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1 Promotion and Rescue of Intracellular Brucella neotomae Replication During 1 Co-Infection With Legionella pneumophila 2 3 Short title: Rescue of Brucella growth by Legionella 4 5 Yoon-Suk Kang 1 & James E. Kirby 1, * 6 7 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 8 Boston, MA, United States of America 9 10 11 *Corresponding author 12 Email: [email protected] 13 14 IAI Accepted Manuscript Posted Online 6 March 2017 Infect. Immun. doi:10.1128/IAI.00991-16 Copyright © 2017 American Society for Microbiology. All Rights Reserved. on April 7, 2019 by guest http://iai.asm.org/ Downloaded from
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1

Promotion and Rescue of Intracellular Brucella neotomae Replication During 1

Co-Infection With Legionella pneumophila 2

3

Short title: Rescue of Brucella growth by Legionella 4

5

Yoon-Suk Kang1 & James E. Kirby1,* 6

7

Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 8

Boston, MA, United States of America 9

10

11

*Corresponding author 12

Email: [email protected] 13

14

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

35

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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

83

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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References 527

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31

47. Choy A, Dancourt J, Mugo B, O'Connor TJ, Isberg RR, Melia TJ, Roy CR. 2012. 662

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32

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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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33

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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34

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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35

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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36

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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