Student (years 4 and up)

PD - Postdoctoral Fellow or PhD equivalent

JR - Posters 1 - 8

(1) Characterization of a novel, multifunctional class of metalloantibiotics against Staphylococcus aureus

Rachel M. Andrews, Whitney Narmore and Frank Wolschendorf

Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL.

One major issue facing medicine today is the increasing preva-lence of antimicrobial resistance among pathogens. This increase in drug resistance combined with a lack of new classes of antim-icrobials in recent decades has made treating infections of patho-gens, like Staphylococcus aureus, difficult. New antimicrobials with multiple functional targets are desperately needed to combat the rise in antimicrobial resistance. Recently, metalloantibiotics were presented as a potential avenue for novel drug development. Metalloantibiotics are compounds which require the presence of a transition metal ion in order to gain or increase the potency of their antimicrobial action. However, the compounds in these studies appear to have a single mode of action: potentiating cop-per toxicity. While this action has relatively broad effects against bacteria, having a single mode of action increases the likelihood of pathogens generating resistance to these compounds. Here we describe a new class of multifunctional metalloantibiotic com-pounds against S. aureus, the benzimidazoles (BZIs), which have different modes of action depending on the interacting transi-tion metal. Members of this class exhibit either micromolar bac-tericidal or nanomolar bacteriostatic antibacterial activity with copper or zinc respectively. Resistant mutants generated against the copper-dependent action of the BZIs are also not more resis-tant to the zinc-dependent action, indicating different mecha-nisms between these activities. This differential activity of the BZIs presents a unique opportunity to advance development of multifunctional antimicrobials to reduce potential antimicrobial resistance by pathogens. Additionally, the BZIs represent, to our knowledge, the first instance of a zinc-dependent antibiotic against a Grampositive bacterium.

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Poster Abstracts

(2) CD4 T cell restricted HIV cryptic epitopes,

an implication of novel antisense proteins

Jacob Files and Paul Goepfert



Background: The alternative reading frames (ARF) of HIV-1 have

been shown to encode and produce peptide antigens of unknown

function, termed cryptic epitopes (CE). Much of the current work

on CE have mainly focused on the recognition of these epitopes by

CD8 T cells, due in part to the fact that CE are often thought to be

products of translational errors and are thus recycled and

presented solely through the MHC-I pathway. This work

investigated whether CD4 T cells could recognize CE.

Method: We generated overlapping peptides to previously

described antisense open reading frames of HIV-1 and optimized it

for CD4 T cell recognition (CD4-CE). Isolated PBMC from chronic

stage HIV-1 infected patients were first depleted of CD8 T cells, the

remaining cells were then stimulated with CD4-CE and tested for

immunogenicity via IFN-γ ELISpot and flow cytometry.

Results: Two of the CD4-CE utilized were found to be

immunogenic and strictly recognized by CD4 T cells within the

small cohort tested (N=20). These results were further confirmed

by flow cytometry, demonstrating CD4 production of effector

cytokines in response to CD4-CE, but a lack of CD8 T cell response

to these epitopes. Conclusion- We show that immune responses to

CE are not only limited to the CD8 branch of T cells but can also

broaden immune responses to the virus by triggering CD4 subsets

as well. Our observation suggests that in addition to ASP, HIV-1

can generate other potential novel and yet unidentified proteins

from its antisense transcripts.

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Poster Abstracts

(3) A structural approach towards understanding

Ccm-mediated capsid remodeling in bacteriophages

Dominik Herrmann, James L. Kizziah, N’toia Hawkins,

Cynthia M. Rodenburg, Peter Prevelige and Terje Dokland



Staphylococcus aureus pathogenicity islands (SaPI) are a class of

phage-induced chromosomal islands that contribute significantly to

horizontal gene transfer and pathogenicity by parasitizing the helper

phage replication machinery [1]. SaPIbov5 encodes for a capsid mor-

phology altering protein Ccm that uniquely remodels the phage cap-

sid to fit only the smaller SaPI genome. It has been shown that dele-

tion of ccm in SaPIbov5 is sufficient to restore the phage titer to wild-

type levels, suggesting that Ccm is primarily responsible for SaPIbov5-

mediated interference [2]. The mechanism by which Ccm remodels

the helper-phage capsid is not yet understood and solving the struc-

ture and determining the stoichiometry of Ccm would provide critical

insight into this question. In this work, Ccm was purified to homoge-

neity by affinity and size exclusion chromatography. This material was

subjected to chemical crosslinking and cryo-EM, followed by refer-

ence-free 2D classification, which showed that Ccm forms a mixture of

pentamers and hexamers. Furthermore, evidence suggests that the

predicted unstructured N-terminal domain of Ccm is important for

solubility, folding and assembly of the Ccm-monomer. Upon removal

of the N-terminal domain, the protein can only be solubilized using

the mild detergent DDM. Analytical size exclusion chromatography

and EM-analysis using negative stain shows that truncated Ccm also

loses its characteristic pentameric/hexameric assembly. Overall, this

work marks a starting point for obtaining a high-resolution structure

of Ccm and provides insight into the function of the N-terminal do-

main, both of which are critical steps towards understanding the

mechanism of capsid remodeling by Ccm.

1. Fillol-Salom, A., et al., Phage-inducible chromosomal islands are ubiqui-

tous within the bacterial universe. The ISME Journal, 2018. 12(9): p. 2114-

2128. 2. Carpena, N., et al., Convergent evolution of pathogenicity islands

in helper cos phage interference. Philosophical Transactions of the Royal

Society B: Biological Sciences, 2016. 371(1707): p. 20150505.

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Poster Abstracts

(4) Understanding the inflammatory status of hearts in

aging animals that leads to cardiac infections

Katherine L. Kruckow1, Cecilia A. Hinojos1,2 and Carlos J. Orihuela1

1. Department of Microbiology, The University of Alabama

at Birmingham, Birmingham, AL. 2. Department of Microbiology

and Immunology, University of Texas Health Science

Center at San Antonio, San Antonio, TX.

The elderly have an increased risk of developing and dying from

pneumonia; specifically, onein-four adults hospitalized for

community-acquired pneumonia experience an adverse cardiac

event, i.e. pneumonia-associated adverse cardiac event (PACE),

such as heart-attacks. In addition, aged compared to young

animals are less protected by the 23-valent pneumovax vaccine

currently given to elderly people. Together, this shows an

importance in understanding why this vulnerable population is

more susceptible to bacterial infections so that new treatments

and preventives can be developed. During normal aging, a state of

chronic inflammation, inflamm-aging, occurs resulting in the

inability to mount an effective immune response when exposed to

an infection. The mechanisms of inflamm-aging are not fully

understood, especially within the heart and could be a potential

reason for increased risk of PACE. It has been shown in the lungs

of aged animals that there are increased levels of bacterial ligands,

PAFR and Laminin receptor (LR), that lead to better endothelial

crossing and macrophages have diminished phagocytosis and elicit

a weak cytokine response to bacterial challenge. To understand

these factors in context of the heart, aged (18 months and older)

and young (3-6 month) hearts were examined for levels PAFR and

LR as well as the activation status MAPK and NFκB, via

immuno-blot and cytokine levels via ELISA. The results of these

studies will help serve as the beginning of understanding how

inflammation in hearts during aging impacts susceptibility to

bacterial infections such as invasive pneumococcal cardiac

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Poster Abstracts

(5) Examining the effect of Hmo1 on transcription

by RNA polymerase I

Abigail K. McConahay, Catherine E. Scull, Andrew M. Clarke,

Dmitry G. Vassylyev and David A. Schneider

Department of Biochemistry and Molecular Genetics,

The University of Alabama at Birmingham, Birmingham, AL

RNA Polymerase I (Pol I) synthesizes ribosomal RNA (rRNA),

which comprises approximately 60% of the ribosome. Upstream

binding factor (UBF) activates transcriptional initiation and

elongation by Pol I in mammals by forming rDNA loops. There is a

potential analog to UBF in yeast, high mobility group protein 1

(Hmo1), which is a histone-like protein that affects Pol I

transcription by an unknown mechanism. The goal of this study is

to use both in vitro and in vivo techniques to gain comprehensive

insight into the function of Hmo1. We have purified Hmo1 and will

add it to in vitro transcription assays to examine whether this

protein directly affects transcription and determine whether

initiation and/or elongation are affected. Additionally, we will use

native elongating transcript sequencing (NET-seq) to analyze the

effect of Hmo1 in vivo, using Saccharomyces cerevisiae. It has been

proposed that Hmo1 may also activate transcription by RNA

Polymerase II (Pol II) by interacting with transcription factor II D,

the first factor recruited to the promoter during transcription by

Pol II. To characterize the effects of Hmo1 on both RNA

Polymerases using NET-seq, we have generated hmo1Δ mutants

that express epitope tags on Pols I or II. The regulation of Pol I and

Pol II transcription by Hmo1 has been established for over a

decade, however, the mechanisms by which it influences these

enzymes remain unknown. This comprehensive study will fully

define the mechanism(s) and conservation of this regulation

during transcription by Pol I and Pol II.

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Poster Abstracts

(6) A proposed role for Interleukin-6 in the

modulation of Th2 asthmatic responses

Erin McLaughlin and Beatriz León-Ruiz



The incidence of allergic asthma in the United States is growing

each year, with the number of people with asthma reaching 25

million in 2018. One proposed explanation for the increase in

asthma in developed countries is the hygiene hypothesis, which

states that a lack of early childhood exposure to high levels of

endotoxin, like lipopolysaccharide (LPS), increases the

susceptibility to developing allergic diseases. However, it is not

known why this protective exposure to endotoxin must occur

during early childhood or by what mechanism endotoxin yields

protection against allergy development. Our lab has previously

examined age-related differences in asthma development

comparing adult and infant C57BL/6 (B6) mice when given house

dust mite (HDM) and varying concentrations of LPS. Preliminary

data suggest low doses of LPS given during HDM sensitization

prevent Th2 allergic responses (a hallmark of asthma pathology) in

adults, while infants still develop robust Th2 responses.

Interestingly, adult B6 mice lacking Interleukin-6 (IL-6) induce

strong Th2 responses, like those of infant B6 mice, when given

HDM with or without LPS, suggesting an important role for IL-6 in

the LPS-mediated suppression of Th2 responses. We hypothesize

that IL-6 influences T cell polarization by modulating the

expression of T-bet, a transcription factor known to inhibit the

development of Th2 cells by promoting Th1 cell development.

Here, we investigate the mechanisms by which IL-6 influences T

cell responses to HDM with or without LPS.

36

Poster Abstracts

(7) TNF deficient mice develop severe colitis via

blockade of the IL-10 receptor

Rachel Q. Muir, Barbara J. Klocke and Craig L. Maynard

Inflammatory bowel disease (IBD) is a relapsing-remitting

condition that affects the gastrointestinal (GI) tract of susceptible

individuals when the immune system mounts an inappropriate

response to intestinal microbes. Currently, there is no cure for

IBD. To date, the most successful treatment to induce lasting

remission, is the administration of a neutralizing antibody to

tumor necrosis factor alpha (TNFα), a pro-inflammatory cytokine

that is highly upregulated in IBD patients. However, 30% of

patients are primary non-responders to anti-TNFα therapy and of

the responders, 30 - 40% lose responsiveness within the first year

of treatment. Interleukin 10 (IL-10) is an anti-inflammatory

cytokine crucial for maintaining gut homeostasis, and

polymorphisms in the IL10 locus increase susceptibility to IBD. To

begin examining the role of TNFα signaling in colitis, we used an

antibody to block the IL-10 receptor (IL-10R) to disrupt gut

homeostasis and induce colonic inflammation in TNF knockout

(Tnf-/-) and wild-type (WT) mice. Interestingly, the Tnf-/- mice

developed more severe colitis according to our key indicators of

colitis – histological inflammation and elevated fecal lipocalin-2 –

as well as an observed expansion of Foxp3+ regulatory T cells.

Additionally, Tnf-/- mice fail to resolve colonic inflammation 30

days after the last administration of the IL-10R blocking antibody.

Our findings demonstrate that in mice with genetic deletion of

TNF, dysregulated IL-10 signaling can still promote severe colitis.

Further, our results suggest a beneficial role for TNFα signaling

during recovery from colitis under these conditions.

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Poster Abstracts

(8) Contribution of Streptococcal pyruvate oxidase (SpxB)

pathway products to biofilm formation of

Streptococcus pneumoniae strains

Sara Stoner, Ashleigh N. Riegler, Terry Brissac

and Carlos J. Orihuela



Biofilm formation plays a key role in Streptococcus pneumoniae

(Spn) pathogenesis by altering metabolism and gene expression to

promote bacterial adhesion to host cells and increase production

of the pore-forming toxin pneumolysin. However, environmental

factors and regulatory pathways responsible for Spn biofilm

formation remain largely unknown. Previous studies have shown

that streptococcal pyruvate oxidase (SpxB) is important for Spn

biofilm formation in vitro and in vivo, although the aspect of SpxB

activity enabling biofilm formation remains unidentified. The goal

of this project was to identify products in the SpxB pathway that

promote biofilm formation. Crystal violet assays were performed

to quantify biofilm growth in multiple Spn serotypes in the

presence of either acetate or hydrogen peroxide - two products of

SpxB activity. Spn serotype TIGR4 ΔSpxB mutant strain showed a

significant increase in biofilm growth at a specific concentration

range of sodium acetate. However, there were no significant

changes in biofilm formation in serotypes D39 and 6A10 in the

presence of varying sodium acetate concentrations. These findings

suggest that acetate produced via the SpxB pathway promotes

biofilm production in a serotype-specific manner. There were no

significant changes in TIGR4 biofilm growth in the presence of

varying hydrogen peroxide concentrations, suggesting that

hydrogen peroxide does not influence biofilm growth in TIGR4.

Overall, these results are important so that we may identify

products of the SpxB pathway that are influencing Spn biofilm

formation and subsequently, pathogenesis. 38

Poster Abstracts

MID - Posters 9 - 16

(9) IL-22 mediated intestinal immunity

against Citrobacter rodentium infection

Baiyi Cai and Casey T. Weaver

Department of Pathology, The University of Alabama


Interleukin 22 (IL-22) is one of the key elements host defense

against attaching effacing pathogen Citrobacter rodentium (C.r.) in

mouse. While it has been shown that IL-22 is indispensable for the

host protection during C.r. infection, little have we known about

the detailed mechanism of how IL-22 induce immunity against

C.r.. Our study finds that IL-22 induce antimicrobial peptides

(AMPs) S100A8/9 and Lipocalin-2 in both epithelial cells and

neutrophils during C.r. infection. Also, IL-22 help with the

recruitment of AMP producing neutrophils to the lamina propria

area during C.r. infection. We conclude that IL-22 mediates

anti-bacterial immunity through the induction of AMPs as well as

recruitment of neutrophils.

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Poster Abstracts

(10) Characterization of newly identified human

neutrophil subsets in HIV-1-infected patients

Marcus D. Davis1, Krystle L. Ong1, Ashley Connelly1, Miguel Melendez-

Ferro1, Harish Pal1, Valeriya Kuznetsova1, Ashish Dhyani1, Richard Hui-

jbregts1, E. Turner Overton2 and Zdenek Hel1

1. Department of Pathology, 2. Department of Medicine,


HIV-1 infection is associated with a breakdown of the gut mucosal

barrier and subsequent microbial translocation to systemic circulation

causing the immune system to be in a chronic inflammatory state.

This chronic inflammatory state eventually puts HIV-infected patients

at a higher risk for development of comorbidities including cardiovas-

cular disease and liver disease. Despite comprehensive studies analyz-

ing HIV-1-infection and its effect on the adaptive immune system, the

role of innate immune system, specifically neutrophils, in mediating

the effect of microbial translocation on disease pathogenesis remains

unknown. Neutrophils are the most abundant circulating population

of leukocytes (50-70%) and the first innate immune responders to

invading pathogens. A significant alteration in their functional capa-

bilities is likely to exert a critical effect on the immune system. ` We

have identified phenotypic changes of total neutrophil population in

HIV-1-infected individuals including changes in surface level expres-

sions of CD16, myeloperoxidase (MPO), LOX-1, and PDL-1, indicating

a unique neutrophil phenotype. We show that these changes in sur-

face marker expression correlate with clinical parameters of liver and

cardiovascular disease progression. Previous studies have shown that

a population of low-density neutrophils (LDNs) copurify in the PBMC

layer after density centrifugation and are expanded during inflamma-

tory conditions. We demonstrate there are two distinct LDN sub-

populations in the PBMC layer each at different stages of maturation

which we define as mature low-density neutrophils (mLDN) and im-

mature low-density neutrophils (imLDN). We developed a method for

monitoring the imLDN subpopulation in whole blood without the

need for centrifugation which allows us to characterize the imLDN

population closer to its in vivo state. Detailed characterization of

neutrophil subpopulations will elucidate the role of neutrophils in

disease progression and the development of comorbidities in HIV-1-

infected individuals and facilitate the pharmacological targeting of

neutrophils or their products in HIV-1-infected individuals. 40

Poster Abstracts

(11) The Role of Ccm in the transformation of

bacteriophage ø12 capsids

N’Toia Hawkins and Terje Dokland



Staphylococcus aureus is a species of bacteria that can cause a

wide range of infections of skin and soft tissues. They are capable

of this through the expression of numerous virulence factors that

are often encoded on mobile genetic elements, such as

pathogenicity islands. Staphylococcus aureus pathogenicity islands

(SaPIs) are normally stably integrated into the bacterial genome,

but can be mobilized by specific “helper” bacteriophages,

including ø12. When SaPIs are packaged, they often cause the

capsids of their helper phage to become smaller. SaPIs that are

mobilized by ø12, such as SaPIbov5, encode an alternative capsid

protein that is required for the change in architecture. However,

the exact mechanism by which capsid size is altered in the ø12

system remains unclear. Using cryo EM, as well as other

biochemical and 3D reconstruction techniques, we were able to

determine the structure of native ø12 capsids. We were also able to

define where the alternative capsid protein is incorporated into

the structure of the smaller capsids containing SaPIbov5.

Investigating the intricacies of capsid shrinkage in this system will

allow us to better understand genetic mobilization and its role in

spreading virulence.

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Poster Abstracts

(12) Repeated low-dose allergen exposure

triggers allergic sensitization

Katundu Katundu and Beatriz León-Ruiz



Exposure to aeroallergens such as house dust mite (HDM) results

in atopic symptoms including asthma in about 10% of individuals.

Type 2 helper T cells (Th2) play a triggering role in the

pathogenesis of HDM allergic asthma. Using an HDM-induced

asthma model in mouse, we have previously published that the

development of HDM-specific Th2 cells that migrate to the lung

and cause pathology, require an initial exposure to HDM or

“sensitization” that involves the priming of IL-4 + T follicular

helper (Tfh) cells in the draining lymph node. Following HDM

challenge, Tfh cells generated during the sensitization phase

differentiated into Th2 cells that homed to the lungs to cause

pathology. The differentiation of IL-4-committed Tfh cells

required sequential Agpresentation by both dendritic cells (DCs)

and B cells. Importantly, repeated allergen exposures, sometimes

during months/years, are required before full-blown allergic

symptoms develop. Here, we analyzed the role of repeated HDM-

allergen exposure in allergen sensitization and later development

of Th2-driven disease. We i.n. sensitized mice with either a single

development of Th2 cells and associated pathology were analyzed

in the lung. We found that repetitive split doses are required to

induce the initial priming IL-4 + Tfh cells after sensitization and

the later development of Th2-driven, allergen-induced airway

inflammation after challenge. Further experiments suggest that

repetitive HDM exposure empowers DCs, but not B cells, to

efficiently prime HDM-specific Th2 responses.

42

Poster Abstracts

(13) Cryo-EM reconstruction of the Staphylococcus aureus

phage 80α baseplate at 3.75Å resolution

James L. Kizziah1, Altaira D. Dearborn2, Keith A. Manning1

and Terje Dokland1

1. Department of Microbiology, University of Alabama at

Birmingham, Birmingham, AL. 2. National Institute of Arthritis ,

Musculoskeletal and Skin Diseases (NIAMS), Bethesda, MD.

Staphylococcus aureus is an opportunistic pathogen and a

common cause of human infections. In S. aureus, horizontal

transfer of genes, including those encoding virulence factors and

antibiotic resistance, usually occurs through bacteriophage

transduction. The S. aureus-infecting phage 80α consists of an

icosahedral capsid containing its DNA genome, a long

non-contractile tail, and an ornate baseplate. It is one of the best

described phages of S. aureus and is representative of other related

S. aureus-infecting phages. The baseplate is the main point of

interaction between the phage and the host, contributes to

determining host specificity, and plays a key role in cell wall

penetration and DNA ejection, though very little is known about

the baseplates of S. aureus-infecting phages. The objective of this

study was to illuminate these essential proesses by determining

the 3D structure of the 80α baseplate. Using cryo-electron

microscopy and single-particle analysis, we generated a 3D

reconstruction of the 80α baseplate at 3.75Å resolution and

modeled several baseplate proteins. The previous crystal structure

of the receptor-binding protein (RBP) from the related phage φ11 is

nearly identical to the structure of 80α RBP, suggesting a

conserved wall teichoic acid-dependent host-binding mechanism.

The major tail protein (MTP) and Dit protein, to which the RBPs

attach, are core baseplate components that establish a continuous,

highly negatively charged β-barrel that likely allows unimpeded

ejection of the DNA upon infection. In addition to RBP, the tail

fiber protein, FibL, likely serves as a secondary receptor binding

protein.

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Poster Abstracts

(14) Microbial-derived antigens are necessary for selection,

but not maintenance of GlcNAc reactive B cells

Austin R. Lenox, J. Stewart New, R. Glenn King and John F. Kearney



Natural antibodies derived from innate-like B lymphocytes play

roles in housekeeping and immune protection through specificity

for evolutionarily conserved epitopes including carbohydrate

moieties. Innate-like B cells reactive to N-acetylglucosamine

(GlcNAc) elicited by immunization with Streptococcus pyogenes

(Group A Streptococcus/GAS) additionally react with endogenous,

cryptic GlcNAc epitopes of the mammalian glycome, however the

relative roles of endogenous and exogenous antigen stimulation in

driving innate-like B cell development remains enigmatic. We

showed by flow cytometric analysis and single-cell immunoglobu-

lin sequencing that under germ-free conditions GAC-reactive B

cells fail to differentiate into mature B-1 B cells and exhibit a loss of

IGHV6-3 expressing B cells clonotypes commonly observed in

conventional mice. In order to investigate the role of microbiota-

derived antigens in the maintenance of mature GAC-reactive B

cells, we treated mice with a combination of amphotericin-B,

vancomycin, neomycin, metronidazole, and ampicillin in order to

deplete the microbiota. The number of GAC-reactive B cells were

slightly decreased in the spleen after microbiota depletion but

remained phenotypically similar to sham-control mice. The

number and phenotype of GAC-reactive B cells in the peritoneal

cavity were unaffected by antibiotic treatment. Total serum and

anti-GAC IgM remained unchanged while total and anti-GAC IgA

decreased with antibiotic treatment. These data show that GACre-

active B cells require the microbiota to be selected into the mature

compartment, but do not rely on antigen stimulation to maintain

number, phenotype, and natural antibody production. Overall, the

data reinforces the importance of early-life microbial exposure in

establishment of the innate-like B cell repertoire.

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Poster Abstracts

(15) Copper’s effect on the growth, shape, and drug

susceptibility of Mycobacterium smegmatis

Jordan Lingo and Frank Wolschendorf



Copper is a transition metal with many biological utilities. It is a

cofactor in several biological enzymes involved in energy

production and antioxidants. However, the highly reactive

properties of copper ions that contribute to these benefits come

with a price: when uncontrolled, copper can cause widespread

cellular damage as it reacts with biomolecules. Thus, all domains

of life have evolved sophisticated copper resistance mechanisms.

Intracellular pathogens, such as Mycobacterium tuberculosis, must

face elevated levels of copper in the phagosomes of innate immune

cells like macrophages. But while copper resistance may be well-

studied, the exact mechanisms of copper toxicity in specific

bacteria remain incompletely defined. In order to address these

gaps in knowledge and uncover how copper can interfere with

mycobacteria, we observed the effects of copper on bacterial

growth and morphology of Mycobacterium smegmatis, a model

organism for M. tuberculosis. We observed that bacteria grown in

copper adapt a distinct and unusual dumbbell-shaped

morphology. This shape is coupled with slowed growth and

division, increased membrane permeability, and greater antibiotic

susceptibility. These data suggest that copper interferes with the

cell envelope homeostasis of mycobacteria.

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Poster Abstracts

(16) Post-transcriptional regulation of Foxp1A is critical for

germinal center T follicular helper cell differentiation

Ryan McMonigle1,2, Yinhu Wang1, Jianlin Geng1,

Hairong Wei3 and Hui Hu1


mingham, Birmingham, AL. 2. Medical Scientist Training Program,


3. Department of Cell, Developmental, and Integrative Biology, The

University of Alabama at Birmingham, Birmingham, AL.

Germinal center (GC) formation during an immune response is

vital for the generation of highaffinity antibodies and long-lived

memory B cells. T follicular helper (Tfh) cells are a subset of CD4 T

lymphocytes critical for GC formation. We have previously identi-

fied transcription factor Foxp1 as a critical negative regulator of

Tfh cell differentiation. Initial Tfh cell differentiation is inhibited

via the sum of constitutively expressed Foxp1A isoform and T cell

receptor stimulation induced Foxp1D isoform. In this study, we

investigated the expression of Foxp1 in CXCR5+ PD1 + precursor

Tfh (pre-Tfh) cells that are dependent on cognate T-B cell

interactions to differentiate into CXCR5hi PD-1 hi GC Tfh cells. We

found that Foxp1D was not induced in pre-Tfh or GC Tfh cells. In

addition, Foxp1A expression was progressively decreased during

pre-Tfh to GC Tfh cell differentiation, without dramatic change in

mRNA expression. Retroviral Foxp1A expression in Foxp1 deficient

T cells substantially blocked pre-Tfh to GC Tfh cell differentiation,

resulting in impaired GC B cell formation. These results suggest

that the post-transcriptional down regulation of Foxp1A is critical

for the differentiation of pre-Tfh to GC-Tfh cells. Elucidating the

signaling pathway responsible for controlling Foxp1A levels may

provide the opportunity to manipulate Tfh responses in vaccine

development and autoimmune disorders. Funding: NIAMS T32

AR069516 UAB MSTP 5T32GM008361-26 NIAID 1R01AI130232.

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Poster Abstracts

SR - POSTERS 17 - 22

(17) Pyrazolopyrimidinones are cupricidal antimicrobials

against S. aureus that disrupt essential membrane functions

Cameron Crawford and Frank Wolschendorf



The treatment of methicillin-resistant Staphylococcus aureus

(MRSA) infections poses a therapeutic challenge. As resistance to

antibiotics spread, our need for antimicrobials with novel modes

of action grows. To achieve this, we must expand our available

therapeutics through costly chemical synthesis, or by repurposing

already synthesized molecules. Metalloantibiotics are a mostly

unexplored reservoir of potent therapeutics, with new inhibitors of

bacterial, fungal, and viral pathogens being discovered inside

previously exhausted libraries. Some of the most potent

metalloantibiotics have utilized copper for their activity, but the

modes of action are often not the same. Herein, we describe the

identification of the pyrazolopyrimidinones (PZPs) using our

previously described copper dependent inhibitor screen against S.

aureus. The most potent PZP with the highest in vitro therapeutic

index, 915, displayed bactericidal properties against MRSA and

biofilms cultures at sub-micromolar concentrations. This

cupricidal activity is founded on the molecule’s ability to

coordinate Cu and induce accumulation of Cu ions inside S. aureus

cells. We demonstrate that exposure to 915+Cu led to an almost

instantaneous collapse of the membrane potential which was

accompanied by a complete depletion of cellular ATP, loss of

cell-associated K+ , a substantial gain of cell associated Na+ , and

an inability to control the influx of protons in slightly acidic

medium, while the integrity of the cell membrane remained intact.

These findings highlight PZP 915 as a novel membrane-active

metalloantibiotic of S. aureus that is likely to target a multiplicity

of membrane associated protein functions rather than imposing

physical damage to the membrane structure.

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Poster Abstracts

(18) HIV-1 envelope N-glycan shield

Audra A. Hargett1, Qing Wei1, Barbora Knoppova2, Stacy Hall1,

Milan Raska1,2, Zina Moldoveanu1, Reda Rawi3, Gwo-Yu Chuang3,

Peter Kwong3, Jan Novak1 and Matthew B. Renfrow1

1. The University of Alabama at Birmingham, Birmingham, AL.

2. Palacky University in Olomouc, Olomouc, Czech Republic.

3. National Institutes of Health, Bethesda, MD.

HIV-1 envelope glycoprotein (Env) gp120/41 trimer, the sole

surface protein of HIV-1 virions, initiates entry of the virus into

host cells and is a target of the host’s immune system.

Approximately 90 N-glycans of gp120/41 trimer form a glycan

shield that is the primary interface between the virus and host im-

mune system. However, how individual N-glycosylation site (NGS)

mutations coordinate to evade the immune system is not well

understood. Using highresolution mass spectrometry, we can track

shifts in N-glycan heterogeneity due to mutations observed in

immune escape viral variants. The shifts in heterogeneity defined a

range of NGS sequons confined to a structural region that we used

for a mutation cluster analysis using the HIV-1 LANL database. We

found a finite number of sequons within a specific microdomain

(high-mannose patch) and created an immune-escape map of con-

served and variable glycans. A 500-ns molecular dynamics

simulation of the gp120/41 trimer variants provided further insight

into the structural dynamics of the glycan movements within each

microdomain that serve to shield Env. We tested how shifting the

number of sequons within one microdomain (highmannose patch)

could affect Env functionality. Four Env gp120 trimeric variants

that have 5, 4, 4, and 3 sequons, respectively, in the high-mannose

patch microdomain were analyzed for their infectivity, ability to

bind CD4 receptor, and sensitivity to antibody neutralization.

These studies provide detail on how we can identify Env N-glycan

microdomains and utilize these microdomains to predict viral-

escape mutations.

48

Poster Abstracts

(19) BK polyomavirus activates the DDR to halt cell cycle progression and enhance viral infection

Justice J.L. and Thompson S.R.

Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL.

Polyomaviruses are small DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the ATM (ataxia telangiectasiamutated) and ATR (ATM-Rad 3 related) DNA damage responses (DDRs), which are needed to prevent severe DNA damage and to promote viral infection. The mechanism by which the host DDR enhances viral infection and the source of the BKPyV-driven host DNA damage that accumulates in DDR deficient cells is poorly understood. Furthermore, the distinction between the roles of ATM and ATR during infection is not defined. We hypothesized that determining the origin of this DNA damage would help elucidate the contribution of the DDR to viral infection. Using a primary kidney cell model we found that the source of the DNA damage was when BKPyV infected cells entered mitosis prematurely. The severe DNA damage was associated with ATR inhibition and resulted from actively replicating host DNA undergoing unregulated mitotic entry, which severely fragments the DNA. By characterizing the cell cycle profile of BKPyV infected cells treated with ATR and ATM inhibitors we found that activation of ATR by BKPyV blocked cell cycle progression by inactivating the mitosis-promoting factor (Cdk1/cyclin B) by inhib-iting Cdk1 through phosphorylation by Wee1. In contrast to ATR, ATM activity was not necessary to block premature mitosis. Rather, ATM activity was required for expedient entry into S phase. Together, the synergistic activation of these two DDR kinases both promoted and maintained BKPyV-mediated S phase to enhance viral production.

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Poster Abstracts

(20) gp44, A minor capsid protein with a major

role in bacteriophage 80α infection

Keith Manning1, Nuria Quiles-Puchalt2, José R. Penadés 2,

and Terje Dokland1


mingham, Birmingham, AL. 2. Institute of Infection, Immunity and

Inflammation, College of Medical, Veterinary and Life Sciences,

University of Glasgow, Glasgow, UK.

Bacteriophage 80α is a prototypical bacteriophage that infects

Staphylococcus aureus. The World Health Organization has rated

S. aureus as a high priority target for the development of novel

antibiotics. Phages provide an alternative to antibiotics for

treatment of bacterial infections. SaPIs are mobile genetic

elements that carry genes encoding antibiotic resistance and viru-

lence factors. When 80α enters the lytic cycle, these SaPIs are

mobilized, and the SaPIs in turn are able to hijack the assembly of

80α virions, creating phage-like transducing particles containing

SaPI DNA. The capsid head of 80α is composed of four proteins:

gp42 (portal), gp44 (minor capsid), gp46 (scaffold), and gp47

(major capsid). We have examined the role of minor capsid

protein gp44 in the phage life cycle. We have determined that

gp44 exists as a dimer with two copies per virion. Homologs of

gp44 exist in a variety of other phages. Gp44 is essential for the

formation of viable phage but is not required for the mobilization

of SaPIs or transduction of plasmids. However, deleting the gene

for gp44 in an 80a lysogen leads to a fully packaged non-infectious

virion and does not directly affect DNA ejection, as shown by in

vitro DNA ejection by heat, or incubation with whole cells.

Lysogenization frequency in the Δ44 mutant is also drastically

reduced. However, infectivity of 80α is partially recovered upon

infection of cells expressing gp44 showing that gp44 serves a role

after injection into the host cell. Furthermore, lytic activity of Δ44

phages is able to be regained post infection when mitomycin C is

added to the culture, suggesting a role for gp44 in the lysis-

lysogeny “decision” upon infection. This is the first time such a

function has been identified for a phage capsid protein. 50

Poster Abstracts

(21) CD8 T cells responding to adapted epitopes are enriched

in chronic HIV infection and induce dendritic cell

maturation with enhanced CD4 infection in vitro

Kai Qin,1 Sushma Boppana,1 Ling Yue,2 Eric Hunter,2

Anju Bansal1 and Paul Goepfert1

1. Department of Medicine, The University of Alabama at

Birmingham, Birmingham, AL. 2. Emory Vaccine Center, Emory

University, Atlanta, GA. 3. Microsoft Research

HIV-1 frequently escapes from CD8 T cell responses via human

leukocyte antigen class I (HLAI) restricted adaptation. We

previously demonstrated that adapted epitopes (AE) compromise

CD8 T cell responses during acute infection and correlate with

poor clinical outcomes. Here, we examined the impact of AE on

CD8 T cells responses and the biological relevance of HIV escape

during chronic HIV infection (CHI). In contrast to acute infection,

NAE and AE response frequency was similar in CHI. Longitudinal

analyses from acute to chronic infection showed increased IFNγ

response frequency and magnitude of AE-specific CD8s. AE-

specific CD8s were more cytotoxic than their NAE counterparts

despite exhibiting lower antigen sensitivity. The former may be

due to the finding that NAE-specific CD8s expressed higher levels

of PD1 and lower level of CD28 and CD57, suggesting a more

exhausted and senescent phenotype. Nevertheless, sequencing

results identified AE-encoding strains as the dominant species

during chronic infection. CD8 responding to AE but not NAE

promote DC maturation which in turn enhanced CD4 T cell

infection in vitro. Taken together, AE-specific CD8s are enriched

during CHI, but despite this seemingly increased immune

pressure, AE showed rare evidence of reversion or additional

escape. Our data suggests that AE-specific CD8s may confer an

advantage to the virus by fostering a DC mediated CD4

trans-infection.

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Poster Abstracts

(22) Type VII secretion substrates control secretion of the

tuberculosis necrotizing toxin

Uday Tak, Dominik Herrmann, Terje Dokland

and Michael Niederweis



Secretion of protein toxins is an ancient mechanism by which

pathogenic bacteria subvert host defenses during colonization and

infection. Therefore the biogenesis of such toxins is of great

interest to understand bacterial physiology, host pathogen

interactions, and to identify targets for antibacterial therapeutics.

Such a toxin was recently identified in Mycobacterium

tuberculosis, named Tuberculosis Necrotizing Toxin (TNT) which

activates necroptosis in macrophages via depletion of host NAD+.

The catalytic activity of TNT is required for host cytotoxicity and

intracellular replication of M. tuberculosis in macrophages, thus

making it an essential cytotoxicity factor. The enzymatic activity of

TNT and subsequent molecular events in the host macrophage

have been characterized in detail, but the secretion of TNT

remained enigmatic. TNT is the surface-exposed C-terminal

domain of the outer membrane protein CpnT (Channel Protein

with Necrosis Inducing Toxin) whose assembly is dependent on a

Type VII Secretion System (T7SS). Thus, CpnT is the first outer

membrane protein translocated by a T7SS, but the mechanism of

how any T7SS substrate crosses the periplasm and reaches the

outer membrane in M. tuberculosis is unknown. In this study we

showed that the T7SS substrates EsxEF, encoded within the cpnT

operon, are required for export and/or assembly of CpnT in in the

outer membrane. Biochemical and structural analysis revealed that

the EsxEF complex forms oligomeric, water-filled channels in lipid

bilayers suggestive of a membranebound periplasmic channel.

Thus, our results identified the first structural components

required for secretion of TNT by M. tuberculosis.

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Poster Abstracts

PD - POSTERS 23 - 32

(23) Dendritic cells regulate CD4 Th1 and Th2

differentiation in cutaneous Leishmaniasis

Natalia Ballesteros, Beatriz León-Ruiz and Frances E. Lund



Leishmania major (Lm) is an intracellular parasite used as model

to study Th1 and Th2 development. Lm induces a protective IFNg-

driven Th1 response in C57BL/6J (B6) mice and non-curing

leishmaniasis in BALB/c mice, which make an IL-4 dominated Th2

response. In this project, we used Lm to assess how dendritic cells

(DCs) regulate Th1 and Th2 development. We previously identified

a population of CXCR5+CCR7lo CD11b+ DCs that localize in the

perifollicular region of the lymph node (LN) following infection

with the Th2-inducing nematode H. polygyrus (Hp), and

demonstrated that altering the placement of these DCs in the LN

by blocking the CXCR5 ligand (CXCL13), was sufficient to prevent

Hp-induced Th2 immunity. Interestingly, following Lm infection,

we observed that migratory DCs from BALB/c mice, but not B6

mice, upregulated CXCR5 and primed Th2 responses. Our previ-

ous data show that neither CXCR5 upregulation by the migratory

DCs nor the initiation of Th2 development following Lm infection

is not intrinsically dependent on the genotype of the DC as DCs

from the genetically susceptible BALB/c strain do not upregulate

CXCR5 and prime Th1 responses when activated in the resistant B6

background. Similarly, B6 DCs upregulate CXCR5 and prime a Th2

response when activated in BALB/c mice. These data therefore

indicate that another BALB/c cell programs DCs to upregulate

CXCR5 and prime Th2 development. Although we have not yet

identified which cell type programs DCs to initiate resistant or non

-protective responses to Lm, our data show that the early

programming cell(s) is bone marrow derived. We are now testing

whether innate lymphoid cells (ILCs) or NK cells regulate the

expression of CXCR5 by DCs thereby allowing for Th2

development. Together, these results contribute to defining how

the earliest innate signals regulate the programming of the

migratory antigen-presenting DCs and allow the DCs to initiate

Th2 immunity.

Poster Abstracts

(24) Invasive pneumococcal disease leads to long-term

cardiac remodeling and dysfunction

Sarah M. Beno, Yong Wang, Griffin M. Wright, Jeevan K. Jadapalli,

Sara N. Stoner, Anukul T. Shenoy, Ganesh V. Halade,

Jessy S. Deshane and Carlos J. Orihuela


at Birmingham, Birmingham, AL

Streptococcus pneumoniae is a Gram-positive opportunistic

bacterial pathogen and the leading cause of infectious death

among the elderly. Pneumococcal pneumonia has been linked to

numerous anatomical site complications. Cardiac insufficiencies

linked to pneumococcal pneumonia have been suspected since the

1930s, and more recent studies have shown up to 20% of adults

hospitalized for Pneumococcal pneumonia later experienced a

cardiac complication, such as congestive heart failure. Using a

mouse model of invasive pneumococcal disease in combination

with immunofluorescence, collagen-specific staining, flow

cytometry, and echocardiography, we assessed organ damage

during acute infection and long-term (up to 3 months post-

infection) cardiac dysfunction and remodeling. A necroptosis-

inhibitor, Ponatinib, was tested as a potential therapeutic.

Following invasive pneumococcal disease, macrophages and

neutrophils are recruited to the heart to fight infection. Levels of

immune cells in the heart remain higher than normal for about

two weeks. Echocardiography shows mice have reduced

myocardial contractility, even three months post-infection. The

decline in ejection fraction lessens when the mice are treated with

Ponatinib. Sirius Red/Fast Green staining, which elucidates the

fraction of collagenous protein, i.e. scar tissue, reveals significantly

higher ratios of scarring in hearts of animals that had severe

pneumococcal infections. Ponatinib used in combination with

ampicillin shows promise as a therapeutic treatment for invasive

pneumococcal disease.

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Poster Abstracts

(25) Dampening of immune responses using tannic

acid-encapsulated antigens

Joseph M. Feduska1, Veronika Kozlovskaya2, Aaron Alford2,

Eugenia Kharlampieva2 and Hubert M. Tse1

1. Department of Microbiology, Comprehensive Diabetes Center,

2. Department of Chemistry, The University of Alabama


In type 1 diabetes (T1D), insulin-secreting β-cells are destroyed by

autoreactive immune cells. T cell activation relies on three signals,

one of which is the synthesis of pro-inflammatory cytokines and

reactive oxygen species. We previously demonstrated that third

signal dissipation impairs autoreactive T cell activation. In this

study, we tested the hypothesis that encapsulation of putative T1D

autoantigens with an antioxidant-containing biomaterial would

induce immune tolerance. We co-cultured bone marrow-derived

dendritic cells (DC) with microcapsules comprised of a neutral

polymer and multilayers of the antioxidant tannic acid (TA).

Induction of a tolerogenic DC phenotype was assessed by

expression of pro-inflammatory cytokine mRNA and secretion of

cytokines by ELISA. DC co-cultured with TA-based microcapsules

displayed decreases in mRNA accumulation of the

pro-inflammatory cytokines Tnfa (55% decrease, p < 0.01) and Il12b

(70% decrease, p < 0.01), and the chemokine Cxcl10 (60% decrease,

p < 0.01). We next investigated whether microcapsules could

abrogate antigenspecific T cell responses using the OT-II mouse,

which expresses a single T cell clone recognizing the OVA323-339

peptide. Flow cytometric analysis showed a significant inhibition

of the T cell activation markers CD25, CD28, CD44, and CD69 (p <

0.001) in splenocytes cocultured with ovalbumin-containing

microcapsules. ELISA showed nearly undetectable levels of IFN-g

(p < 0.01) for cells cultured with microcapsules compared to oval-

bumin protein. These data show that microcapsules can effectively

blunt the proinflammatory third signal in an antigen-specific

manner. As a T cell-mediated disease, inhibition of autoreactive T

cells represents an attractive potential therapy for T1D.

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Poster Abstracts

(26)The tuberculosis necrotizing toxin induces

lethal oxidative stress in macrophages

David Pajuelo1, Norberto Gonzalez-Juarbe2 and Michael Niederweis1

1. Department of Microbiology, The University of Alabama

at Birmingham, Birmingham, AL. 2. J. Craig Venter

Institute, Rockville, MD.

Alveolar macrophages are the primary responders to lung

infections with Mycobacterium tuberculosis (Mtb). It has been

shown that Mtb induces a programmed necrosis (i.e. necroptosis)

which enhances mycobacterial replication and dissemination. The

tuberculosis necrotizing toxin TNT is the major bacterial factor

which initiates necroptosis by activating RIPK3 through depletion

of cellular NAD+. Accumulation of reactive oxygen species (ROS)

has also been shown to play a key role in necroptotic cell death of

Mtb-infected macrophages. However, a direct role for TNT in the

generation of oxidative stress was not known. Here we describe

that TNT is a major contributor to ROS accumulation in both Mtb

-infected macrophages and Jurkat T-cells expressing the tnt gene.

TNT-induced NAD+ depletion in macrophages led to generation of

cellular ROS and enhanced cell death. In addition, RIPK3/MLKL

activation by TNT was found to be essential for the increased

oxidative stress observed during Mtb infection. Our results suggest

that replenishment of NAD+, inhibition of necroptosis and/or

neutralization of oxidative stress are promising targets of

host-directed therapeutic approaches against tuberculosis.

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Poster Abstracts

(27) Construction of single-chain MspA nanopore

for DNA sequencing

Mikhail Pavlenok, Dominik Herrmann and Michael Niederweis


at Birmingham , Birmingham, AL.

Nanopore sequencing is a novel single molecule DNA sequencing

method which is inexpensive, fast, capable of long reads and

retains epigenetic information. In this method ionic current is

measured while single-stranded DNA (ssDNA) is electrophoreti-

cally translocated through a nanometer-scale pore. Each passing

nucleotide blocks current with a characteristic amplitude and

duration which are used to identify DNA sequence. The

Mycobacterium smegmatis porin A (MspA) is an octameric, chan-

nel-forming protein with a short and narrow constriction zone

which is ideal for nanopore DNA sequencing. However, wild-type

MspA does not translocate DNA. We identified key positions in

MspA and constructed MspA mutants capable of ssDNA

translocation. Due to octameric symmetry of MspA, precise

control of any amino acid residue in the functional pore is

challenging. Therefore, we constructed a single-chain mspA

(scmspA) gene where all eight subunits are connected by regions

encoding a peptide linker. In addition, to control the pore’s

stoichiometry and thereby the size of the constriction zone, we

constructed truncated scMspA variants with the number of linked

subunits ranging from three to seven. These proteins were purified

from E. coli. Each of these scMspA variants formed functional

channels in lipid bilayer experiments indicating that linking

subunits does not change the channel-forming properties of

MspA. Consistent with the functional experiments, negative-

staining electron microscopy demonstrated that scMspA has a

central water-filled channel. In conclusion, we have created an

efficient platform for fine-tuning DNA translocation through

scMspA nanopore which enables precise control of the chemistry

and the subunit composition of the MspA.

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Poster Abstracts

(28) Structural and biochemical studies of major transcrip-tional regulators of Mycobacterium tuberculosis provide

novel insights into their mechanism of functioning

Ritesh Rajesh Sevalkar1, Prabhat Ranjan Singh1, Ranjeet Singh1, Suruchi Singh1, Vinay K. Nandicoori2, Subramanian Karthikeyan1

and Dibyendu Sarkar1

1. CSIR-Institute of Microbial Technology, Sector 39 A, Chandigarh, India. 2. National Institute of Immunology,

Aruna Asaf Ali Marg, New Delhi, India.

M. tuberculosis (Mtb), as one of the most successful intracellular human pathogens, encounters various stressful environment during its complicated life cycle. Thus, relatively quick readjust-ment to such varying environmental conditions and remarkably effective adaptation of Mtb are strikingly critical for the survival and growth of tubercle bacilli. It is believed that ineffective adaptive response is controlled by coordinated regulation of stress proteins, failure to which results in elevated immune recognition with reduced survival during chronic infections. Thus, our work is focused on understanding how major transcriptional regulators, like virulence regulator PhoP is functioning as a global coordinator of mycobacterial stress response. Remarkably, we find that PhoP impacts on global regulation of heat-shock proteins, which protect Mtb against stress generated by macrophages during infection. Our results identify and demonstrate that unconventional protein-protein interactions in addition to classical DNA-protein interactions control complex mechanisms of expression of heat-shock proteins, an essential pathogenic determinant of Mtb. In addition to this, we have characterized an essential MerR-family of transcriptional regulator, Mtb Rv1828. With the objective of whether we can use and develop this essential transcriptional regulator as a possible drug-target, we solved the crystal structure of the C-terminal domain of Rv1828 and showed that the N-terminal domain of Rv1828 is responsible for its specific DNA binding properties. Together, our results identify important mechanistic features suggesting its functional role as an important regulator of Mtb. Using structural coordinates of the regulator, further effort is underway to probe whether small molecules against Mtb can be effectively designed to target this essential transcription factor.

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Poster Abstracts

(29) CD4+ T cell-derived Interleukin-21 promotes the

clearance of enteropathogenic bacteria via enhancement

of humoral immunity and inhibition of T follicular

regulatory cell development

Daniel J. Silberger1, Carlene L. Zindl1, Carson E. Moseley1,

Jeffrey R. Singer1, Craig L. Maynard1, Bruce A. Vallance4,

James J. Moon5, Casey D. Morrow5, David A. Randolph2

and Casey T. Weaver1

1. Department of Pathology, 2. Department of Pediatrics, 3. Cell,

Developmental, and Integrative Biology, The University of Alabama

at Birmingham, Birmingham, AL. 4. Department of Pediatrics, Uni-

versity of British Columbia, Vancouver, British Columbia, Canada.

5. Center for Immunology and Inflammatory Diseases, Massachu-

setts General Hospital, Harvard Medical School, Boston, MA.

Interleukin-21 (IL-21) is important in T-dependent antibody

responses and mediates host protection against opportunistic

infections in humans. Despite studies indicating a role for IL-21

during intestinal inflammation, precisely how it affects homeosta-

sis and host-defense in the intestinal mucosa is not clear. Using

bone marrow chimeric mice, we found that expression of the IL-

21R by hematopoietic cells was required for the efficient clearance

of the enteropathogen C. rodentium. Using a novel dual IL-21

reporter/conditional deletion mouse, we show that CD4+ T cells

are the dominant source of IL-21 and that IL-21-producing CD4+ T

cells are required the efficient clearance of C. rodentium. Mice

with a conditional deletion of Il21 from T cells (Il21CKO mice) had

exacerbated colitis, increased systemic bacterial dissemination,

decreased germinal center (GC) B cells, decreased plasmablasts,

and reduced C. rodentium-specific IgG relative to littermate con-

trol animals. In association with impaired humoral immunity,

Il21CKO mice exhibited dysregulated T cell development charac-

terized by a reduced ratio of T follicular helper (Tfh) cells to

Foxp3+ T follicular regulatory (Tfr) cells in the draining MLN

following infection. Similarly, Il21r– /– mice displayed increased

follicular Foxp3+ cells and Tfr cells in the draining MLNs during

infection. These findings demonstrate that T cells are the primary

source of IL-21 during enteropathogenic infection and that IL-21

promotes the clearance of enteropathogens via enhancement of

humoral immunity and inhibition of Tfr cell development.

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Poster Abstracts

(30) Type 1 diabetes-associated IFIH1 SNP, A946T,

in Coxsackievirus B infection

Jared Taylor and Hubert M. Tse



Type 1 diabetes (T1D) is a multifactorial autoimmune disease with

a strong genetic basis. However, evidence suggests a role for

environmental factors such as viral infections in triggering disease

onset. One viral infection that is highly correlated with T1D is the

Coxsackievirus B (CVB). Sensing of CVB is mediated by the

cytosolic sensor of dsRNA, melanoma differentiation-associated

protein 5 (MDA-5), which is encoded by the IFIH1 gene.

Stimulation of MDA-5 results in the production of type I IFNs,

which initiate the antiviral response. Single nucleotide

polymorphisms in the IFIH1 gene such as rs1990760, which results

in a non-synonymous mutation that changes alanine at position

946 to a threonine, is highly associated with increased risk for T1D.

Studies in human PBMCs and mice have demonstrated that the

A946T SNP results in an increased sensitivity to viral ligands and

subsequently a stronger downstream IFN response. However, it is

unclear what effect this SNP has on the function of antigen-

presenting cells such as macrophages, which play a key role in

inducing adaptive immune responses. We hypothesize that the

A946T SNP leads to an exaggerated response to CVB infection by

macrophages, which results in a stronger IFN response. We

predict that the A946T SNP results in an exacerbated type I IFN

response that will enhance the ability of macrophages to activate

autoreactive CD4+ and CD8+ T cells in response to diabetogenic

CVB infections. This study aims to increase our knowledge of how

genetic susceptibility and environmental factors together

contribute to disease.

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Poster Abstracts

(31) A tale of two infections: Allergic airway disease impairs

the host immune response to bacterial infection in a mouse

model of chronic airway allergen sensitization

A. H. Totten, L. Xiao, D. Briles, J. Y. Hale, T. R. Schoeb,

A. S. Alishlash, K.B. Waites and T. P. Atkinson

Rationale: Mycoplasma pneumoniae (Mpn) and Streptococcus

pneumoniae (Spn) are important causes of pneumonia in children.

Asthmatics are at greater risk of invasive infection with both

pathogens. We predicted that allergic airway disease might

decrease the ability of the host to mount an effective immune

response to respiratory bacterial infections. We explored this

hypothesis using a mouse model of chronic airway allergen

sensitization and infection.

Methods: BALB/cJ mice were sensitized with ovalbumin (OVA) to

induce allergic airway inflammation, and then infected with Mpn

or Spn. Lung histopathologic analysis in H&E and PAS-stained

sections, cytokine mRNA and protein levels in bronchoalveolar

lavage fluid (BALF) and pulmonary function testing were carried

out on different treatment groups at 30 days post-infection.

Results: OVA-sensitized groups had increased levels of T2 cyto-

kines (IL-4, IL-5, IL13) in BAL fluid and increased histopathologic

scores compared to allergen-naïve groups. The IgG antibody

response to Mpn infection was reduced by ~50% in OVA-

sensitized mice, and a similar, though lower level of impairment

was seen in Spn infected mice. OVA-sensitized, Mpn-infected ani-

mals had significantly decreased BAL cytokine protein concentra-

tions (IL-5, IL-6, IL-13) compared to uninfected, OVA-sensitized

animals, and a similar though lesser reduction was seen in OVA-

sensitized Spn-infected mice. OVA-sensitized, Mpn-infected mice

exhibited significantly decreased responses during graded

methacholine challenge compared to all other groups.

Conclusions: Prior OVA sensitization impairs the immune

response to Mpn infection, and to a lesser extent, Spn infection.

Mpn infection ablates airway responsiveness to methacholine and

T2 cytokine levels in a mouse model of chronic airway

sensitization and infection.

Poster Abstracts

(32) Genome-wide assessment for iron utilization in Mycobacterium tuberculosis

Lei Zhang and Michael Niederweis

Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL.

Iron is essential for almost all life. The human pathogen Mycobac-terium tuberculosis (Mtb) requires iron for its growth and intracellular survival. So far as we know, Mtb can utilize siderophore and heme-based iron sources. However, the process of iron uptake and utilization in Mtb is largely uncharacterized. In this study, we constructed high density Mtb transposon libraries (~75% coverage) under different iron sources and performed a transposon-insertion sequencing (Tn-Seq) which could systematically identify the obligate genes for iron acquisition and utilization by Mtb in different iron sources. Interestingly, the Tn-Seq analysis revealed a unknown function protein Rv0455c which may be involved in the process of siderophore secretion in Mtb. We deleted rv0455c gene from Mtb genome and the rv0455c deletion mutant is hypersensitive to exogenous mycobactin and carboxymycobactin. The whole cell lipidomic analysis further confirmed that loss of Rv0455c leads to decreased production of mycobactins and increased accumulation of carboxymycobactins in Mtb. Additionally, the subcellular localization showed that Rv0455c is a 14-kDa secreted protein. In the future, we will characterize the function of Rv0455c by in vitro and in vivo experi-ments.

62

Saturday Night Buffet Menu

A vegetarian option will be available

to those with dietary restrictions

63

Notes

64

The real

voyage of

discovery

consists not

in seeking

new

landscapes

but in

having new

eyes.

~ Marcel

Proust

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