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Promotion and Rescue of Intracellular Brucella neotomae Replication During 1
Co-Infection With Legionella pneumophila 2
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Short title: Rescue of Brucella growth by Legionella 4
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Yoon-Suk Kang1 & James E. Kirby1,* 6
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Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 8
Boston, MA, United States of America 9
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*Corresponding author 12
Email: [email protected] 13
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IAI Accepted Manuscript Posted Online 6 March 2017Infect. Immun. doi:10.1128/IAI.00991-16Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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Abstract 15
We established a new Brucella neotomae in vitro model system for study of type IV secretion 16
system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a 17
rodent pathogen and, unlike B. abortus, melitensis, and suis, has not been observed to infect 18
humans. It therefore can be handled more facilely using biosafety level 2 practices. More 19
particularly, using a series of novel fluorescent protein and lux operon reporter systems to 20
differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent 21
intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae 22
exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated 23
phagosome maturation. These findings recapitulate prior observations for human pathogenic 24
Brucella spp. In addition, during co-infection experiments with L. pneumophila, we found that 25
defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline 26
levels of intracellular replication of wild type B. neotomae significantly stimulated by co-27
infection with wild type, but not T4SS mutant L. pneumophila. Using confocal microscopy, it 28
was determined that intracellular co-localization of B. neotomae and L. pneumophila was 29
required for rescue and that colocalization came at a cost to L. pneumophila fitness. These 30
findings were not completely expected based on known temporal and qualitative differences in 31
intracellular life cycles of these two pathogens. Taken together, we have developed a new 32
system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent 33
interplay between Brucella and Legionella during macrophage co-infection. 34
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Introduction 36
Brucella are Gram-negative α-Proteobacteria that cause chronic, systemic infections in 37
mammals and zoonotic infections in humans (1). These pathogens are known to infect the 38
reticuloendothelial system and proliferate significantly in macrophage rich organs such as liver, 39
spleen, and bone marrow. Chronic, often debilitating bloodstream infection is typical. In 40
humans, chronic course punctuated by spikes in body temperature is underscored by the 41
descriptive name, undulant fever, given to disease caused by these organisms. Endovascular 42
infection and osteomyelitis are concerning sequelae. Humans may acquire Brucella from 43
airborne exposure related to high quantities of organisms shed from birthing livestock or from 44
ingesting unpasteurized dairy products. 45
B. abortus, B. melitensis, and B. suis, the species responsible for human zoonotic 46
infection, are facultative intracellular pathogens (1). Intracellular growth is thought critical to 47
successful infection of the host. More particularly, each of these pathogens deploys a type IV 48
secretion system (T4SS), a molecular syringe, to inject virulence factors into host cells and 49
establish a productive intracellular growth niche (2, 3). Specifically, through use of its T4SS, 50
each Brucella species orchestrates remodeling of its phagosome which proceeds in temporal 51
fashion to take on properties first of endosomal compartments; then of endoplasmic reticulum (4), 52
where intracellular growth initially takes place; and finally of autophagosomes, during 53
completion of an intracellular replication cycle (5). Virulence factors injected by the T4SS, that 54
presumably are responsible for this altered phagosome maturation, are still being defined. 55
Based on infectivity, respiratory mode of acquisition, and chronic and potentially life 56
threatening disease manifestations, zoonotic Brucella species are generally classified as biosafety 57
level 3 pathogens. B. abortus, B. melitensis, and B. suis are also considered potential biothreat 58
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agents, and are designated as Overlap Select Agents by the Human Health Services and United 59
States Department of Agriculture (http://www.selectagents.gov/SelectAgentsandToxinsList.html) 60
and as Category B Priority Agents by the United States National Institutes for Allergy and 61
Infectious Diseases (https://www.niaid.nih.gov/research/emerging-infectious-diseases-62
pathogens). The need for select agent and biosafety level 3 precautions has made research on this 63
fascinating genus more difficult. Ideally, development of new models using species that 64
recapitulate infection at the cellular level, but which do not infect humans, would be ideal. 65
Currently, three Brucella species are designated as biosafety level 2 pathogens by the American 66
Type Culture Collection (http://www.atcc.org) (B. neotomae and B. ovis) and the Czech 67
Collection of Microorganisms (http://www.sci.muni.cz/ccm/i) (B. microti). B. neotaomae and B. 68
microti are rodent pathogens. B. ovis is primarily a sheep pathogen. Notably, B. neotomae is not 69
generally considered a human pathogen (6, 7), yet still shares high genetic homology with 70
zoonotic Brucella species (8, 9). As such, its use as a model pathogen may hold promise. 71
Therefore, our goal was to establish Brucella neotomae (Bn) as a new model system for 72
investigation of in vitro pathogenesis and to take advantage of its biosafety level 2 status to 73
accelerate experimental work. To this end, we examined T4SS dependence of intracellular 74
growth. Furthermore, we explored interaction with several pathogens during co-infection 75
experiments, most importantly, the T4SS-dependent pathogen, Legionella pneumophila (Lp). Of 76
note, L. pneumophila also alters normal endocytic trafficking through use of an unrelated T4SS 77
and, in doing so, establishes an endoplasmic reticulum-associated replicative niche with 78
morphological similarity to the Brucella replicative vacuole (10-12). Complementary 79
experiments using novel reporter strains revealed an unexpected and dramatic co-operative 80
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interaction between Legionella pneumophila and Brucella neotomae during co-infection 81
experiments. 82
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Materials & Methods 84
Bacterial strains and cell lines. Bacterial strains, plasmids, and eukaryotic cell lines used in 85
this study are listed in Table 1. Escherichia coli NEB-5α, NEB-10β (New England Biolabs, 86
Ipswich, MA), and BW25113 (E. coli Genetic Stock Center, Yale University, New Haven, CT) 87
(13) were grown at 37 °C with shaking in Luria broth (LB) medium (BD, Franklin Lakes, NJ). 88
Brucella neotomae 5K33 was obtained from BEI Resources (NIAID, NIH) and used for strain 89
construction. Brucella strains were grown at 37 °C in a humidified incubator (5% CO2) in 90
trypticase soy broth (TSB) medium (BD). Staphylococcus aureus 25923 (American Type Culture 91
Collection, Manassas, VA) was cultured at 37 °C in LB medium with 5% CO2. Legionella 92
pneumophila Lp02 flaA (thyA, hsdR, and rpsL) and Lp03 flaA (Lp02 dotA flaA) strains (14-16) 93
were grown at 37 °C on buffered charcoal yeast extract (BCYE) (17) medium supplemented with 94
100μg/ml thymidine. Bacterial optical density was monitored at 600 nm using a DU 800 95
spectrophotometer (Beckman Coulter, Brea, CA) or an Epoch microtiter plater reader (BioTek, 96
Winooski, VT). 97
E. coli and B. neotomae were grown with 100 µg/ml spectinomycin (Spec), 20 µg/mL 98
phleomycin (Phl), 50 µg/ml nourseothricin (Ntc), 50 µg/ml kanamycin (Km), 50 µg/ml 99
hygromycin (Hyg), or 100 µg/ml ampicillin (Amp) to select for cognate resistance markers. 100
BCYE was supplemented with 200 µg/ml Phl or 50 μg/ml Ntc to select for resistance markers in 101
L. pneumophila strains. Murine J774A.1 (ATCC TIB-67) and human THP-1 (ATCC TIB-202) 102
macrophage cell lines were passaged in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) 103
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containing 9 % iron-supplemented, heat-inactivated calf serum (GemCell, Gemini Bio-Products, 104
West Sacramento, CA) (referred to hereafter as RPMI) in a humidified, 5% CO2 incubator at 105
37 °C. 106
107
Construction of bio-reporter expression or transposon plasmids. Plasmid isolation, gel 108
electrophoresis, transformation, and PCR amplification of DNA were performed as previously 109
described (18). The broad-host-range plasmid, pAT28 (19), was used as a vector backbone for 110
plasmid-borne, bio-reporter expression in Gram-negative and Gram-positive bacterial cells (see 111
Fig. S1). The S. aureus codon-optimized eGFP gene (SaEGFP), synthesized de novo as a gBlock 112
by Integrated DNA Technologies (Coralville, IA), and GP-specific lux operon (luxABCDE)(20) 113
from plasmid, pMV306G13+Lux (21), obtained from Addgene (Cambridge, MA), were PCR 114
amplified using extended PCR primers for incorporation of a ribosome-binding sequence 115
(AGGAGG-) and EcoRI, and SalI restriction sites at 5' and 3’ends, respectively. These gene 116
fragments were then cloned into corresponding restrictions sites in pAT28 to create promoterless 117
versions of these reporters. The constructs were then further cut with EcoRI, dephosphorylated, 118
and ligated to a synthetic proD promoter sequence (22) with compatible cohesive ends. The 119
presence and orientation of the proD promoter was confirmed by Sanger sequencing. 120
The transposon plasmid, pMOD3 (Epicentre Biotechnologies, Madison, WI), 121
diagrammed in Fig S1, was used for chromosomal integration of bio-reporters. The tdTomato, 122
mClover, mWasabi, mCardinal, and mNeptune2 fluorescent proteins were codon-optimized for 123
high-level expression in E. coli and through selective elimination of very rare frequency codons 124
in Brucella neotomae (Bn). The genes were then synthesized de novo as gBlock fragments for 125
cloning into pMOD3-based transposons. The proD/tdTomato-nat vector and lux operon vectors 126
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were first constructed in a single step using the Gibson assembly method (23) from three 127
separate DNA fragments: (a) a pMOD3 vector fragment containing R6Kγori, AmpR, the ColE1 128
ori, and bordering 19 bp-Mosaic End sequences; (b) a nat fragment encoding a codon-optimized 129
nourseothricin resistance gene; and (c) a codon-optimized tdTomato gene or a PCR-amplified 130
luxCDABE fragment from pUWGR4 (24). Gibson assembly was performed using a commercial 131
Gibson assembly kit (New England Biolabs) and a mixture of 0.1 pmol of vector fragment, 0.3 132
pmol of NAT, and 0.3 pmol of bio-reporter sequence. Following incubation at 50 °C for 30 133
minutes, each vector construct mixture was transformed into E. coli NEB-5α. Other synthetic 134
reporter genes were PCR amplified and cloned directly into the pMOD3 vector using EcoRI and 135
SalI restriction sites. Selectable markers, nat (conferring nourseothricin resistance), Sh ble 136
(conferring phleomycin resistance) and hph (conferring hygromycin resistance) were inserted 137
downstream of reporters using SalI and HindIII sites to create an operon structure. The proD 138
promoter, EcoRI fragment was inserted upstream of fluorescent protein reporter operons in 139
designated vectors. 140
141
Chromosomal integration of reporter transposons. Following construction, transposon 142
reporter DNA sequence was isolated by digestion of the pMOD3 plasmids with PvuII, which 143
cuts immediately outside of the 19 bp Mosaic Ends of the transposon constructs. Reporter 144
transposons were then gel purified using a Gel Extraction Kit (Qiagen, Valencia, CA). 200 ng of 145
purified transposon were combined and incubated with EZ-Tn5™ transposase (Epicentre, 146
Madison, WI) per the manufacturer's instructions. 1 µL of each transposome complex was then 147
incubated with electrocompetent bacteria on ice for 5 min (E. coli, Lp02, and Lp03) or 30 min (B. 148
neotomae) and electroporated into respective host bacterial cells. Electroporation was performed 149
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with 1-mm gap cuvettes using a Bio-Rad MicroPulser (Bio-Rad, Hercules, CA) with settings of 150
1.5 kV, 25 µF, and 200 Ω for E. coli; 1.8 kV, 25 µF, and 200 Ω for Lp; and 2.0 kV, 25 µF, and 151
400 Ω for Bn. After electroporation, 1 mL of appropriate media was added to the electroporation 152
mixture, and bacterial cells were incubated at 37 °C for 1 hr (E. coli), 7 hr (Lp), or 24 hr (Bn) to 153
allow for transposition and resistance marker expression. Bacterial suspensions were spread on 154
agar plates containing appropriate selection agents to isolate transposon integrants in different 155
species of interest. 156
157
Generation of T4SS deletion mutant in B. neotomae and complementation. To generate the 158
ΔvirB4 in-frame deletion mutant in Bn, we cloned two regions of genomic DNA bracketing the 159
N- and C-terminal regions of the virB4 gene using the overlap extension PCR method (25). The 160
in-frame deletion construct obtained was digested with BamHI and SacI; ligated into the pSR47s 161
suicide vector (26); and transformed into EC100D pir+ (Epicenter Technologies). The resulting 162
strain was conjugated with Bn through triparental mating by mixing suspensions of the donor, 163
recipient, and an E. coli strain containing the pRK600 helper plasmid (27) on a solid agar surface 164
and incubating for 24 hours at 37 °C. The mating mixture was then spread on trypticase soy agar 165
(TSA) plates containing 50 µg/mL kanamycin and 50 µg/mL aztreonam to select for the 166
recipient and plasmid integrants. The transconjugant integration was confirmed by PCR 167
amplification. Counterselection for the double reciprocal recombinatorial deletion of the virB4 168
gene was accomplished on TSA plates containing 10 % sucrose. After 5 days of incubation, the 169
colonies were screened by PCR for loss of the kanamycin resistance and generation of the 170
chromosomal ΔvirB4 in-frame deletion. 171
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For complementation of the virB4 gene deletion, the amplified virB4 DNA fragment was 172
digested with XbaI and EcoRV and ligated with identically digested pBMTL2 vector (28) 173
obtained from Addgene. The constructed complementation plasmid was electroporated into a 174
ΔvirB4 transposant expressing the proD/tdTomato reporter. The complemented strain was 175
selected on a TSA plate containing kanamycin and nourseothricin. For complementation 176
experiments, the strain was grown overnight in liquid medium containing the same antibiotics 177
prior to infection of J774A.1 or THP-1 cells. 178
179
LAMP-1 and calreticulin colocalization experiments. LAMP-1 (from plasmid, Lamp1-YFP, 180
Addgene #1816 (29)) and calreticulin (from plasmid, pmTurquoise2-ER, Addgene #36204 (30)) 181
genes were cloned, and ligated to the mTurquoise2 fluorescent protein reporter, also from 182
pmTurquoise2-ER, to create fusion proteins. The fusions were inserted into the BamHI/EcoRI 183
restriction sites of the pRetroX-Tet-Off Advanced Vector (Clontech, Mountain View, CA), 184
replacing the rtTA-advanced gene. The corresponding retroviral constructs were then transfected 185
into the Gryphon Ecotropic Packaging Cell Line for Retrovirus (Allele Biotech, San Diego, CA) 186
using Lipofectamine LTX (ThermoFisher Scientific, Waltham, MA) according to the 187
manufacturer's instructions. Three days after transfection, viral supernatant was harvested and 188
incubated with J774A.1 macrophages for 3 days. Macrophages were then washed twice with 189
PBS and replated in 6-well plates with RPMI medium. G-418 (Sigma Aldrich, St. Louis, MO) at 190
1,000 μg ml-1 final concentration was added to each well for selection. Individual colonies 191
expressing LAMP-1 or calreticulin fluorescent reporter fusion proteins were expanded and 192
frozen in RPMI containing 10 % dimethyl sulfoxide. Each cell line was passaged in RPMI 193
containing 500 μg ml-1 G-418 until use in experiments. 194
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195
Cell-culture microplate infection experiments. The murine J774A.1 macrophage cell line was 196
seeded into white, flat-bottom, 96-well plates (Greiner Bio-One, Monroe, NC) at a density of 1 x 197
105 cells per well. THP-1 suspension cells were seeded in the same way with the addition of 100 198
nM 1,25-dihydroxyvitamin D3 to induce macrophage differentiation. One day following cell 199
attachment, macrophage cells were co-infected at a multiplicity of infection (MOI) of 1 with 200
luminescent Bn wt or Bn ΔvirB4, and fluorescent-Lp02, Lp03, or S. aureus cells. Plates were 201
then immediately centrifuged at 930 x g for ten minutes to synchronize bacterial infection. Four 202
hours post-infection, gentamicin (100 µg ml-1 final concentration) was added to wells, and plates 203
were incubated for an additional hour. Plates were then washed two times with PBS without 204
Ca2+ or Mg2+. Immediately afterwards, day 0 luminescence or fluorescence was measured. 205
Macrophages were then further incubated and assessed for luminescence and fluorescence at 206
indicated time points using an EnVision multi-mode reader (PerkinElmer, Akron, OH). Further 207
details on instrument settings are supplied in supplementary materials. Alternatively, 208
macrophage wells were lyzed at indicated time points with 0.2% saponin in PBS, and serial 209
dilutions were plated on media for colony forming unit (CFU) determination . 210
211
Confocal Microscopy. J774A.1 cells were cultured on 12 mm-round glass cover slips (Warner 212
Instruments, Hamden, CT) in Corning 3513 Costar 12-well plates (Fisher Scientific, Waltham, 213
MA). After reaching 70 to 80 % confluence, macrophages were infected at an MOI of 1 with 214
either single or combinations of bacterial species. Plates were then immediately centrifuged for 215
10 min at 930 x g to synchronize bacterial invasion. After four hours of incubation, 100 µg ml-1 216
gentamicin was added to wells for 1 hour to kill extracellular bacteria. Each coverslip was then 217
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washed three times with PBS without Ca2+ and Mg2+, and fresh RPMI was then added. 218
Coverslips were harvested at 4 h, 24 h, and 48 h post-infection, and fixed at room temperature 219
for 30 min with 10 % formalin buffered with 1X PBS containing Ca2+ and Mg2+. After washing 220
3 times with PBS, coverslips were incubated with PBS containing 0.2 % Triton-X 100 for 15 min. 221
Coverslips were then washed 3 times with PBS and mounted with ProLong Gold (Invitrogen, 222
Carlsbad, CA). Images were acquired with a Zeiss LSM 880 confocal microscope (Carl Zeiss 223
Microscopy, Thornwood, NY) using the eGFP settings for detection of mClover or mWasabi; the 224
tdTomato settings for tdTomato; and mPlum settings for mCardinal or mNeptune2. Area 225
measurements were determined using the microscope's ZEN blue or lite software (Carl Zeiss). In 226
these experiments, co-infected J774A.1 cells were chosen randomly, and the area (nm2) of the 227
LCV and BCV was calculated based on the contours of respective reporter signal in confocal 228
images. 229
For experiments investigating co-localization of Bn with LAMP-1 and calreticulin, we 230
similarly infected J774A.1 LAMP-1::mTurquoise2 or J774A.1 mTurquoise2::calreticulin 231
transfectants at an MOI of 1. At indicated time points, coverslips were fixed as described above. 232
Images were evaluated for colocalization using the Zeiss LSM 880 tdTomato and eCFP confocal 233
settings for detection of bacteria and mTurquoise2 fusion protein signal, respectively. 234
235
Statistical analysis Statistical significance was determined in Prism 7 (Graphpad, Inc., La Jolla, 236
CA) using the two-tailed, Mann-Whitney U test for continuous data and Fisher's Exact test for 237
categorical data. A P < 0.01 was considered statistically significant. 238
239
Results 240
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Intracellular growth and type IV secretion system-dependence of B. neotomae. 241
To establish a biosafety level 2 Brucella in vitro model system, we considered use of 242
three available species: B. neotomae, B. microti, and B. ovis. All organisms are facultative 243
intracellular pathogens. However, B. ovis and the two available B. microti species did not grow 244
discernibly more in the presence of J774A.1 macrophage cells than in tissue culture medium 245
alone (Chiaraviglio and Kirby, data not shown). In contrast, B. neotomae grew >50-fold more in 246
the presence of eukaryotic cells. This intracellular growth selectivity will presumably provide 247
advantage in studying intracellular growth phenotypes in long-term tissue culture experiments 248
where adventitious growth of extracellular organisms may confound experimental interpretation. 249
Furthermore, in contrast to B. neotomae and human pathogenic Brucella species, B. ovis lacks O-250
polysaccharide side chains potentially making its use as a model pathogen less desirable (31-33). 251
Therefore, B. neotomae was chosen for further model development. 252
Notably, in an experiment in which infected J744A.1 macrophages were treated with 253
gentamicin after a short infection period to kill extracellular organisms, intracellular bacterial 254
colony forming units increased in number by approximately ~30-fold during a 48 h incubation 255
period (Fig. 1A). To enable more facile analysis of this intracellular growth, we created a bio-256
reporter toolkit to label Brucella with optimized, spectrally distinct reporters. In this way, we 257
would be able to conveniently track intracellular growth using fluorescence or luminescence 258
output, and study cell biology and interaction with other pathogens expressing complementary 259
reporters. Further details and validation of reporter constructs are provided in Supplemental 260
Materials, Supplemental Methods, Supplemental Tables S1-S2, and Supplemental Figures S1-S4. 261
Briefly, both bacterial lux operon and fluorescent proteins constructs were driven by a strong 262
ProD promoter. This previously described synthetic, insulated promoter, originally developed 263
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for high level expression in E. coli (22), was found to drive substantial reporter expression in 264
both Gram-negative and -positive organisms from several different species. The codon-265
optimized reporters (including mClover, tdTomato, mNeptune2) and the Photorhabdus 266
luminescens lux operon were linked in synthetic operons to non-antibiotic selectable markers to 267
allow selection in the several species ultimately examined. The reporters were either inserted via 268
transposition in single copy into the bacterial genome of target organisms or expressed from a 269
multi-copy, broad-host-range plasmid (S. aureus only). 270
Using these reporter strains, we first tested whether a single copy proD:lux and 271
proD:tdTomato reporters could be used to characterize intracellular growth of Brucella neotomae 272
(Bn) and to test whether this growth was dependent on its T4SS. Notably, the virB4 gene was 273
shown previously to be absolutely required for T4SS function in other Brucella species (2, 3, 34). 274
We therefore created a virB4 in-frame deletion mutant through standard double reciprocal 275
homologous recombination as previously described (35). Wt and ΔvirB4 mutant strains were 276
then marked through transposition with lux operon and tdTomato reporter constructs, and 277
intracellular growth phenotypes were evaluated in murine and human macrophage cell lines, 278
J774A.1 and THP-1. After initial infection, extracellular bacteria were killed through treatment 279
with gentamicin to prevent extracellular replication in surrounding medium. Therefore, increase 280
in reporter output over time should predominantly reflect intracellular growth alone. 281
Notably, a > 4.5 to 8.6-fold increase in luminescence and fluorescence signal was 282
observed during a 48 hour infection with wild type, reporter-marked organisms (Fig. 1B,C). In 283
contrast, only a relatively small increase in luminescence and fluorescence signal was observed 284
during infection with the isogenic Bn ΔvirB4 mutant. Importantly, complementation of the 285
ΔvirB4 mutant with a plasmid expressing a cloned virB4 gene rescued the intracellular growth 286
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defect (Fig 1C, VirB4c), supporting linkage of the ΔvirB4 mutation with the observed 287
intracellular growth defects. These results indicate that the virB4 gene is required for 288
intracellular growth of Bn and that Bn is a type IV secretion system (T4SS)-dependent pathogen. 289
We suspect that the lower absolute increase in reporter signal (Fig. 1B, C) compared to CFU 290
counts (Fig. 1A) in parallel assays likely reflects compression of reporter output during the 291
intracellular growth cycle. Despite compression of scale, RLU or RFU measurements and CFU 292
for Bn wt were highly correlated (R2 of 0.98 and 0.96, respectively). Therefore, the reporter-293
labeled organisms provide a facile and predictive tool for real-time assessment of intracellular 294
bacterial growth (36, 37). 295
Based on known temporal association of phagosomes containing human pathogenic 296
Brucella spp. with endosomal and endoplasmic reticulum markers (3-5, 38), colocalization 297
phenotypes of Bn wt and ΔvirB were likewise examined. Colocalization was assessed using 298
permanently transfected J774A.1 cell lines expressing LAMP-1 (a late endosomal and lysosomal 299
associated protein)::mTurquoise2 or mTurquoise2::calreticulin (an endoplasmic reticulum 300
associated protein) fusion proteins, respectively. As described previously for human pathogenic 301
species, wt Bn colocalized at early time points with the late endosomal marker, LAMP-1 (Fig 302
2A). At later time points, colocalization with LAMP-1 significantly decreased and association 303
with the endoplasmic reticulum marker, calreticulin, significantly increased (P < 0.0001 at 24 304
and 48 h post infection) compared to the Bn ΔvirB4 strain (Fig. 2A, B). In contrast, the Bn 305
ΔvirB4 strain exhibited high colocalization with LAMP-1 and low colocalization with 306
calreticulin throughout the 48 h infection period. Representative colocalization images are 307
shown in Fig 2C. 308
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We next considered whether co-infection with other pathogens might influence 309
intracellular replication of Bn, testing pairwise co-infection of macrophages with fluorescent 310
protein reporter-labeled Legionella pneumophila (Lp), Bn, and Staphylococcus aureus. 311
Although not generally considered an intracellular pathogen, S. aureus was included based on 312
reports that it can take up prolonged residence in macrophage vacuoles (8, 39, 40) and might 313
therefore potentially also interact with Lp or Bn. As shown in Fig. 3, we simultaneously co-314
infected macrophages with Bn proD/tdTomato and S. aureus pAT28-proD/eGFP; Bn 315
proD/tdTomato and Lp02 proD/mClover; or S. aureus pAT28-proD/eGFP and Lp02 316
proD/mNeptune2. At 24 h post-infection with an MOI of 10, we easily visualized by confocal 317
microscopy two distinct bacterial pathogens within eukaryotic cells for each pairing. 318
Interestingly, Bn and Lp02, and Bn and S. aureus co-localized with some frequency, presumably 319
within the same phagosomal compartment. This contrasted with the S. aureus and Lp02 co-320
infection where co-localization was vanishingly rare. These results implied that pathogens in the 321
first two pairings may occupy the same intracellular niche at some point during their intracellular 322
life cycle and therefore may potentially influence one another. 323
324
Co-infection with Lp rescues intracellular, replication-defective Bn ΔvirB4 and promotes 325
growth of wt Bn 326
We therefore made further use of reporter-labeled organisms to investigate the reciprocal 327
influence of pathogen pairings on intracellular growth. J774A.1 macrophages were co-infected 328
at an MOI of 1 with luminescent Bn wt or an isogenic ΔvirB4 mutant, and either S. aureus 329
(pAT28-proD/eGFP) or L. pneumophila Lp02 or Lp03 (proD-mClover transposants). Lp02 and 330
Lp03 are previously well-characterized, isogenic strains of Lp that are T4SS competent and 331
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incompetent, respectively (15, 16). Luminescence and fluorescence were recorded during a 48 332
hour infection using a multi-mode microplate reader (Fig. 4A). Notably, Lp02, but not Lp03, 333
significantly stimulated growth of luminescent Bn wt or ΔvirB4 strains conferring a 12.7- or 334
24.7-fold increase (P= 0.002) in growth signal, respectively, at 24 h post infection (p.i.) and a 335
11.4- or 48.9-fold increase (P = 0.002) in growth signal, respectively, at 48 h p.i. relative to 336
infection with either Bn wt or ΔvirB4 strains alone. In contrast, Bn and ΔvirB4 did not 337
differentially affect growth of fluorescent protein reporter-labeled Lp02 or Lp03 (Fig 4B). In 338
addition, S. aureus did not affect reporter signal from Bn, nor did Bn wt or ΔvirB4 affect S. 339
aureus reporter signal (data not shown). In a parallel experiment using the same assay 340
conditions and assessed by CFU determination (Fig 4D), growth of Bn ΔvirB4 was stimulated 341
1000-fold during co-infection with Lp02, and conversely was not stimulated during co-infection 342
with either Lp03 or S. aureus. These observations provide evidence that reporter strain results 343
reflect true rescue and stimulation of intracellular replication, rather than an adventitious effect 344
on reporter signal. 345
We next considered whether stimulation of Bn intracellular growth by Lp02 might be 346
dependent on colocalization in the same host cell. If this were so, then growth stimulation 347
presumably would be enhanced by increasing the ratio of Lp02 to Bn during macrophage 348
infections. Of interest, when the Bn wt strain (MOI 1) was co-infected with Lp02 (MOI of 1, 2, 349
5 and 10), the intracellular growth of Bn increased by 17.2-, 26.5-, 34.7-, or 43.9-fold, 350
respectively, at 24 h p.i. (Fig 4C). However, more impressively, when the intracellular growth 351
defective ΔvirB4 mutant was co-infected with Lp02 (MOI of 1, 2, 5 and 10), its growth was 352
stimulated by 26.1-, 46.8-, 86.2-, or 96.5-fold, respectively. Taken together these observations 353
suggested a potential role for interaction of Lp02 and Bn within the same host cell. 354
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To begin to address potential mechanisms underlying stimulation of Bn intracellular 355
growth by Lp02, Brucella (proD/tdTomato transposant) and Legionella (proD/mClover 356
transposant) co-infections of J774A.1 macrophages were analyzed by confocal microscopy. 357
Enhanced co-localization of Bn and ΔvirB4 with Lp02, but not Lp03, was apparent at all time 358
points based on overlapping signal observed in merged images (Fig. 5). Importantly, single 359
infection of J774A.1 cells with reporter-labeled Bn or Lp verified absence of signal bleed 360
through between red and green fluorescent protein reporter signals using available confocal 361
lasers and adjustable bandpass optics (Fig. S4), thereby ruling out colocalization signal arising 362
artifactually from a single reporter. 363
Quantitative assessment of bacterial uptake and co-localization in confocal images was 364
then performed. Interestingly, percent of J774A.1 cells infected by Bn wt or ΔvirB4 mutant 365
(17 %) (Fig. 6A) was significantly increased by approximately 2-fold during co-infection with 366
Lp02, but not Lp03, at 4 hours p.i. (P < 0.001, Fisher's exact test based on >180 J774A.1 cells 367
scored per condition). In contrast, uptake of Lp02 or Lp03 was decreased to only a marginal 368
degree by co-infection with Bn wt or ΔvirB4 (Fig. 6B). Therefore, Lp02 infection appeared to 369
stimulate uptake of Bn. 370
Furthermore, co-infection of individual macrophages at four hours p.i. was a significantly 371
more frequent occurrence with either Bn wt or ΔvirB4, and Lp02 pathogen pairings than with Bn 372
wt or ΔvirB4, and Lp03 pathogen pairings (1.8 and 1.5 fold-increase, respectively, P < 0.01, Fig. 373
6C). An even greater disparity in co-infection rates at late time points likely reflects a balance 374
between replication, infection of additional macrophages, and, for Lp03, organism clearance. 375
In co-infected macrophages, we next evaluated the frequency of subcellular co-376
localization of Bn and Lp (Fig. 5, 6D), based on overlapping fluorescent signal from their 377
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respective tdTomato and mClover fluorescent protein reporters. To determine percent co-378
localization, the numbers of overlapping organisms or phagosomes were summed for the 379
numerator and total numbers of individual bacteria or phagosomes of either co-infecting species 380
were summed for the denominator. Interestingly, the percent co-localization of Bn wt or ΔvirB4 381
with Lp02 appeared to peak at 4 h p.i. and decreased over time, from approximately 20% at 4 h 382
p.i. to 10 % at 48 p.i. In contrast, colocalization with Lp03 cells was negligible at all time points. 383
In pairwise comparisons at individual time points, co-localization of Bn species with Lp02 was 384
significantly greater (P < 0.01) than co-localization of Bn species with Lp03 (with the exception 385
of ΔvirB4 co-localization at 48 hours p.i.). Therefore, Lp02 appeared to stimulate increased rates 386
of both co-infection and co-localization. 387
Co-infection and co-localization results suggested that Lp02 may potentially act either in 388
trans (within the same cell) or in cis (in the same phagosome) to promote growth of Bn. To 389
distinguish between the possibilities, we took advantage of the observation that Bn ΔvirB4 by 390
itself does not replicate intracellularly and therefore is found primarily as single discrete 391
organisms twenty-four hours post infection (Fig. S4). We hypothesized that if Lp02 acted 392
primarily in cis, then clusters (aggregates of ≥ 4 organisms or ≥ 3µm in diameter) of Bn ΔvirB4, 393
a measure of intracellular growth, would preferentially be found when Lp02 and Bn were co-394
localized in the same vacuole. Alternatively, if Lp02 acted in trans then Bn ΔvirB4 clusters 395
should be randomly distributed in the host cell without a specific spatial relationship with Lp02-396
containing vacuoles (LCV). 397
To distinguish between these possibilities, co-infected cells were examined by confocal 398
microscopy. If Brucella-containing vacuole (BCV) cluster distribution were random, we 399
predicted in two dimensional images that overlap between and LCV and BCV should occur 400
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roughly at a frequency similar to the average percent surface area occupied by BCV and LCV 401
within coinfected host cells. In contrast, if overlap were non-random then we would expect a 402
much higher percentage colocalization. In experiments, 73% of Bn ΔvirB4 clusters overlapped 403
with LCV at 24 h p.i. (Fig 6E). This compared with an average 24% total percent surface area 404
occupied by BCV and LCV (95% confidence interval of 18-30%, from n=14 randomly selected 405
co-infected cells). Therefore, observed co-localization appeared far from random compared with 406
predictions for random overlap (P < 0.0001, even more conservatively considering 50% random 407
overlap as the null hypothesis in contingency analysis). 408
As overlap was non-random, we next considered the relationship of colocalization to 409
BCV size. Specifically, we used confocal morphometric analysis to compare the area of cis and 410
trans BCV in co-infected cells (Fig 6F). Of note, a randomly selected group of cis BCV clusters 411
(n=14) were approximately ten times the area of trans (n=13) clusters (median of 1.14E+09 nm2 412
versus 6.67E+07 nm2, respectively, P < 0.001). Therefore, co-localization was associated with 413
significantly greater BCV size. 414
Lastly, we made the reciprocal observation that BCV clusters interfere with growth of 415
LCV in co-infected cells (Fig 6F). Specifically, LCV were significantly smaller (~6-fold) in area 416
when co-infected macrophages contained any Bn ΔvirB4 clusters than when co-infected 417
macrophages only contained BCV with ≤ 3 organisms (P < 0.0001). This suggested that co-418
infection rescued and promoted Bn growth at a cost to Lp. 419
420
Discussion 421
Here we characterize an in vitro infection model using the desert wood rat pathogen (41), 422
B. neotomae. Importantly, the model organism, Bn, recapitulated host cell infection dynamics 423
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previously described for B. abortus, melitensis, and suis (2-5, 24, 42). First, Bn showed robust 424
T4SS-dependent intracellular growth in macrophage cells lines. Second, Bn demonstrate early, 425
T4SS-dependent temporal colocalization of Bn with the late endosomal and lysosomal marker, 426
LAMP-1, and later association with endoplasmic reticulum marker, calreticulin. 427
Notably, the T4SS-dependent phagosome maturation pathway described here for Bn and 428
previously for other Brucella spp. has both overlap with and differences from that of another 429
T4SS-dependent pathogen, Lp. Both pathogens primarily infect phagocytic cells. For Bn, 430
phagosomes initially mature along the endocytic route, for about 8 hours initially associating 431
with high frequency with LAMP-1. During this time, its T4SS is induced by acidic pH (43) 432
leading to translocation of effectors into the eukaryotic cytoplasm and remodeling of the BCV 433
into a replicative vacuole (rBCV) derived from endoplasmic reticulum. In contrast, for Lp, the 434
T4SS is primed to intervene during initial phagocytosis (44, 45), and the phagosome rapidly 435
takes on characteristics of an exocytic, endoplasmic reticulum-associated vacuole (46). Later, 436
Brucella's replicative niche is converted into an autophagy-like vacuole (aBCV) for completion 437
of the intracellular life cycle and spread to other host cells (4, 5). In contrast, Lp appears to 438
prevent actively later autophagic maturation of its vacuole through action of specific T4SS-439
translocated effectors (47). Nevertheless, despite qualitative and temporal differences in 440
phagosome maturation, both organisms appear to share a morphologically similar endoplasmic 441
reticulum-associated replicative niche. Therefore, from a theoretical perspective, it was therefore 442
possible that Lp and Bn could interact during co-infection. 443
Fascinatingly, our co-infection data demonstrated that T4SS-competent Lp02 promoted 444
intracellular growth of wild-type Bn and rescued intracellular growth of Bn ΔvirB4 (Figs. 4-6). 445
Furthermore, these two pathogens colocalized to a significant extent in the same vacuole. 446
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Colocalization in the same phagosome, rather than co-infection of the same host cell, was 447
associated with Bn ΔvirB4 replication (Fig. 6F). Therefore, Lp02 appeared to act in cis to 448
promote growth of Bn. In contrast, T4SS-defective, Lp03, was incapable of promoting or 449
rescuing Bn intracellular growth and did not appear to co-localize with Bn at an appreciable 450
frequency at time points examined. 451
Bn conversely was not able to rescue replication-defective Lp03. Therefore, rescue and 452
growth promotion was unidirectional. We speculate that unidirectional rescue may relate to 453
either lack of sufficient comingling of BCV with Lp03 LCV or deficiency in Bn effector 454
functionality to fully support the requirements for intracellular Lp replication. Both possibilities 455
are consistent with prior observation of the ability of Legionella longbeacheae to rescue T4SS-456
incompetent Lp (48) when colocalized in the same vacuole or wild type B. melitensis to rescue 457
an isogenic virB mutant during co-infection experiments (49). 458
Our results with Lp02 contrast with the apparent non-interaction during Legionella and 459
Coxiella burnetii co-infection experiments, where no colocalization was observed despite a much 460
closer phylogenetic relationship and a highly conserved T4SS shared by the two organisms (50). 461
These contrasting observations may relate to the distinct lysosomal replicative niche used by 462
Coxiella, as opposed to the morphologically similar ER-associated niche used by both Lp and Bn, 463
and/or inability of Lp superinfection, described in these former studies, to access pre-existing 464
Coxiella phagosomes. 465
Interestingly, co-localization of Bn and Lp was greater than might be expected four hours 466
after infection. Specifically, during single infection, the majority of BCV show LAMP-1 467
positivity 4 hours post-infection (4). In contrast, few LCV associate with LAMP-1 during this 468
same period (51). Therefore, our observation of high co-localization frequency early in infection 469
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suggests that co-infection potentially redirects divergent early intracellular trafficking patterns. 470
Alternatively, the modestly enhanced invasion observed for Bn during co-infection with Lp02 471
may foster colocalization during initial phagocytosis. 472
We also make further note of the new bio-reporter tool kit that was created to support our 473
studies as described and characterized in the supplementary material. This toolkit supported 474
spectrally distinct luminescent and fluorescent real-time microplate and microscopy-based 475
readout during single and polymicrobial infection experiments. Based on the Tn5-based 476
hyperactive in vitro transposase-based system and broadly expressed ProD promoter driven 477
reporter expression, the toolkit should prove broadly applicable to a wide variety of Gram-478
negative and -positive bacterial species (52). In addition, the available non-antibiotic selectable 479
markers encoding resistance to nourseothricin, (53, 54), phleomycin (55), and hygromycin 480
provide multiple options for selection without imparting resistance to therapeutic antibiotics. 481
Such antibiotic resistance when not normally found in pathogens under study might otherwise 482
limit therapeutic options and is potentially proscribed under "dual use research of concern" (56) 483
policies (http://www.phe.gov/s3/dualuse/documents/durc-policy.pdf). 484
We note several potential limitations of the experimental system. Luminescence and 485
fluorescence measures did not show complete quantitative correspondence to CFU 486
measurements. Specifically, dynamic range of the reporters was somewhat compressed. This 487
dynamic range compression may relate to differences in the underlying measurements: CFU 488
indicating the number of viable bacteria and plating efficiency; luminescence reporter output 489
integrating expression of luciferase and substrate, and cellular ATP levels; and fluorescence 490
reflecting reporter expression. Furthermore, microplate measurements may be affected by optics 491
and dynamic range of detection technology. Nevertheless, qualitatively and quantitatively, 492
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reporter output showed a high degree of correlation with CFU analysis. Also we make note of 493
the ~2 fold increase of Bn ΔvirB4 CFU during 48 hour single infection experiments and 494
corresponding increase observed for luminescence and fluorescent measurements (Figs. 1 and 4). 495
However, microscopically, we did not appreciate co-incident intracellular replication of this 496
strain. We therefore speculate these small increases do not represent true intracellular growth, 497
but rather may relate to the ability of extracellular organisms, either surviving gentamicin 498
treatment or released into the extracellular space post gentamicin treatment, to grow slowly in 499
tissue culture medium. Indeed, the extracellular growth phenomenon was very apparent with B. 500
ovis and B. microti (data not shown) and was one motivation for use of B. neotomae in 501
establishing a model system. 502
Of note, B. neotomae is designated by the ATCC as a biosafety level 2 pathogen. 503
Nevertheless, as little work has been performed with this organism, our own recommended 504
practice, based on the known respiratory route of laboratory-acquired infection with human 505
pathogenic species, is to perform experimental manipulations with care in a biosafety cabinet and 506
make use of gasketed safety carriers or sealed rotors during centrifugation steps. 507
In summary, we provide evidence for a model system for studying Brucella pathogenesis 508
that recapitulates several prominent cell biological features observed with human pathogenic 509
species. In particular, Brucella neotomae provides benefit in allowing research to be performed 510
in biosafety level 2 facilities. By thus facilitating experimental work, this model could be used 511
as a first step in identifying pathogenic traits and thereby streamline later investigation and 512
confirmation using select agent pathogenic species. The utility of the model is underscored by 513
the use of Bn in combination with a new bioreporter toolkit to reveal dramatic and unexpected 514
interaction between two type IV secretion system-dependent pathogens, Lp and Bn. Future 515
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exploration of the crosstalk between these organisms may help elucidate important aspects of 516
brucella pathogen-host cell interaction. 517
518
Acknowledgments. We thank Dr. Gireesh Rajashekara (Dept. of Veterinary Preventive 519
Medicine, The Ohio State University) for generously providing pUWGR4 plasmid; E. Coli 520
Genetic Stock Center (New Haven, CT) for providing the BW25113 E. coli strain; and the 521
Harvard-ICCB Longwood Screening facility for use of the Envision instrument. We would also 522
like to thank Lucius Chiaraviglio, Jennifer Tsang and Kenneth P. Smith for critical reading of the 523
manuscript. 524
525
526
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55. Gatignol A, Durand H, Tiraby G. 1988. Bleomycin resistance conferred by a drug-681
binding protein. FEBS Lett 230:171-175. 682
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56. Casadevall A, Dermody TS, Imperiale MJ, Sandri-Goldin RM, Shenk T. 2015. Dual-683
Use Research of Concern (DURC) Review at American Society for Microbiology 684
Journals. MBio 6:e01236. 685
57. Brown L, Cai T, DasGupta A. 2001. Interval Estimation for a Binomial Proportion. 686
Statist. Sci. 16:101-133. 687
58. Andrews HL, Vogel JP, Isberg RR. 1998. Identification of linked Legionella 688
pneumophila genes essential for intracellular growth and evasion of the endocytic 689
pathway. Infect Immun 66:950-958. 690
691
692
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Figure Legends 693
694
Fig. 1. Intracellular growth of wt and ΔvirB4 B. neotomae (A) Colony forming units from 695
lysed macrophages per microplate well at indicated times post infection. (B) Intracellular growth 696
of lux operon-labeled B. neotomae wt and ΔvirB4 mutant. Mouse J774A.1 or human THP-1 697
macrophage cell lines adherent to 96-well plates were infected with luminescent Brucella cells at 698
an MOI 1, treated with gentamicin to kill extracellular bacteria, and incubated for indicated time 699
points. (C) Intracellular replication of proD/tdTomato-expressing Bn wt, ΔvirB4 mutant, and 700
ΔvirB4/virB4 (virB4c) complemented strains in J774A.1 cells. Data shown are the mean ± S.D. 701
of at least three replicates for experiments performed in parallel, and are representative of two 702
independent experiments. 703
704
Figure 2. Phagosome trafficking. (A) Percent of phagosomes containing ProD:tdTomato Bn 705
wt or ΔvirB4 cololocalizing with LAMP-1::mTurquoise2-labeled compartments during infection 706
of J744A.1 cells. (B) Percent of phagosomes containing ProD:tdTomato Bn wt or ΔvirB4 707
cololocalizing with mTurquoise2::calreticulin-labeled compartments during infection of J744A.1 708
cells. Total scored events for each data point were used for Fisher's exact test contingency 709
analysis described in the text. (C) Representative confocal laser scanning microscope images of 710
ProD:tdTomato Bn wt or ΔvirB4 colocalizing with either LAMP-1::mTurquoise2 or 711
mTurquoise2::calreticulin at 24 hours p.i. For each panel, lower right inset shows Bn signal 712
pseudocolored red; upper right inset shows eukaryotic fusion protein signal pseudocolored green, 713
and left panel shows an enlarged, merged image of the two signals superimposed on a 714
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differential interference contrast (DIC) image. Arrows indicate examples of co-localization 715
events associated with a yellow merged overlap signal. Scale bar, 5µm. 716
717
718
Fig. 3. Intracellular colocalization of bacterial pathogens. Confocal laser scanning 719
microscope images of J774A.1 cells co-infected for 24 hours with proD/tdTomato-expressing 720
Brucella neotomae (Bn) and eGFP expressing-Staphylococcus aureus (SAU); proD/tdTomato-721
expressing Bn and proD/mClover expressing L. pneumophila, Lp02; or eGFP-expressing SAU 722
and proD/mNeptune2-expressing Lp02. Pseudocolored confocal images from green (mClover), 723
and red (tdTomato) or far-red channels (mNeptune2) superimposed on differential interference 724
contrast (DIC) images demonstrated distinct fluorescent signals originating from the different co-725
infecting intracellular pathogens. Co-localization is indicated by yellow coloration in the merged 726
images (arrows highlighting examples). The images shown are representative of at least 20 cells 727
imaged from two independent experiments. Scale bar, 5µm. 728
729
Fig. 4. Use of luminescence and fluorescence reporters to independently assess intracellular 730
growth of co-infecting pathogens. Intracellular growth was simultaneously assessed by 731
luminescence (A) and fluorescence (B) during macrophage infection at an MOI of 1 in 96-well 732
microplate format for co-infections with luminescent Bn (wt or ΔvirB4 strains) and fluorescent 733
Lp02, Lp03, or S. aureus. Shown are the mean and S.D. of results from 6 replicate wells per 734
data point and are representative of two independent experiments. (C) Effect of Lp02 MOI on 735
its ability to stimulate intracellular replication of Bn wt and ΔvirB4 mutant. J774A.1 736
macrophages were infected with luminescent Bn at an MOI 1 and Lp02 at MOI's of 1, 2, 5, or 10 737
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for indicated times. Shown are the mean and S.D. of results from 6 replicate wells per data point, 738
and are representative of two independent experiments. (D) Intracellular growth in macrophages 739
of the Bn ΔvirB4 strain co-infected with Lp02, Lp03, or S. aureus was assessed by colony 740
forming unit determination. Shown are the mean and S.D. of results from 6 replicate wells per 741
data point. 742
743
Fig. 5. Co-localization of Bn and Lp during macrophage co-infection. (A) Co-infection of 744
J774A.1 macrophages with Bn wt or ΔvirB4 expressing proD/tdTomato, and Lp02 or Lp03 745
expressing proD/mClover. At indicated time points, cells were fixed and examined by confocal 746
microscopy. Shown are merged pseudocolored images obtained using red (tdTomato) and green 747
(mClover) settings. Bar size, 5µm. Areas enclosed in circles are magnified 3X in insets to 748
demonstrate either presence (yellow merged signal) or absence of pathogen species overlap. 749
750
Figure 6. Quantitative Co-Infection Analysis. (A) The percent of J774A.1 cells infected by 751
Bn wt or ΔvirB4 at 4 h p.i. in the presence or absence of co-infection with Lp02 or Lp03, or 752
alternatively (B) the percent of J774A.1 cells infected with Lp02 or Lp03 at 4 h p.i. in the 753
presence or absence of Bn wt or ΔvirB4 mutant. Data shown are means and S.D. of data 754
obtained from two-independent experiments with at least fifty J774A.1 cells examined in each 755
replicate. Scored events from both trials were combined for Fisher's exact test contingency 756
analysis described in the text. (C) J774A.1 cells were infected with indicated Bn and Lp strains 757
at an MOI of 1. The percent of J774A.1 cells co-infected with both indicated Bn and Lp strains 758
is shown. Data points are mean percent co-infection ± S.D. of data obtained from two 759
independent experiments with a minimum of 50 host cells scored in each. Scored events from 760
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both trials were combined for Fisher's exact test contingency analysis described in the text. (D) 761
Co-localization of indicated strains of Bn and Lp were compared at different times post infection. 762
Co-localization percentage was calculated by examining co-infected macrophages using confocal 763
microscopy, counting the number of phagosomes with co-localized pathogens (yellow merged 764
signal) as the numerator and counting all phagosomes with Bn and/or Lp as the denominator. 765
Data shown are means ± S.D. of two-independent experiments - with an average of > 150 cells 766
scored in total per data point. Total scored events for each data point were used for Fisher's 767
exact test contingency analysis described in the text. (E) Relationship of Bn ΔvirB4 bacterial 768
clusters to Lp02 in co-infected J774A.1 cells 24 h post infection. Clusters (defined as a Bn 769
ΔvirB4-containing vacuole or BCV containing ≥ 4 Bn bacteria or ≥ 3µm in diameter) that 770
overlapped with Lp02-containing vacuoles (LCV) were considered "cis"; those that did not were 771
considered "trans", and rare clusters present in a macrophage without apparent LCV were 772
considered "null". Results shown are cumulative percentage data from 108 randomly selected 773
clusters scored over two independent co-infection experiments with 95% confidence intervals 774
shown calculated using the Wilson/Brown method (57). (F) Mean surface area ± S.D. of Bn 775
ΔvirB4 clusters found in cis or trans to Lp02, or in cells without apparent LCV (null) 24 h p.i.; or 776
mean surface area ± S.D. of LCV in host cells 24 h p.i. containing any number of ΔvirB4 clusters 777
or in host cells only containing BCV with < 4 ΔvirB4 bacteria, designated as "single". 778
779
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780
Table 1. Bacterial strains and plasmids 781 Strain or plasmid Relevant markers and characteristics Reference or source Bacteria: Brucella neotomae 5K33 Parent biosafety level 2 rodent pathogen BEI Resources ΔvirB4 virB4 in-frame deletion mutant of B. neotomae 5K33 This study Bn (proD/tdtomato-nat) Transposon mutant of 5K33 having proD/tdtomato-nat genes This study
ΔvirB4 (proD/tdtomato-nat) Transposon mutant of ΔvirB4 having proD/tdtomato-nat genes This study
virB4c (proD/tdtomato-nat) Complemented strain of ΔvirB4 (proD/tdtomato-nat) containing pBMTL2-virB4 This study
Bn(Lux-nat) Transposon mutant of Bn expressing Lux operon-nat genes This study
ΔvirB4 (Lux-nat) Transposon mutant of ΔvirB4 expressing Lux operon-nat genes This study
Legionella pneumophila Lp02fla Philadelphia 1, thyA rpsL hsdR flaA (14) Lp03fla thyA rpsL hsdR dotA03 flaA, Dot/Icm translocation deficient (14) Lp02fla (mClover-nat or Sh ble) Transposon mutant expressing mClover-nat or mClover-Sh ble This study Lp03fla (mClover-nat or Sh ble) This study Lp02fla (mWasabi-nat or Sh ble) Transposon mutant expressing mWasabi-nat or mWasabi-Sh ble This study Lp03fla (mWasabi-nat or Sh ble) This study Lp02fla (mCardinal-nat or Sh ble) Transposon mutant expressing mCardinal-nat or mCardinal-Sh ble This study Lp03fla (mCardinal-nat or Sh ble) This study Lp02fla (mNeptune2-nat or Sh ble) Transposon mutant expressing mNeptune2-nat or mNeptune2-Sh ble This study Lp03fla (mNeptune2-nat or Sh ble) This study Lp02fla (proD/mClover-nat) Transposon mutant expressing proD/mClover-nat This study Lp03fla (proD/mClover-nat) This study Lp02fla (proD/mNeptune2-nat) Transposon mutant expressing proD/mNeptune2-nat This study Lp03fla (proD/mNeptune2-nat) This study Lp02fla (proD/tdTomato-nat) Transposon mutant expressing proD/tdTomato-nat This study Lp03fla (proD/tdTomato-nat) This study
Staphylococcus aureus 25923 Wild type, Biosafety Level 2 pathogen ATCC Sa (pAT28-SaEGFP) S. aureus with pAT28-SaEGFP This study Sa (pAT28-proD/SaEGFP) S. aureus with pAT28-proD/SaEGFP This study Sa (pAT28-GPLux) S. aureus with pAT28-GPLux This study Sa (pAT28-proD/GPLux) S. aureus with pAT28-proD/GPLux This study
Escherichia coli
EC100D pir+ F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1
araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG pir
+(DHFR)
Epicentre
NEB-5α fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 NEB
NEB-10β Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (Str
R) rph spoT1 Δ(mrr-
hsdRMS-mcrBC) NEB
BW25113 lacI+rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 rph-1 Δ(araB–
D)567 Δ(rhaD–B)568 ΔlacZ4787(::rrnB-3) hsdR514 rph-1 (13)
BW25113 (Lux-nat) Transposant expressing Lux operon-nat genes This study BW25113 (proD/Lux-nat) Transposon with proD/Lux operone-nat genes This study MT607 Triparental E. coli strain containing the pRK600 helper plasmid (27)
Brucella microti CCM 4915 Czech collection of micro-organisms
Brucella microti CCM 4916 As Above Brucella ovis NR-682 BEI Resources
Eukaryote: J774a.1 Mouse macrophage, ATCC TIB-67 ATCC THP-1 Human monocyte, ATCC TIB-202 ATCC
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Gryphon cells Gryphon ecotropic cell line for retrovirus packaging Allele Biotechnology
Plasmid: pSR47s R6K, sacB, Km
R, suicide vector (58)
pSR47s-virB4 pSR47s vector having in-frame deletion region of the Bn virB4 gene This study pBMTL2 Km
R, broad host range vector (28)
pBMTL2-virB4 pBMTL2 vector including whole virB4 gene This study pMV306G13+Lux pMV306 vector having Gram Positive optimized lux operon (21) pAT28 Spec
R, Gram-Positive shuttle vector (19)
pAT28-SaEGFP pAT28 vector with promoterless eGFP (Gram Positive optimization) This study
pAT28-proD/SaEGFP pAT28 vector with eGFP (Gram Positive optimization) under the control of proD promoter This study
pAT28-GPLux pAT28 vector with promoterless Gram-positive organized Lux operon This study
pAT28-proD/GPLux pAT28 vector with Gram-positive organized Lux operon with proD promoter This study
pMOD3 EZ-Tn5 pMOD (R6Kγori/MCS) transposon vector, AmpR Epicentre, (52)
Lamp1-YFP Mammalian expression vector with LAMP-1, NeoR or KanR Addgene (29) pmTurquoise2ER Mammalian expression vector with calreticulin::mTurquoise2 fusion, KanR Addgene (30) pRetroX-Tet-Off Advanced Retroviral vector for mammalian expression, NeoR or KanR Clontech pRetroX-mTurquoise2::ER pRetroX Vector expressing mTurquoise2 and calreticulin fusion protein This study pRetroX-Lamp1::mTurquoise2 pRetroX Vector expressing LAMP-1 and mTurquoise2 fusion protein This study pMOD3-Lux-nat pMOD3 with promoterless lux operon and nat selectable marker This study pMOD3-proD/Lux-nat pMOD3 with proD promoter, Lux operon and nat selectable marker This study pMOD3-tdTomato-nat pMOD3 with promoterless tdTomato and nat selectable marker This study pMOD3-proD/tdTomato-nat(hph) pMOD3 with proD tdTomato and nat or hph selectable markers This study pMOD3-mClover-nat (Sh ble) pMOD3 with promoterless mClover and nat or Sh ble selectable markers This study pMOD3-proD/mClover/nat pMOD3 with proD promoter, mClover and nat selectable marker This study pMOD3-mWasabi-nat (Sh ble) pMOD3 with promoterless mWasabi and nat or Sh ble selectable markers This study
pMOD3-mCardinal-nat (Sh ble) pMOD3 with promoterless mCardinal and nat Sh ble selectable markers This study
pMOD3-mNeptune2-nat (Sh ble) pMOD3 with promoterless mNeptune2 and nat or Sh ble selectable markers This study
pMOD3-proD/mNeptune2-nat pMOD3 with proD promoter, mNeptune2 and nat selectable marker This study
NEB: New England Biolabs; ATCC: American Type Culture Collection; nat: nourseothricin 782
acetyltransferase; Sh ble: phleomycin resistance gene; hph: hygromycin phosphotransferase 783
gene. 784
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C
Post-Infection (hr)
Rela
tive F
luo
rescen
ce wt
virB4
virB4c
A
CF
U
wt
DvirB4
Post Infection (hr)
B
Post Infection (hr)
Rela
tive L
um
inescen
ce
wt J774A.1
DvirB4 J774A.1
wt THP-1
DvirB4 THP-1
48244
10000
48244
1 106
0 4 24 48
105
106
107
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4 24 480%
20%
40%
60%
80%
100%
Post Infection (hr)
Calr
eti
cu
lin
Co
localizati
on
wt
DvirB4
4 24 480%
20%
40%
60%
80%
100%
Post Infection (hr)
LA
MP
-1 C
olo
calizati
on wt
DvirB
BA
C
LAMP-1
ΔvirB4
Calreticulin
ΔvirB4
LAMP-1
Bn WT
Calreticulin
Bn WT
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Merge
Bn SAU
Bn Lp02
SAULp02
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Bn
Bn & Sa
Bn & Lp02
Bn & Lp03
virB4
virB4 & Sa
virB4 & Lp02
virB4 & Lp03
A
4 24 48
104
105
106
Post Infection (hr)
Rela
tive L
um
inescen
ce
Lp02 & Bn
Lp03 & Bn
Lp02 & DvirB
Lp03 & DvirB
B
4 24 48104
105
Post Infection (hr)
Rela
tive F
luo
rescen
ce
C
Bn
virB4
0 1 2 5 10103
104
105
106
Lp02 MOIR
ela
tive L
um
inescen
ce
0 4 24 48
105
106
107
108
109
Post Infection (hr)
CF
U
virB4
virB4 & Sa
virB4 & Lp02
virB4 & Lp03
D
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4h 24h 48h
Bn wt
Lp02
Bn wt
Lp03
ΔvirB4Lp02
ΔvirB4Lp03
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Bn+Lp02
Bn+Lp03
ΔvirB4+Lp02
ΔvirB4+Lp03
4 24 480%
10%
20%
30%
40%
Post Infection (hr)
Co
-In
fecti
on
Rate
A B
C
Contr
ol
Lp02
Lp03
0%
10%
20%
30%
40%
50%
Bn
up
take d
uri
ng
co
infe
cti
on
Bn
ΔvirB4
contr
olBn
ΔvirB4
0%
10%
20%
30%
40%
50%
Lp
up
take d
uri
ng
co
infe
cto
in
Lp02
Lp03
4 24 480%
10%
20%
30%
Post Infection (hr)
Co
localizati
on
D
E F
cis
tran
snull
ΔvirB4
clust
er
ΔvirB4
single
0
2 107
4 107
6 107
8 107
are
a (
nm
2)
BCV size LCV size
**
cis
tran
snull
0%
20%
40%
60%
80%
100%
Perc
en
t o
f Δv
irB
4 c
luste
rsw
ith
in
dic
ate
d r
ela
tio
nsh
ip t
o L
CV
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