Virtual Special Issue:

Bacterial

Pathogenesis

July 2012

Contents

Overview

Molecular Microbiology publishes papers that lead to a deeper understanding of molecular principles underlying microbiological processes, including those mediating interactions between microbes and their eukaryotic hosts.

This Virtual Special Issue on Bacterial Pathogenesis is a collection of articles that reveal mechanistic insight into how bacterial virulence factors are produced, secreted, and modified, how they interact with host cells and influence host responses, and how the genes encoding them are regulated. This Special Issue is designed to highlight Molecular Microbiology’s contributions to the field of bacterial pathogenesis and to stimulate scientific discussion in this area.

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo.

Alonzo, F., III, Benson, M.A., Chen, J., Novick, R.P., Shopsin, B., and Torres, V.J. (2012).

Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU.

Anderson, D.M., Schmalzer, K.M., Sato, H., Casey, M., Terhune, S.S., Haas, A.L., Feix, J.B., and Frank, D.W. (2011).

Involvement of multiple distinct Bordetella receptor proteins in the utilization of iron liberated from transferrin by host catecholamine stress hormones.

Armstrong, S.K., Brickman, T.J., and Suhadolc, R.J. (2012).

Cover Image by Michael Henderson and Peggy Cotter.

Contents

Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization.

Barrero-Tobon, A.M., and Hendrixson, D.R. (2012).

Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.

Bottai, D., Di Luca, M., Majlessi, L., Frigui, W., Simeone, R., Sayes, F., Bitter, W., Brennan, M.J., Leclerc, C., Batoni, G., et al. (2012).

The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer.

Diepold, A., Wiesand, U., and Cornelis, G.R. (2011).

Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.

Georgiadou, M., Castagnini, M., Karimova, G., Ladant, D., and Pelicic, V. (2012).

Characterization of the Group A Streptococcus Mga virulence regulator reveals a role for the C-terminal region in oligomerization and transcriptional activation.

Hondorp, E.R., Hou, S.C., Hempstead, A.D., Hause, L.L., Beckett, D.M., and McIver, K.S. (2012).

Arginine catabolic mobile element encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines.

Joshi, G.S., Spontak, J.S., Klapper, D.G., and Richardson, A.R. (2011).

Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes.

Pich, O.Q., Carpenter, B.M., Gilbreath, J.J., and Merrell, D.S. (2012).

A glycine betaine importer limits Salmonella stress resistance and tissue colonization by reducing trehalose production.

Pilonieta, M.C., Nagy, T.A., Jorgensen, D.R., and Detweiler, C.S. (2012).

Contents

The novel Listeria monocytogenes bile sensor BrtA controls expression of the cholic acid efflux pump MdrT.

Quillin, S.J., Schwartz, K.T., and Leber, J.H. (2011).

Either periplasmic tethering or protease resistance is sufficient to allow a SodC to protect Salmonella enterica serovar Typhimurium from phagocytic superoxide.

Rushing, M.D., and Slauch, J.M. (2011).

The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.

Shaw, D.K., Hyde, J.A., and Skare, J.T. (2011).

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

Strotmeier, J., Gu, S., Jutzi, S., Mahrhold, S., Zhou, J., Pich, A., Eichner, T., Bigalke, H., Rummel, A., Jin, R., et al. (2011).

Carbon storage regulator A (CsrABb) is a repressor of Borrelia burgdorferi flagellin protein FlaB.

Sze, C.W., Morado, D.R., Liu, J., Charon, N.W., Xu, H., and Li, C. (2011).

Regulation of Helicobacter pylori adherence by gene conversion.

Talarico, S., Whitefield, S.E., Fero, J., Haas, R., and Salama, N.R. (2012).

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst.

Talà, A., Monaco, C., Nagorska, K., Exley, R.M., Corbett, A., Zychlinsky, A., Alifano, P., and Tang, C.M. (2011).

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH.

Taylor, J.L., De Silva, R.S., Kovacikova, G., Lin, W., Taylor, R.K., Skorupski, K., and Kull, F.J. (2012).

The promoter architectural landscape of the Salmonella PhoP regulon.

Zwir, I., Latifi, T., Perez, J.C., Huang, H., and Groisman, E.A. (2012).

Summary

Bloodstream infection with Staphylococcus aureus is common and can be fatal.

However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined.

We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice.

We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue.

Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils.

The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis.

Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo.

Alonzo, F., III, Benson, M.A., Chen, J., Novick, R.P., Shopsin, B., and Torres, V.J.

Volume 83, Issue 2, January 2012, Pages: 423–435

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Summary

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients.

Our research has focused on the structure–activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa.

ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU.

Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system.

The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.

Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU.

Anderson, D.M., Schmalzer, K.M., Sato, H., Casey, M., Terhune, S.S., Haas, A.L., Feix, J.B., and Frank, D.W.

Volume 82, Issue, 6 , December 2011, Pages: 1454–1467

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Summary

Bordetella bronchiseptica is a pathogen that can acquire iron using its native alcaligin siderophore system, but can also use the catechol xenosiderophore enterobactin via the BfeA outer membrane receptor.

Transcription of bfeA is positively controlled by a regulator that requires induction by enterobactin. Catecholamine hormones also induce bfeA transcription and B. bronchiseptica can use the catecholamine noradrenaline for growth on transferrin.

In this study, B. bronchiseptica was shown to use catecholamines to obtain iron from both transferrin and lactoferrin in the absence of siderophore. In the presence of siderophore, noradrenaline augmented transferrin utilization by B. bronchiseptica, as well as siderophore function in vitro.

Genetic analysis identified BfrA, BfrD and BfrE as TonB-dependent outer membrane catecholamine receptors.

The BfeA enterobactin receptor was found to not be involved directly in catecholamine utilization; however, the BfrA, BfrD and BfrE catecholamine receptors could serve as receptors for enterobactin and its degradation product 2,3-dihydroxybenzoic acid. Thus, there is a functional link between enterobactin-dependent and catecholamine-dependent transferrin utilization.

This investigation characterizes a new B. bronchiseptica mechanism for iron uptake from transferrin that uses host stress hormones that not only deliver iron directly to catecholamine receptors, but also potentiate siderophore activity by acting as iron shuttles.

Involvement of multiple distinct Bordetella receptor proteins in the utilization of iron liberated from transferrin by host catecholamine stress hormones.

Armstrong, S.K., Brickman, T.J., and Suhadolc, R.J.

Volume 84, Issue 3, May 2012, Pages: 446–462

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Summary

The flagellum of Campylobacter jejuni provides motility essential for commensal colonization of the intestinal tract of avian species and infection of humans resulting in diarrhoeal disease.

Additionally, the flagellar type III secretion system has been reported to secrete proteins such as CiaI that influence invasion of human intestinal cells and possibly pathogenesis. The flagellar regulatory system ultimately influences σ28 activity required for expression of the FlaA major flagellin and other flagellar filament proteins.

In this work, we discovered that transcription of ciaI and four genes we propose annotating as feds (for flagellar coexpressed determinants) is dependent upon σ28, but these genes are not required for motility. Instead, the Feds and CiaI are involved in commensal colonization of chicks, with FedA additionally involved in promoting invasion of human intestinal cells.

We also discovered that the major flagellin influences production, stability or secretion of σ28-dependent proteins.

Specific transcriptional and translational mechanisms affecting CiaI were identified and domains of CiaI were analysed for importance in commensalism or invasion.

Our work broadens the genes controlled by the flagellar regulatory system and implicates this system in co-ordinating production of colonization and virulence determinants with flagella, which together are required for optimal interactions with diverse hosts.

Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization.

Barrero-Tobon, A.M., and Hendrixson, D.R.

Volume 84, Issue 2, April 2012, Pages: 352–369

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Summary

The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1–ESX-5).

While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses.

To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA5, eccD5, rv1794 and esxM genes or the ppe25-pe19 region.

Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family.

Strikingly, the MtbeccD5ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics.

When the virulence of the five mutants was evaluated, the MtbeccD5ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model.

Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation

Bottai, D., Di Luca, M., Majlessi, L., Frigui, W., Simeone, R., Sayes, F., Bitter, W., Brennan, M.J., Leclerc, C., Batoni, G., et al. (2012).

Volume 83, Issue 6, March 2012, Pages: 1195–1209

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Summary

YscV (FlhA in the flagellum) is an essential component of the inner membrane (IM) export apparatus of the type III secretion injectisome.

It contains eight transmembrane helices and a large C-terminal cytosolic domain.

YscV was expressed at a significantly higher level than the other export apparatus components YscR, YscS, YscT, and YscU, and YscV-EGFP formed bright fluorescent spots at the bacterial periphery, colocalizing in most cases with YscC-mCherry.

This suggested that YscV is the only protein of the export apparatus that oligomerizes. Oligomerization required the cytosolic domain of YscV, as well as YscR, -S, -T, but no other Ysc protein, indicating that an IM platform can assemble independently from the membrane-ring forming proteins YscC, -D, -J. However, in the absence of YscC, -D, -J, this IM platform moved laterally at the bacterial surface.

YscJ, but not YscD could be recruited to the IM platform in the absence of the secretin YscC. As YscJ was shown earlier to be also recruited by the outer membrane (OM) platform made of YscC and YscD, we infer that assembly of the injectisome proceeds through the independent assembly of an IM and an OM platform that merge through YscJ.

The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer

Diepold, A., Wiesand, U., and Cornelis, G.R.

Volume 82, Issue 2, October 2011, Pages: 502–514

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Summary

The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system.

In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery.

Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis.

We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact.

We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM–PilN interactions and pilus assembly.

Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.

Georgiadou, M., Castagnini, M., Karimova, G., Ladant, D., and Pelicic, V.

Volume 84, Issue 5, June 2012, Pages: 857–873

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Summary

The Group A Streptococcus (GAS) is a strict human pathogen that causes a broad spectrum of illnesses.

One of the key regulators of virulence in GAS is the transcriptional activator Mga, which co-ordinates the early stages of infection.

Although the targets of Mga have been well characterized, basic biochemical analyses have been limited due to difficulties in obtaining purified protein.

In this study, high-level purification of soluble Mga was achieved, enabling the first detailed characterization of the protein. Fluorescence titrations coupled with filter-binding assays indicate that Mga binds cognate DNA with nanomolar affinity.

Gel filtration analyses, analytical ultracentrifugation and co-immunoprecipitation experiments demonstrate that Mga forms oligomers in solution.

Moreover, the ability of the protein to oligomerize in solution was found to correlate with transcriptional activation; DNA binding appears to be necessary but insufficient for full activity.

Truncation analyses reveal that the uncharacterized C-terminal region of Mga, possessing similarity to phosphotransferase system EIIB proteins, plays a critical role in oligomerization and in vivo activity.

Mga from a divergent serotype was found to behave similarly, suggesting that this study describes a general mechanism for Mga regulation of target virulence genes within GAS and provides insight into related regulators in other Gram-positive pathogens.

Characterization of the Group Characterization of the Group A Streptococcus regulator reveals a role for the C-terminal region in oligomerization and transcriptional activation

Hondorp, E.R., Hou, S.C., Hempstead, A.D., Hause, L.L., Beckett, D.M., and McIver, K.S.

Volume 83, Issue 5, March 2012, Pages: 953–967

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Summary

Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis.

Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine.

Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiological concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine sensitivity.

However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of community-associated methicillin-resistant S. aureus (CA-MRSA).

We identified one gene within the USA-300-specific arginine catabolic mobile element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection; however, the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine hypersensitivity, a peculiar trait of S. aureus.

Arginine catabolic mobile element encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines

Joshi, G.S., Spontak, J.S., Klapper, D.G., and Richardson, A.R.

Volume 82, Issue 1, October 2011, Pages: 9–20

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Summary

In Helicobacter pylori, iron balance is controlled by the Ferric uptake regulator (Fur), an iron-sensing repressor protein that typically regulates expression of genes implicated in iron transport and storage.

Herein, we carried out extensive analysis of Fur-regulated promoters and identified a 7-1-7 motif with dyad symmetry (5′-TAATAATnATTATTA-3′), which functions as the Fur box core sequence of H. pylori.

Addition of this sequence to the promoter region of a typically non-Fur regulated gene was sufficient to impose Fur-dependent regulation in vivo.

Moreover, mutation of this sequence within Fur-controlled promoters negated regulation. Analysis of the H. pylori chromosome for the occurrence of the Fur box established the existence of well-conserved Fur boxes in the promoters of numerous known Fur-regulated genes, and revealed novel putative Fur targets.

Transcriptional analysis of the new candidate genes demonstrated Fur-dependent repression of HPG27_51, HPG27_52, HPG27_199, HPG27_445, HPG27_825 and HPG27_1063, as well as Fur-mediated activation of the cytotoxin associated gene A, cagA (HPG27_507).

Furthermore, electrophoretic mobility shift assays confirmed specific binding of Fur to the promoters of each of these genes.

Future experiments will determine whether loss of Fur regulation of any of these particular genes contributes to the defects in colonization exhibited by the H. pylori fur mutant.

Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes

Pich, O.Q., Carpenter, B.M., Gilbreath, J.J., and Merrell, D.S.

Volume 84, Issue 5, June 2012, Pages: 921–941

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Summary

Mechanisms by which Salmonella establish chronic infections are not well understood.

Microbes respond to stress by importing or producing compatible solutes, small molecules that stabilize proteins and lipids. The Salmonella locus opuABCD (also called OpuC) encodes a predicted importer of the compatible solute glycine betaine.

Under stress conditions, if glycine betaine cannot be imported, Salmonella enterica produce the disaccharide trehalose, a highly effective compatible solute.

We demonstrate that strains lacking opuABCD accumulate more trehalose under stress conditions than wild-type strains.

ΔopuABCD mutant strains are more resistant to high-salt, low-pH and -hydrogen peroxide, conditions that mimic aspects of innate immunity, in a trehalose-dependent manner. In addition, ΔopuABCD mutant strains require the trehalose production genes to out-compete wild-type strains in mice and macrophages.

These data suggest that in the absence of opuABCD, trehalose accumulation increases bacterial resistance to stress in broth and mice.

Thus, opuABCD reduces bacterial colonization via a mechanism that limits trehalose production. Mechanisms by which microbes limit disease may reveal novel pathways as therapeutic targets.

A glycine betaine importer limits Salmonella stress resistance and tissue colonization by reducing trehalose production

Pilonieta, M.C., Nagy, T.A., Jorgensen, D.R., and Detweiler, C.S.

Volume 84, Issue 2, April 2012, Pages: 296–309

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Summary

Mammalian bile has potent anti-microbial activity, yet bacterial pathogens of the gastrointestinal tract and hepatobiliary system nonetheless persist and replicate within bile-rich environments.

Listeria monocytogenes, a Gram-positive pathogen, encounters bile at three stages throughout its infectious cycle in vivo: in the gut during initial infection, in the liver where it replicates robustly and in the gallbladder, from which it can return to the intestine and thence to the environment.

The mechanisms by which L. monocytogenes senses mammalian bile and counteracts its bactericidal effects are not fully understood. In this report, we have determined the L. monocytogenes bile-induced transcriptome, finding that many critical virulence factors are regulated by bile.

Among these, the multidrug efflux pumps MdrM and MdrT, previously shown to be critical for the bacterial provocation of a pathogenesis-promoting host innate immune response, are robustly and specifically induced by the bile component cholic acid.

This induction is mediated by BrtA, the first identified L. monocytogenes sensor of bile, which loses the ability to bind to and repress the mdrT promoter in the presence of cholic acid.

We show that MdrT can export cholic acid, and that ΔmdrT bacteria are significantly attenuated both in vitro when exposed to cholic acid or bile, and in vivo in the gallbladders and livers of infected mice.

The novel Listeria monocytogenes bile sensor BrtA controls expression of the cholic acid efflux pump MdrT

Quillin, S.J., Schwartz, K.T., and Leber, J.H.

Volume 81, Issue 1, July 2011, Pages: 129–142

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Summary

Salmonella Typhimurium combats phagocytic superoxide by producing the periplasmic superoxide dismutase, SodCI. The homologous protein, SodCII, is also produced during infection, but does not contribute to virulence.

The proteins physically differ in that SodCI is dimeric, protease resistant and non-covalently tethered within the periplasm. Conversely, SodCII is a protease-sensitive monomer that is released normally from the periplasm by osmotic shock.

To identify which properties correlate with virulence, we constructed over 20 enzymatically functional hybrid SodC proteins and assayed them for protease susceptibility, release by osmotic shock, multimerization and affinity for metal cofactors. Protease susceptibility maps to the C-terminus of SodCII, while SodCI residues 120–131 are required for tethering.

A protease-resistant SodCII hybrid was able to substitute for SodCI during infection. Interestingly, a tethered but protease-sensitive SodCII hybrid was also able to confer protection. Thus, either tethering or protease resistance is sufficient for a SodC to function during infection.

These results support our model that in the macrophage, the outer membrane of Salmonella is partially disrupted by antimicrobial peptides. Periplasmic proteins, including SodCII, are released and/or phagocytic proteases gain access. SodCI is both tethered within the periplasm and protease resistant, thereby surviving to detoxify superoxide.

Either periplasmic tethering or protease resistance is sufficient to allow a SodC to protect Salmonella enterica serovar Typhimurium from phagocytic superoxide

Rushing, M.D., and Slauch, J.M.

Volume 82, Issue 4, November 2011, Pages: 952–963

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Summary

The etiological agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes genus and, if untreated, can cause significant morbidity in affected individuals.

Recent reports have shown that polyunsaturated fatty acids in the B. burgdorferi cell envelope are potential targets for oxidative damage, which can be lethal.

How B. burgdorferi responds to this assault is not known.

Herein we report evidence that bb0646 codes for a lipase that is located within the bosR operon and that has specificity for both saturated and polyunsaturated fatty acids.

Specifically, strains harbouring mutated copies of the lipase, either in the form of an insertionally inactivated construct or site-directed mutations within the active site, demonstrated attenuated lipolytic and haemolytic phenotypes when compared with the isogenic parent and trans-complements.

In vivo analysis showed that while the bb0646 mutant remains infectious, the spirochaetal load is significantly lower than both the isogenic parent and the complemented mutant strains.

Taken together, these data demonstrate that BB0646 is a broad substrate specific lipase that contributes to lipolytic and haemolytic activity in vitro and is required for optimal B. burgdorferi infection.

The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease

Shaw, D.K., Hyde, J.A., and Skare, J.T.

Volume 83, Issue 2, January 2012, Pages: 319–334

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Summary

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons.

Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein.

Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid.

The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively.

Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction.

Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites.

These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites

Strotmeier, J., Gu, S., Jutzi, S., Mahrhold, S., Zhou, J., Pich, A., Eichner, T., Bigalke, H., Rummel, A., Jin, R., et al.

Volume 81, Issue 1, July 2011, Pages: 143–156

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Summary

The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive.

A recent study reported that the increased level of BB0184 (CsrABb; a homologue of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi.

In this report, we deciphered the regulatory role of CsrABb on FlaB synthesis and the mechanism involved by analysing two mutants, csrABb

- (a deletion mutant of csrABb) and csrABb+ (a mutant conditionally

overexpressing csrABb).

We found that FlaB accumulation was significantly inhibited in csrABb+ but was substantially increased in

csrABb-. In contrast, the levels of other flagellar proteins remained unchanged.

Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrABb in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine–Dalgarno sequence.

We found that CsrABb bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrABb specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.

Carbon storage regulator A (CsrABb) is a repressor of Borrelia burgdorferi flagellin protein FlaB

Sze, C.W., Morado, D.R., Liu, J., Charon, N.W., Xu, H., and Li, C.

Volume 82, Issue 4, November 2011, Pages: 851–864

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Summary

Genetic diversification of Helicobacter pylori adhesin genes may allow adaptation of adherence properties to facilitate persistence despite host defences.

The sabA gene encodes an adhesin that binds sialyl-Lewis antigens on inflamed gastric tissue.

We found variability in the copy number and locus of the sabA gene and the closely related sabB and omp27 genes due to gene conversion among 51 North American paediatric H. pylori strains.

We determined that sabB to sabA gene conversion is predominantly the result of intra-genomic recombination and RecA, RecG and AddA influence the rate at which it occurs.

Although all clinical strains had at least one sabA gene copy, sabA and sabB were lost due to gene conversion at similar rates in vitro, suggesting host selection to maintain the sabA gene.

sabA gene duplication resulted in increased SabA protein production and increased adherence to sialyl-Lewis antigens and mouse gastric tissue.

In conclusion, gene conversion is a mechanism for H. pylori to regulate sabA expression level and adherence.

Regulation of Helicobacter pylori adherence by gene conversion

Talarico, S., Whitefield, S.E., Fero, J., Haas, R., and Salama, N.R.

Volume 84, Issue 6, June 2012, Pages: 1050–1061

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Summary

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells.

Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire l-glutamate through its GltT uptake system.

We demonstrate that the uptake of available l-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst.

In the meningococcus, l-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide.

We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst

Talà, A., Monaco, C., Nagorska, K., Exley, R.M., Corbett, A., Zychlinsky, A., Alifano, P., and Tang, C.M.

Volume 81, Issue 5, September 2011, Pages: 1330–1342

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Summary

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB.

AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression.

We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations.

Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA.

We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen.

These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals.

We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding.

Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH

Taylor, J.L., De Silva, R.S., Kovacikova, G., Lin, W., Taylor, R.K., Skorupski, K., and Kull, F.J.

Volume 83, Issue 3, February 2012, Pages: 457–470

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Summary

The DNA-binding protein PhoP controls virulence and Mg2+ homeostasis in the Gram-negative pathogen Salmonella enterica serovar Typhimurium.

PhoP regulates expression of a large number of genes that differ both in their ancestry and in the biochemical functions and physiological roles of the encoded products. This suggests that PhoP-regulated genes are differentially expressed.

To understand how a bacterial activator might generate varied gene expression behaviour, we investigated the cis-acting promoter features (i.e. the number of PhoP binding sites, as well as their orientation and location with respect to the sites bound by RNA polymerase and the sequences that constitute the PhoP binding sites) in 23 PhoP-activated promoters.

Our results show that natural PhoP-activated promoters utilize only a limited number of combinations of cis-acting features – or promoter architectures.

We determine that PhoP activates transcription by different mechanisms, and that ancestral and horizontally acquired PhoP-activated genes have distinct promoter architectures.

The promoter architectural landscape of the Salmonella PhoP regulon

Zwir, I., Latifi, T., Perez, J.C., Huang, H., and Groisman, E.A.

Volume 84, Issue 3, May 2012, Pages: 463–485

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