CELLULAR MICROBIOLOGY: PATHOGEN-HOST CELL MOLECULAR INTERACTIONS

crossm Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila Yoon-Suk Kang, James E. Kirby Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

ABSTRACT

Received 29 November 2016 Returned for modification 20 December 2016 Accepted 28 February 2017 Accepted manuscript posted online 6 March 2017 Citation Kang Y-S, Kirby JE. 2017. Promotion and rescue of intracellular Brucella neotomae replication during coinfection with Legionella pneumophila. Infect Immun 85:e00991-16. https://doi.org/10.1128/IAI.00991-16. Editor Craig R. Roy, Yale University School of Medicine Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to James E. Kirby, [email protected].

KEYWORDS Brucella, Legionella pneumophila, coinfection, fluorescent image analysis, intracellular pathogens, pathogenesis, phagosomes, reporter genes, type IV secretion system

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rucella species are Gram-negative Alphaproteobacteria that cause chronic, systemic infections in mammals and zoonotic infections in humans (1). These pathogens are known to infect the reticuloendothelial system and proliferate significantly in macrophage-rich organs such as liver, spleen, and bone marrow. Chronic, often debilitating bloodstream infection is typical. In humans, a chronic course punctuated by spikes in body temperature is underscored by the descriptive name, undulant fever, given to disease caused by these organisms. Endovascular infection and osteomyelitis are concerning sequelae. Humans may acquire Brucella from airborne exposure related to large quantities of organisms shed from birthing livestock or from ingesting unpasteurized dairy products. B. abortus, B. melitensis, and B. suis, the species responsible for human zoonotic infection, are facultative intracellular pathogens (1). Intracellular growth is thought critical to successful infection of the host. More particularly, each of these pathogens deploys a type IV secretion system (T4SS), a molecular syringe, to inject virulence May 2017 Volume 85 Issue 5 e00991-16

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factors into host cells and establish a productive intracellular growth niche (2, 3). Specifically, through use of its T4SS, each Brucella species orchestrates remodeling of its phagosome, which proceeds in a temporal fashion to take on properties first of endosomal compartments, then of endoplasmic reticulum (ER) (4), where intracellular growth initially takes place, and finally of autophagosomes, during completion of an intracellular replication cycle (5). Virulence factors injected by the T4SS, which presumably are responsible for this altered phagosome maturation, are still being defined. Based on infectivity, respiratory mode of acquisition, and chronic and potentially life-threatening disease manifestations, zoonotic Brucella species are generally classified as biosafety level 3 pathogens. B. abortus, B. melitensis, and B. suis are also considered potential biothreat agents and are designated overlap select agents by the Human Health Services and United States Department of Agriculture (http://www .selectagents.gov/SelectAgentsandToxinsList.html) and as category B priority agents by the United States National Institutes for Allergy and Infectious Diseases (https:// www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens). The need for select agent and biosafety level 3 precautions has made research on this fascinating genus more difficult. Ideally, development of new models using species that recapitulate infection at the cellular level, but which do not infect humans, would be ideal. Currently, three Brucella species are designated biosafety level 2 pathogens by the American Type Culture Collection (http://www.atcc.org) (B. neotomae and B. ovis) and the Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/i) (B. microti). B. neotaomae and B. microti are rodent pathogens. B. ovis is primarily a sheep pathogen. Notably, B. neotomae is not generally considered a human pathogen (6, 7), yet still shares high genetic homology with zoonotic Brucella species (8, 9). As such, its use as a model pathogen may hold promise. Therefore, our goal was to establish Brucella neotomae as a new model system for investigation of in vitro pathogenesis and to take advantage of its biosafety level 2 status to accelerate experimental work. To this end, we examined T4SS dependence of intracellular growth. Furthermore, we explored interaction with several pathogens during coinfection experiments—most importantly, the T4SS-dependent pathogen, Legionella pneumophila. Of note, L. pneumophila also alters normal endocytic trafficking through use of an unrelated T4SS and, in doing so, establishes an endoplasmic reticulum-associated replicative niche with morphological similarity to the Brucella replicative vacuole (10–12). Complementary experiments using novel reporter strains revealed an unexpected and dramatic cooperative interaction between Legionella pneumophila and Brucella neotomae during coinfection experiments. RESULTS Intracellular growth and type IV secretion system dependence of B. neotomae. To establish a biosafety level 2 Brucella in vitro model system, we considered use of three available species: B. neotomae, B. microti, and B. ovis. All organisms are facultative intracellular pathogens. However, B. ovis and the two available B. microti species did not grow discernibly more in the presence of J774A.1 macrophage cells than in tissue culture medium alone (L. Chiaraviglio and J. E. Kirby, data not shown). In contrast, B. neotomae grew ⬎50-fold more in the presence of eukaryotic cells. This intracellular growth selectivity will presumably provide an advantage in studying intracellular growth phenotypes in long-term tissue culture experiments where adventitious growth of extracellular organisms may confound experimental interpretation. Furthermore, in contrast to B. neotomae and human-pathogenic Brucella species, B. ovis lacks O-polysaccharide side chains, potentially making its use as a model pathogen less desirable (13–15). Therefore, B. neotomae was chosen for further model development. Notably, in an experiment in which infected J744A.1 macrophages were treated with gentamicin after a short infection period to kill extracellular organisms, intracellular bacterial CFU increased in number by approximately ⬃30-fold during a 48-h incubation period (Fig. 1A). To enable more facile analysis of this intracellular growth, we created a bio-reporter toolkit to label Brucella with optimized, spectrally distinct reporters. In May 2017 Volume 85 Issue 5 e00991-16

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Post-Infection (hr) FIG 1 Intracellular growth of wild-type and ΔvirB4 mutant B. neotomae. (A) CFU from lysed macrophages per microplate well at indicated times postinfection. (B) Intracellular growth of lux operon-labeled B. neotomae wild-type (wt) and ΔvirB4 mutant. Cells of the mouse J774A.1 or human THP-1 macrophage cell lines adherent to 96-well plates were infected with luminescent Brucella cells at an MOI of 1, treated with gentamicin to kill extracellular bacteria, and incubated for the indicated time points. (C) Intracellular replication of proD/tdTomato-expressing B. neotomae wild-type, ΔvirB4 mutant, and ΔvirB4/virB4 (virB4c) complemented strains in J774A.1 cells. The data shown are the mean ⫾ standard deviation (SD) from at least three replicates for experiments performed in parallel and are representative of two independent experiments.

this way, we would be able to conveniently track intracellular growth using fluorescence or luminescence output and to study cell biology and interaction with other pathogens expressing complementary reporters. Further details and validation of reporter constructs are provided in the supplemental materials and methods, Tables S1 and S2, and Fig. S1 to S4 in the supplemental material. Briefly, both the bacterial lux operon and fluorescent protein constructs were driven by a strong proD promoter. This previously described synthetic, insulated promoter, originally developed for high-level expression in Escherichia coli (16), was found to drive substantial reporter expression in May 2017 Volume 85 Issue 5 e00991-16

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both Gram-negative and -positive organisms from several different species. The codonoptimized reporters (including mClover, tdTomato, and mNeptune2) and the Photorhabdus luminescens lux operon were linked in synthetic operons to nonantibiotic selectable markers to allow selection in the several species ultimately examined. The reporters were either inserted via transposition in single copy into the bacterial genome of target organisms or expressed from a multicopy, broad-host-range plasmid (Staphylococcus aureus only). Using these reporter strains, we first tested whether single-copy proD::lux and proD::tdTomato reporters could be used to characterize intracellular growth of Brucella neotomae and to test whether this growth was dependent on its T4SS. Notably, the virB4 gene was shown previously to be absolutely required for T4SS function in other Brucella species (2, 3, 17). We therefore created a virB4 in-frame deletion mutant through standard double reciprocal homologous recombination as previously described (18). Wild-type and ΔvirB4 mutant strains were then marked through transposition with lux operon and tdTomato reporter constructs, and intracellular growth phenotypes were evaluated in the murine and human macrophage cell lines J774A.1 and THP-1. After initial infection, extracellular bacteria were killed through treatment with gentamicin to prevent extracellular replication in the surrounding medium. Therefore, increase in reporter output over time should predominantly reflect intracellular growth alone. Notably, a ⬎4.5- to 8.6-fold increase in luminescence and fluorescence signal was observed during a 48-h infection with wild-type, reporter-marked organisms (Fig. 1B and C). In contrast, only a relatively small increase in luminescence and fluorescence signal was observed during infection with the isogenic B. neotomae ΔvirB4 mutant. Importantly, complementation of the ΔvirB4 mutant with a plasmid expressing a cloned virB4 gene rescued the intracellular growth defect (Fig. 1C; VirB4c), supporting linkage of the ΔvirB4 mutation with the observed intracellular growth defects. These results indicate that the virB4 gene is required for intracellular growth of B. neotomae and that B. neotomae is a type IV secretion system (T4SS)-dependent pathogen. We suspect that the lower absolute increase in reporter signal (Fig. 1B and C) compared to CFU counts (Fig. 1A) in parallel assays likely reflects compression of reporter output during the intracellular growth cycle. Despite compression of scale, relative light unit (RLU) or relative fluorescence unit (RFU) measurements and CFU for the B. neotomae wild type were highly correlated (R2 values of 0.98 and 0.96, respectively). Therefore, the reporterlabeled organisms provide a facile and predictive tool for real-time assessment of intracellular bacterial growth (19, 20). Based on known temporal association of phagosomes containing human-pathogenic Brucella spp. with endosomal and endoplasmic reticulum markers (3–5, 21), colocalization phenotypes of the B. neotomae wild type and ΔvirB mutant were likewise examined. Colocalization was assessed using permanently transfected J774A.1 cell lines expressing LAMP-1::mTurquoise2 or mTurquoise2::calreticulin fusion proteins, respectively. (LAMP-1 is a late endosomal and lysosome-associated protein, and calreticulin is an endoplasmic reticulum-associated protein.) As described previously for human-pathogenic species, wildtype B. neotomae colocalized at early time points with the late endosomal marker LAMP-1 (Fig. 2A). At later time points, colocalization with LAMP-1 significantly decreased and association with the endoplasmic reticulum marker calreticulin significantly increased (P ⬍ 0.0001 at 24 and 48 h postinfection) compared to the B. neotomae ΔvirB4 strain (Fig. 2A and B). In contrast, the B. neotomae ΔvirB4 strain exhibited high colocalization with LAMP-1 and low colocalization with calreticulin throughout the 48-h infection period. Representative colocalization images are shown in Fig. 2C. We next considered whether coinfection with other pathogens might influence intracellular replication of B. neotomae, testing pairwise coinfection of macrophages with fluorescent protein reporter-labeled Legionella pneumophila, B. neotomae, and Staphylococcus aureus. Although not generally considered an intracellular pathogen, S. aureus was included based on reports that it can take up prolonged residence in macrophage vacuoles (8, 22, 23) and might therefore potentially also interact with L. May 2017 Volume 85 Issue 5 e00991-16

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FIG 2 Phagosome trafficking. (A) Percentage of phagosomes containing the proD/tdTomato B. neotomae (Bn) wild type (wt) or ΔvirB4 mutant cololocalizing with LAMP-1::mTurquoise2-labeled compartments during infection of J744A.1 cells. (B) Percentage of phagosomes containing proD/tdTomato B. neotomae wild-type or ΔvirB4 mutant cololocalizing with mTurquoise2::calreticulin-labeled compartments during infection of J744A.1 cells. Total scored events for each data point were used for Fisher’s exact test contingency analysis as described in the text. (C) Representative confocal laser scanning microscope images of cells of the proD/tdTomato B. neotomae wild type or ΔvirB4 mutant colocalizing with either LAMP-1::mTurquoise2 or mTurquoise2::calreticulin at 24 h p.i. For each panel, the lower right inset shows B. neotomae signal pseudocolored red, the upper right inset shows eukaryotic fusion protein signal pseudocolored green, and the left panel shows an enlarged, merged image of the two signals superimposed on a differential interference contrast (DIC) image. Arrows indicate examples of colocalization events associated with a yellow merged overlap signal. Scale bars, 5 ␮m.

pneumophila or B. neotomae. As shown in Fig. 3, we simultaneously coinfected macrophages with B. neotomae(proD/tdTomato) and S. aureus(pAT28-proD/eGFP), B. neotomae(proD/tdTomato) and Lp02(proD/mClover), or S. aureus(pAT28-proD/eGFP) and Lp02(proD/mNeptune2). At 24 h postinfection with a multiplicity of infection (MOI) of 10, we easily visualized by confocal microscopy two distinct bacterial pathogens within eukaryotic cells for each pairing. Interestingly, B. neotomae and Lp02 and B. neotomae and S. aureus colocalized with some frequency, presumably within the same phagosomal compartment. This contrasted with the S. aureus and Lp02 coinfection, where colocalization was vanishingly rare. These results implied that pathogens in the first two pairings may occupy the same intracellular niche at some point during their intracellular life cycle and therefore may potentially influence one another. Coinfection with L. pneumophila rescues intracellular, replication-defective B. neotomae ⌬virB4 mutant and promotes growth of wild-type B. neotomae. We thereMay 2017 Volume 85 Issue 5 e00991-16

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FIG 3 Intracellular colocalization of bacterial pathogens. Shown are confocal laser scanning microscope images of J774A.1 cells coinfected for 24 h with proD::tdTomato-expressing Brucella neotomae (Bn) and eGFP-expressing Staphylococcus aureus (SAU), proD/tdTomato-expressing B. neotomae and proD/ mClover-expressing L. pneumophila Lp02, or eGFP-expressing S. aureus and proD/mNeptune2-expressing Lp02. Pseudocolored confocal images from green (mClover) and red (tdTomato) or far-red (mNeptune2) channels superimposed on differential interference contrast (DIC) images demonstrated distinct fluorescent signals originating from the different coinfecting intracellular pathogens. Colocalization is indicated by yellow coloration in the merged images (arrows highlight examples). The images shown are representative of at least 20 cells imaged from two independent experiments. Scale bars, 5 ␮m.

fore made further use of reporter-labeled organisms to investigate the reciprocal influence of pathogen pairings on intracellular growth. J774A.1 macrophages were coinfected at an MOI of 1 with the luminescent B. neotomae wild type or an isogenic ΔvirB4 mutant, and either S. aureus(pAT28-proD/eGFP) or L. pneumophila Lp02(proD/ mClover) or Lp03(proD/mClover) transposants. Lp02 and Lp03 are previously wellcharacterized, isogenic strains of L. pneumophila that are T4SS competent and incompetent, respectively (24, 25). Luminescence and fluorescence were recorded during a 48-h infection using a multimode microplate reader (Fig. 4A). Notably, Lp02, but not Lp03, significantly stimulated growth of luminescent B. neotomae wild-type or ΔvirB4 strains conferring a 12.7- or 24.7-fold increase (P ⫽ 0.002) in growth signal, respectively, at 24 h postinfection (p.i.) and an 11.4- or 48.9-fold increase (P ⫽ 0.002) in growth signal, respectively, at 48 h p.i. relative to infection with either the B. neotomae wild-type or ΔvirB4 mutant strain alone. In contrast, the B. neotomae wild type and the ΔvirB4 mutant did not differentially affect growth of fluorescent protein reporterlabeled Lp02 or Lp03 (Fig. 4B). In addition, S. aureus did not affect reporter signal from B. neotomae, nor did the B. neotomae wild type or ΔvirB4 mutant affect S. aureus reporter signal (data not shown). In a parallel experiment using the same assay conditions and assessed by CFU determination (Fig. 4D), growth of the B. neotomae ΔvirB4 mutant was stimulated 1,000-fold during coinfection with Lp02 and conversely was not stimulated during coinfection with either Lp03 or S. aureus. These observations provide evidence that reporter strain results reflect true rescue and stimulation of intracellular replication, rather than an adventitious effect on reporter signal. We next considered whether stimulation of B. neotomae intracellular growth by Lp02 might be dependent on colocalization in the same host cell. If this were so, then growth stimulation presumably would be enhanced by increasing the ratio of Lp02 to B. neotomae during macrophage infections. Of interest, when the B. neotomae wildtype strain (MOI of 1) was coinfected with Lp02 (MOI of 1, 2, 5, and 10), the intracellular growth of B. neotomae increased by 17.2-, 26.5-, 34.7-, or 43.9-fold, respectively, at 24 h p.i. (Fig. 4C). However, more impressively, when the intracellular growth-defective ΔvirB4 mutant was coinfected with Lp02 (MOI of 1, 2, 5, and 10), its growth was stimulated by 26.1-, 46.8-, 86.2-, or 96.5-fold, respectively. Taken together, these obMay 2017 Volume 85 Issue 5 e00991-16

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FIG 4 Use of luminescence and fluorescence reporters to independently assess intracellular growth of coinfecting pathogens. Intracellular growth was simultaneously assessed by luminescence (A) and fluorescence (B) during macrophage infection at an MOI of 1 in a 96-well microplate format for coinfections with luminescent B. neotomae (Bn) (wild-type [wt] or ΔvirB4 mutant strains) and fluorescent Lp02, Lp03, or S. aureus. Shown are the mean and SD of results from 6 replicate wells per data point; results are representative of two independent experiments. (C) Effect of Lp02 MOI on its ability to stimulate intracellular replication of the B. neotomae wild type and ΔvirB4 mutant. J774A.1 macrophages were infected with luminescent B. neotomae at an MOI of 1 and Lp02 at MOI of 1, 2, 5, or 10 for the indicated times. Shown are the mean and SD of results from 6 replicate wells per data point; results are representative of two independent experiments. (D) Intracellular growth in macrophages of the B. neotomae ΔvirB4 strain coinfected with Lp02, Lp03, or S. aureus was assessed by CFU determination. Shown are the mean and SD of results from 6 replicate wells per data point.

servations suggested a potential role for interaction of Lp02 and B. neotomae within the same host cell. To begin to address potential mechanisms underlying stimulation of B. neotomae intracellular growth by Lp02, Brucella(proD/tdTomato transposant) and Legionella (proD/mClover transposant) coinfections of J774A.1 macrophages were analyzed by confocal microscopy. Enhanced colocalization of the B. neotomae wild type and the ΔvirB4 mutant with Lp02, but not Lp03, was apparent at all time points based on overlapping signal observed in merged images (Fig. 5). Importantly, single infection of J774A.1 cells with reporter-labeled B. neotomae or L. pneumophila verified the absence of signal bleed through between red and green fluorescent protein reporter signals using available confocal lasers and adjustable bandpass optics (Fig. S4), thereby ruling out colocalization signal arising artifactually from a single reporter. Quantitative assessment of bacterial uptake and colocalization in confocal images was then performed. Interestingly, the percentage of J774A.1 cells infected by the B. neotomae wild type or ΔvirB4 mutant (17%) (Fig. 6A) was significantly increased by approximately 2-fold during coinfection with Lp02, but not Lp03, at 4 h p.i. (P ⬍ 0.001; Fisher’s exact test based on ⬎180 J774A.1 cells scored per condition). In contrast, uptake of Lp02 or Lp03 was decreased to only a marginal degree by coinfection with the B. neotomae wild type or ΔvirB4 mutant (Fig. 6B). Therefore, Lp02 infection appeared to stimulate uptake of B. neotomae. Furthermore, coinfection of individual macrophages at 4 h p.i. was a significantly more frequent occurrence with either the B. neotomae wild-type or ΔvirB4 mutant and May 2017 Volume 85 Issue 5 e00991-16

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FIG 5 Colocalization of B. neotomae and L. pneumophila during macrophage coinfection. Coinfection of J774A.1 macrophages with the B. neotomae wild type (Bn wt) or ΔvirB4 mutant expressing proD/ tdTomato and L. pneumophila Lp02 or Lp03 expressing proD/mClover. At indicated time points, cells were fixed and examined by confocal microscopy. Shown are merged pseudocolored images obtained using the red (tdTomato) and green (mClover) settings. Size bars, 5 ␮m. Areas enclosed in circles are magnified 3⫻ in insets to demonstrate either presence (yellow merged signal) or absence of pathogen species overlap.

Lp02 pathogen pairings than with the B. neotomae wild-type or ΔvirB4 mutant and Lp03 pathogen pairings (1.8- and 1.5-fold increases, respectively; P ⬍ 0.01) (Fig. 6C). An even greater disparity in coinfection rates at late time points likely reflects a balance between replication, infection of additional macrophages, and, for Lp03, organism clearance. In coinfected macrophages, we next evaluated the frequency of subcellular colocalization of B. neotomae and L. pneumophila (Fig. 5 and 6D), based on overlapping fluorescent signal from their respective tdTomato and mClover fluorescent protein reporters. To determine the percentage of colocalization, the numbers of overlapping organisms or phagosomes were summed for the numerator, and total numbers of individual bacteria or phagosomes of either coinfecting species were summed for the denominator. Interestingly, the percentage of colocalization of the B. neotomae wild type or ΔvirB4 mutant with Lp02 appeared to peak at 4 h p.i. and decreased over time, from approximately 20% at 4 h p.i. to 10% at 48 p.i. In contrast, colocalization with Lp03 cells was negligible at all time points. In pairwise comparisons at individual time points, colocalization of B. neotomae species with Lp02 was significantly greater (P ⬍ 0.01) than colocalization of B. neotomae species with Lp03 (with the exception of ΔvirB4 mutant colocalization at 48 h p.i.). Therefore, Lp02 appeared to stimulate increased rates of both coinfection and colocalization. Coinfection and colocalization results suggested that Lp02 may potentially act either in trans (within the same cell) or in cis (in the same phagosome) to promote growth of B. neotomae. To distinguish between the possibilities, we took advantage of the observation that the B. neotomae ΔvirB4 mutant by itself does not replicate intracellularly and therefore is found primarily as single discrete organisms 24 h postinfection (Fig. S4). We hypothesized that if Lp02 acted primarily in cis, then clusters (aggregates of ⱖ4 organisms or ⱖ3 ␮m in diameter) of the B. neotomae ΔvirB4 cells, a measure of intracellular growth, would preferentially be found when Lp02 and B. neotomae were colocalized in the same vacuole. Alternatively, if Lp02 acted in trans, May 2017 Volume 85 Issue 5 e00991-16

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then B. neotomae ΔvirB4 cell clusters should be randomly distributed in the host cell without a specific spatial relationship with Lp02-containing vacuoles (LCV). To distinguish between these possibilities, coinfected cells were examined by confocal microscopy. If the Brucella-containing vacuole (BCV) cluster distribution were random, we predicted in two-dimensional images that the overlap between LCV and BCV should occur roughly at a frequency similar to the average percentage of surface area occupied by BCV and LCV within coinfected host cells. In contrast, if overlap were nonrandom, then we would expect a much higher percentage of colocalization. In experiments, 73% of B. neotomae ΔvirB4 cell clusters overlapped with LCV at 24 h p.i. (Fig. 6E). This compared with an average 24% total surface area occupied by BCV and LCV (95% confidence interval of 18 to 30% from n ⫽ 14 randomly selected coinfected cells). Therefore, the observed colocalization appeared far from random compared with predictions for random overlap (P ⬍ 0.0001, even more conservatively considering 50% random overlap as the null hypothesis in contingency analysis). As overlap was nonrandom, we next considered the relationship of colocalization to BCV size. Specifically, we used confocal morphometric analysis to compare the areas of cis and trans BCV in coinfected cells (Fig. 6F). Of note, a randomly selected group of cis BCV clusters (n ⫽ 26) were approximately 6 times the area of trans clusters (n ⫽ 14) (P ⬍ 0.0001). Therefore, colocalization was associated with significantly greater BCV size. Finally, we made the reciprocal observation that BCV clusters interfere with growth of LCV in coinfected cells (Fig. 6F). Specifically, LCV were significantly smaller (⬃6-fold) in area when coinfected macrophages contained any B. neotomae ΔvirB4 cell clusters than when coinfected macrophages only contained BCV with ⱕ3 organisms (P ⬍ 0.0001). This suggested that coinfection rescued and promoted B. neotomae growth at a cost to L. pneumophila. DISCUSSION Here we characterize an in vitro infection model using the desert wood rat pathogen B. neotomae (26). Importantly, the model organism, B. neotomae, recapitulated host cell infection dynamics previously described for B. abortus, B. melitensis, and B. suis (2–5, 27, 28). First, B. neotomae showed robust T4SS-dependent intracellular growth in macrophage cells lines. Second, B. neotomae demonstrated early, T4SS-dependent temporal colocalization of B. neotomae with the late endosomal and lysosomal marker LAMP-1 and later association with the endoplasmic reticulum marker calreticulin. Notably, the T4SS-dependent phagosome maturation pathway described here for B. neotomae and previously for other Brucella spp. has both overlap and differences from that of another T4SS-dependent pathogen, L. pneumophila. Both pathogens primarily infect phagocytic cells. For B. neotomae, phagosomes initially mature along the endocytic route for about 8 h, initially associating with high frequency with LAMP-1. During this time, its T4SS is induced by acidic pH (29) leading to translocation of effectors into the eukaryotic cytoplasm and remodeling of the BCV into a replicative vacuole (rBCV) derived from endoplasmic reticulum. In contrast, for L. pneumophila, the T4SS is primed to intervene during initial phagocytosis (30, 31), and the phagosome rapidly takes on characteristics of an exocytic, endoplasmic reticulum-associated vacuole (32). Later, Brucella’s replicative niche is converted into an autophagy-like vacuole (aBCV) for completion of the intracellular life cycle and spread to other host cells (4, 5). In contrast, L. pneumophila appears to prevent actively later autophagic maturation of its vacuole through action of specific T4SS-translocated effectors (33). Nevertheless, despite qualitative and temporal differences in phagosome maturation, both organisms appear to share a morphologically similar endoplasmic reticulum-associated replicative niche. Therefore, from a theoretical perspective, it was possible that L. pneumophila and B. neotomae could interact during coinfection. Fascinatingly, our coinfection data demonstrated that T4SS-competent Lp02 promoted intracellular growth of wild-type B. neotomae and rescued intracellular growth of the B. neotomae ΔvirB4 mutant (Fig. 4 to 6). Furthermore, these two pathogens May 2017 Volume 85 Issue 5 e00991-16

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colocalized to a significant extent in the same vacuole. Colocalization in the same phagosome, rather than coinfection of the same host cell, was associated with B. neotomae ΔvirB4 replication (Fig. 6F). Therefore, Lp02 appeared to act in cis to promote growth of B. neotomae. In contrast, T4SS-defective strain Lp03 was incapable of promoting or rescuing B. neotomae intracellular growth and did not appear to colocalize with B. neotomae at an appreciable frequency at the time points examined. B. neotomae conversely was not able to rescue replication-defective Lp03. Therefore, rescue and growth promotion were unidirectional. We speculate that unidirectional rescue may relate to either lack of sufficient comingling of BCV with Lp03 LCV or deficiency in B. neotomae effector functionality to fully support the requirements for intracellular L. pneumophila replication. Both possibilities are consistent with prior observation of the ability of Legionella longbeachae to rescue T4SS-incompetent L. pneumophila (34) when colocalized in the same vacuole or wild-type B. melitensis to rescue an isogenic virB mutant during coinfection experiments (35). Our results with Lp02 contrast with the apparent noninteraction during Legionella and Coxiella burnetii coinfection experiments, where no colocalization was observed despite a much closer phylogenetic relationship and a highly conserved T4SS shared by the two organisms (36). These contrasting observations may relate to the distinct lysosomal replicative niche used by Coxiella, as opposed to the morphologically similar ER-associated niche used by both L. pneumophila and B. neotomae and/or the inability of L. pneumophila superinfection, described in these former studies, to access preexisting Coxiella phagosomes. Interestingly, colocalization of B. neotomae and L. pneumophila was greater than might be expected 4 h after infection. Specifically, during single infection, the majority of BCV show LAMP-1 positivity 4 h postinfection (4). In contrast, few LCV associate with LAMP-1 during the same period (37). Therefore, our observation of high colocalization frequency early in infection suggests that coinfection potentially redirects divergent early intracellular trafficking patterns. Alternatively, the modestly enhanced invasion observed for B. neotomae during coinfection with Lp02 may foster colocalization during initial phagocytosis. We also make further note of the new bio-reporter tool kit that was created to support our studies as described and characterized in the supplemental material. This toolkit supported spectrally distinct luminescent and fluorescent real-time microplate and microscopy-based readout during single and polymicrobial infection experiments. Based on the Tn5-based hyperactive in vitro transposase-based system and broadly expressed proD promoter-driven reporter expression, the toolkit should prove broadly applicable to a wide variety of Gram-negative and -positive bacterial species (38). In addition, the available nonantibiotic selectable markers encoding resistance to nourseothricin (39, 40), phleomycin (41), and hygromycin provide multiple options for selection without imparting resistance to therapeutic antibiotics. Such antibiotic resistance when not normally found in pathogens under study might otherwise limit therapeutic options and is potentially proscribed under “dual use research of concern” (42) policies (http://www.phe.gov/s3/dualuse/documents/durc-policy.pdf). We note several potential limitations of the experimental system. Luminescence and fluorescence measures did not show complete quantitative correspondence to CFU measurements. Specifically, the dynamic range of the reporters was somewhat compressed. This dynamic range compression may relate to differences in the underlying measurements: CFU indicating the number of viable bacteria and plating efficiency, luminescence reporter output integrating expression of luciferase and substrate and cellular ATP levels, and fluorescence reflecting reporter expression. Furthermore, microplate measurements may be affected by optics and the dynamic range of the detection technology. Nevertheless, qualitatively and quantitatively reporter output showed a high degree of correlation with CFU analysis. Also we make note of the ⬃2-fold increase of B. neotomae ΔvirB4 CFU during 48-h single-infection experiments and the corresponding increase observed for luminescence and fluorescent measurements (Fig. 1 and 4). However, microscopically, we did not appreciate coincident May 2017 Volume 85 Issue 5 e00991-16

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intracellular replication of this strain. We therefore speculate these small increases do not represent true intracellular growth but rather may relate to the ability of extracellular organisms, either surviving gentamicin treatment or released into the extracellular space post-gentamicin treatment, to grow slowly in tissue culture medium. Indeed, the extracellular growth phenomenon was very apparent with B. ovis and B. microti (data not shown) and was one motivation for use of B. neotomae in establishing a model system. Of note, B. neotomae is designated by the ATCC as a biosafety level 2 pathogen. Nevertheless, as little work has been performed with this organism, our own recommended practice, based on the known respiratory route of laboratory-acquired infection with human-pathogenic species, is to perform experimental manipulations with care in a biosafety cabinet and make use of gasketed safety carriers or sealed rotors during centrifugation steps. In summary, we provide evidence for a model system for studying Brucella pathogenesis that recapitulates several prominent cell biological features observed with human-pathogenic species. In particular, Brucella neotomae provides benefit in allowing research to be performed in biosafety level 2 facilities. By thus facilitating experimental work, this model could be used as a first step in identifying pathogenic traits and thereby streamline later investigation and confirmation using select agent pathogenic species. The utility of the model is underscored by the use of B. neotomae in combination with a new bio-reporter toolkit to reveal dramatic and unexpected interaction between two type IV secretion system-dependent pathogens: L. pneumophila and B. neotomae. Future exploration of the cross talk between these organisms may help elucidate important aspects of Brucella pathogen-host cell interaction. MATERIALS AND METHODS Bacterial strains and cell lines. The bacterial strains, plasmids, and eukaryotic cell lines used in this study are listed in Table 1. Escherichia coli NEB-5␣, NEB-10␤ (New England BioLabs, Ipswich, MA), and BW25113 (E. coli Genetic Stock Center, Yale University, New Haven, CT) (43) were grown at 37°C with shaking in Luria broth (LB) medium (BD, Franklin Lakes, NJ). Brucella neotomae 5K33 was obtained from BEI Resources (NIAID, NIH) and used for strain construction. Brucella strains were grown at 37°C in a humidified incubator (5% CO2) in Trypticase soy broth (TSB) medium (BD). Staphylococcus aureus 25923 (American Type Culture Collection, Manassas, VA) was cultured at 37°C in LB medium with 5% CO2. Legionella pneumophila Lp02 flaA (thyA hsdR rpsL) and Lp03 flaA (Lp02 dotA flaA) strains (24, 25, 44) were grown at 37°C on buffered charcoal yeast extract (BCYE) (45) medium supplemented with 100 ␮g/ml thymidine. Bacterial optical density was monitored at 600 nm using a DU 800 spectrophotometer (Beckman Coulter, Brea, CA) or an Epoch microtiter plater reader (BioTek, Winooski, VT). E. coli and B. neotomae were grown with 100 ␮g/ml spectinomycin (Spec), 20 ␮g/ml phleomycin (Phl), 50 ␮g/ml nourseothricin (Ntc), 50 ␮g/ml kanamycin (Km), 50 ␮g/ml hygromycin (Hyg), or 100 ␮g/ml ampicillin (Amp) to select for cognate resistance markers. BCYE was supplemented with 200 ␮g/ml Phl or 50 ␮g/ml Ntc to select for resistance markers in L. pneumophila strains. The murine J774A.1 (ATCC TIB-67) and human THP-1 (ATCC TIB-202) macrophage cell lines were passaged in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) containing 9% iron-supplemented, heat-inactivated calf serum (GemCell; Gemini Bio-Products, West Sacramento, CA) (referred to here as RPMI) in a humidified 5% CO2 incubator at 37°C. Construction of bio-reporter expression or transposon plasmids. Plasmid isolation, gel electrophoresis, transformation, and PCR amplification of DNA were performed as previously described (46). The broad-host-range plasmid pAT28 (47) was used as a vector backbone for plasmid-borne, bio-reporter expression in Gram-negative and Gram-positive bacterial cells (Fig. S1). The S. aureus codon-optimized enhanced green fluorescent protein gene (SaEGFP), synthesized de novo as a genomic block (gBlock) by Integrated DNA Technologies (Coralville, IA), and GFP-specific lux operon (luxABCDE) (48) from plasmid pMV306G13⫹Lux (49), obtained from Addgene (Cambridge, MA), were PCR amplified using extended PCR primers for incorporation of a ribosome-binding sequence (AGGAGG-) and EcoRI and SalI restriction sites at 5= and 3= ends, respectively. These gene fragments were then cloned into corresponding restriction sites in pAT28 to create promoterless versions of these reporters. The constructs were then further cut with EcoRI, dephosphorylated, and ligated to a synthetic proD promoter sequence (16) with compatible cohesive ends. The presence and orientation of the proD promoter were confirmed by Sanger sequencing. The transposon plasmid pMOD3 (Epicentre Biotechnologies, Madison, WI), diagrammed in Fig. S1, was used for chromosomal integration of bio-reporters. The tdTomato, mClover, mWasabi, mCardinal, and mNeptune2 fluorescent proteins were codon optimized for high-level expression in E. coli and through selective elimination of very rare frequency codons in Brucella neotomae. The genes were then synthesized de novo as gBlock fragments for cloning into pMOD3-based transposons. The proD/ tdTomato-nat vector and lux operon vectors were first constructed in a single step using the Gibson May 2017 Volume 85 Issue 5 e00991-16

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TABLE 1 Bacterial strains and plasmids used in this studya Strain or plasmid Bacterial strains Brucella neotomae 5K33 ΔvirB4 strain proD/tdtomato-nat strain ΔvirB4(proD/tdtomato-nat) strain virB4c(proD/tdtomato-nat) strain lux-nat strain ΔvirB4(lux-nat) strain Legionella pneumophila Lp02fla Lp03fla Lp02fla(mClover-nat or Shble) Lp03fla(mClover-nat or Shble) Lp02fla(mWasabi-nat or Shble) Lp03fla(mWasabi-nat or Shble) Lp02fla(mCardinal-nat or Shble) Lp03fla(mCardinal-nat or Shble) Lp02fla(mNeptune2-nat or Shble) Lp03fla(mNeptune2-nat or Shble) Lp02fla(proD/mClover-nat) Lp03fla(proD/mClover-nat) Lp02fla(proD/mNeptune2-nat) Lp03fla(proD/mNeptune2-nat) Lp02fla(proD/tdTomato-nat) Lp03fla(proD/tdTomato-nat) Staphylococcus aureus 25923 pAT28-SaEGFP strain pAT28-proD/SaEGFP strain pAT28-GPLux strain pAT28-proD/GPLux strain Escherichia coli EC100D pir⫹ NEB-5␣ NEB-10␤ BW25113 BW25113(lux-nat) BW25113(proD/lux-nat) MT607

Relevant markers and characteristics

Source or reference

Parent biosafety level 2 rodent pathogen virB4 in-frame deletion mutant of B. neotomae 5K33 Transposon mutant of 5K33 having proD/tdtomato-nat genes Transposon mutant of ΔvirB4 strain having proD/tdtomato-nat genes Complemented strain of ΔvirB4(proD/tdtomato-nat) strain containing pBMTL2-virB4 Transposon mutant of B. neotomae expressing lux operon-nat genes Transposon mutant of ΔvirB4 strain expressing lux operon-nat genes

BEI Resources This study This study This study This study This study This study

Philadelphia 1, thyA rpsL hsdR flaA thyA rpsL hsdR dotA03 flaA, Dot/Icm translocation deficient Transposon mutant expressing mClover-nat or mClover-Shble

44 44 This This This This This This This This This This This This This This

Transposon mutant expressing mWasabi-nat or mWasabi-Shble Transposon mutant expressing mCardinal-nat or mCardinal-Shble Transposon mutant expressing mNeptune2-nat or mNeptune2-Shble Transposon mutant expressing proD/mClover-nat Transposon mutant expressing proD/mNeptune2-nat Transposon mutant expressing proD/tdTomato-nat

study study study study study study study study study study study study study study

Wild-type, biosafety level 2 pathogen S. aureus with pAT28-SaEGFP S. aureus with pAT28-proD/SaEGFP S. aureus with pAT28-GPLux S. aureus with pAT28-proD/GPLux

ATCC This study This study This study This study

F⫺ mcrA Δ(mrr-hsdRMS-mcrBC) ␾80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK ␭⫺ rpsL (Strr) nupG pir⫹(DHFR) fhuA2 Δ(argF-lacZ)U169 phoA glnV44 ␾80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 Δ(ara-leu)7697 araD139 fhuA ΔlacX74 galK16 galE15 e14-␾80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (Strr) rph spoT1 Δ(mrr-hsdRMS-mcrBC) lacI⫹ rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 rph-1 Δ(araB–D)567 Δ(rhaD–B)568 ΔlacZ4787(::rrnB-3) hsdR514 rph-1 Transposant expressing lux operon-nat genes Transposon with proD/lux operon-nat genes Triparental E. coli strain containing pRK600 helper plasmid

Epicentre

Brucella microti CCM 4915

NEB NEB 43 This study This study 53

Czech Collection of Microorganisms Czech Collection of Microorganisms

CCM 4916

Brucella ovis NR-682

BEI Resources

Eukaryotic cell lines J774a.1 THP-1 Gryphon

Mouse macrophage, ATCC TIB-67 Human monocyte, ATCC TIB-202 Gryphon ecotropic cell line for retrovirus packaging

Plasmids pSR47s

R6K, sacB Kmr, suicide vector

ATCC ATCC Allele Biotechnology

58

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TABLE 1 (Continued) Strain or plasmid pSR47s-virB4 pBMTL2 pBMTL2-virB4 pMV306G13⫹Lux pAT28 pAT28-SaEGFP pAT28-proD/SaEGFP pAT28-GPLux pAT28-proD/GPLux pMOD3 Lamp1-YFP pmTurquoise2ER pRetroX-Tet-Off Advanced pRetroX-mTurquoise2::ER pRetroX-Lamp1::mTurquoise2 pMOD3-Lux-nat pMOD3-proD/Lux-nat pMOD3-tdTomato-nat pMOD3-proD/tdTomato-nat(hph) pMOD3-mClover-nat (Shble) pMOD3-proD/mClover/nat pMOD3-mWasabi-nat (Shble) pMOD3-mCardinal-nat (Shble) pMOD3-mNeptune2-nat (Shble) pMOD3-proD/mNeptune2-nat

Relevant markers and characteristics pSR47s vector having in-frame deletion region of B. neotomae virB4 gene Kmr, broad-host-range vector pBMTL2 vector including whole virB4 gene pMV306 vector having Gram-positive optimized lux operon Specr, Gram-positive shuttle vector pAT28 vector with promoterless eGFP (Gram-positive optimization) pAT28 vector with eGFP (Gram-positive optimization) under control of proD promoter pAT28 vector with promoterless Gram-positive organized lux operon pAT28 vector with Gram-positive organized lux operon with proD promoter EZ-Tn5 pMOD (R6K␥ ori/MCS) transposon vector, Ampr Mammalian expression vector with LAMP-1, Neor or Kanr Mammalian expression vector with calreticulin::mTurquoise2 fusion, Kanr Retroviral vector for mammalian expression, Neor or Kanr pRetroX vector expressing mTurquoise2::calreticulin fusion protein pRetroX vector expressing LAMP-1::mTurquoise2 fusion protein pMOD3 with promoterless lux operon and nat selectable marker pMOD3 with proD promoter, lux operon, and nat selectable marker pMOD3 with promoterless tdTomato and nat selectable marker pMOD3 with proD tdTomato and nat or hph selectable markers pMOD3 with promoterless mClover and nat or Shble selectable markers pMOD3 with proD promoter, mClover, and nat selectable marker pMOD3 with promoterless mWasabi and nat or Shble selectable markers pMOD3 with promoterless mCardinal and nat or Shble selectable markers pMOD3 with promoterless mNeptune2 and nat or Shble selectable markers pMOD3 with proD promoter, mNeptune2, and nat selectable marker

Source or reference This study 54 This study 49 47 This study This study This study This study Epicentre (38) Addgene (55) Addgene (56) Clontech This study This study This study This study This study This study This study This study This study This study This study This study

aNEB,

New England Biolabs; ATCC, American Type Culture Collection; nat, nourseothricin acetyltransferase gene; Shble, phleomycin resistance gene from Streptoalloteichus hindustanus; hph, hygromycin phosphotransferase gene; DHFR, dihydrofolate reductase; MCS, multiple cloning site.

assembly method (50) from three separate DNA fragments: (i) a pMOD3 vector fragment containing R6K␥ ori, Ampr, and the ColE1 ori and bordering 19-bp mosaic end sequences; (ii) a nat fragment containing a codon-optimized nourseothricin resistance gene; and (iii) a codon-optimized tdTomato gene or a PCR-amplified luxCDABE fragment from pUWGR4 (27). Gibson assembly was performed using a commercial Gibson assembly kit (New England BioLabs) and a mixture of 0.1 pmol of vector fragment, 0.3 pmol of NAT, and 0.3 pmol of bio-reporter sequence. Following incubation at 50°C for 30 min, each vector construct mixture was transformed into E. coli NEB-5␣. Other synthetic reporter genes were PCR amplified and cloned directly into the pMOD3 vector using EcoRI and SalI restriction sites. The selectable markers nat, conferring nourseothricin resistance, Shble (i.e., ble gene originally isolated from Streptoalloteichus hindustanus), conferring phleomycin resistance, and hph, conferring hygromycin resistance, were inserted downstream of reporters using SalI and HindIII sites to create an operon structure. The proD promoter EcoRI fragment was inserted upstream of fluorescent protein reporter operons in designated vectors. Chromosomal integration of reporter transposons. Following construction, transposon reporter DNA sequence was isolated by digestion of the pMOD3 plasmids with PvuII, which cuts immediately outside the 19-bp mosaic ends of the transposon constructs. Reporter transposons were then gel purified using a gel extraction kit (Qiagen, Valencia, CA). Two hundred nanograms of purified transposon was combined and incubated with EZ-Tn5 transposase (Epicentre, Madison, WI) per the manufacturer’s instructions. One microliter of each transposome complex was then incubated with electrocompetent bacteria on ice for 5 min (E. coli, Lp02, and Lp03) or 30 min (B. neotomae) and electroporated into the respective host bacterial cells. Electroporation was performed with 1-mm-gap cuvettes using a Bio-Rad MicroPulser (Bio-Rad, Hercules, CA) with settings of 1.5 kV, 25 ␮F, and 200 ⍀ for E. coli, 1.8 kV, 25 ␮F, and 200 ⍀ for L. pneumophila, and 2.0 kV, 25 ␮F, and 400 ⍀ for B. neotomae. After electroporation, 1 ml of appropriate medium was added to the electroporation mixture, and bacterial cells were incubated at 37°C for 1 h (E. coli), 7 h (L. pneumophila), or 24 h (B. neotomae) to allow for transposition and resistance marker expression. Bacterial suspensions were spread on agar plates containing appropriate selection agents to isolate transposon integrants in different species of interest. Generation of T4SS deletion mutant in B. neotomae and complementation. To generate the ΔvirB4 in-frame deletion mutant in B. neotomae, we cloned two regions of genomic DNA bracketing the N- and C-terminal regions of the virB4 gene using the overlap extension PCR method (51). The in-frame deletion construct obtained was digested with BamHI and SacI, ligated into the pSR47s suicide vector (52), and transformed into EC100D pir⫹ (Epicenter Technologies). The resulting strain was conjugated with B. neotomae through triparental mating by mixing suspensions of the donor, recipient, and an E. coli strain containing the pRK600 helper plasmid (53) on a solid agar surface and incubating the mixture for 24 h at 37°C. The mating mixture was then spread on Trypticase soy agar (TSA) plates containing 50 ␮g/ml kanamycin and 50 ␮g/ml aztreonam to select for the recipient and plasmid integrants. The transconjugant integration was confirmed by PCR amplification. Counterselection for the double recipMay 2017 Volume 85 Issue 5 e00991-16

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rocal recombinatorial deletion of the virB4 gene was accomplished on TSA plates containing 10% sucrose. After 5 days of incubation, the colonies were screened by PCR for loss of the kanamycin resistance and generation of the chromosomal ΔvirB4 in-frame deletion. For complementation of the virB4 gene deletion, the amplified virB4 DNA fragment was digested with XbaI and EcoRV and ligated with identically digested pBMTL2 vector (54) obtained from Addgene. The constructed complementation plasmid was electroporated into a ΔvirB4 transposant expressing the proD/tdTomato reporter. The complemented strain was selected on a TSA plate containing kanamycin and nourseothricin. For complementation experiments, the strain was grown overnight in liquid medium containing the same antibiotics prior to infection of J774A.1 or THP-1 cells. LAMP-1 and calreticulin colocalization experiments. LAMP-1 (from plasmid Lamp1-YFP; Addgene 1816 [55]) and calreticulin (from plasmid pmTurquoise2-ER; Addgene 36204 [56]) genes were cloned and ligated to the mTurquoise2 fluorescent protein reporter, also from pmTurquoise2-ER, to create fusion proteins. The fusions were inserted into the BamHI/EcoRI restriction sites of the pRetroX-Tet-Off Advanced vector (Clontech, Mountain View, CA), replacing the rtTA-advanced gene. The corresponding retroviral constructs were then transfected into the Gryphon ecotropic packaging cell line for retrovirus (Allele Biotech, San Diego, CA) using Lipofectamine LTX (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Three days after transfection, viral supernatant was harvested and incubated with J774A.1 macrophages for 3 days. Macrophages were then washed twice with phosphatebuffered saline (PBS) and replated in 6-well plates with RPMI medium. G-418 (Sigma-Aldrich, St. Louis, MO) at a final concentration of 1,000 ␮g ml⫺1 was added to each well for selection. Individual colonies expressing LAMP-1 or calreticulin fluorescent reporter fusion proteins were expanded and frozen in RPMI containing 10% dimethyl sulfoxide. Each cell line was passaged in RPMI containing 500 ␮g ml⫺1 G-418 until use in experiments. Cell culture microplate infection experiments. The murine J774A.1 macrophage cell line was seeded into white, flat-bottom, 96-well plates (Greiner Bio-One, Monroe, NC) at a density of 1 ⫻ 105 cells per well. THP-1 suspension cells were seeded in the same way with the addition of 100 nM 1,25dihydroxyvitamin D3 to induce macrophage differentiation. One day following cell attachment, macrophage cells were coinfected at a multiplicity of infection (MOI) of 1 with luminescent B. neotomae wild-type or B. neotomae ΔvirB4 mutant cells and fluorescent Lp02, Lp03, or S. aureus cells. Plates were then immediately centrifuged at 930 ⫻ g for 10 min to synchronize bacterial infection. Four hours postinfection, gentamicin (final concentration, 100 ␮g ml⫺1) was added to wells, and plates were incubated for an additional hour. Plates were then washed two times with PBS without Ca2⫹ or Mg2⫹. Immediately afterwards, day 0 luminescence or fluorescence was measured. Macrophages were then further incubated and assessed for luminescence and fluorescence at indicated time points using an EnVision multimode reader (PerkinElmer, Akron, OH). Further details on instrument settings are supplied in the supplemental material. Alternatively, macrophage wells were lysed at indicated time points with 0.2% saponin in PBS, and serial dilutions were plated on medium for CFU determination. Confocal microscopy. J774A.1 cells were cultured on 12-mm-round glass coverslips (Warner Instruments, Hamden, CT) in Corning 3513 Costar 12-well plates (Fisher Scientific, Waltham, MA). After reaching 70 to 80% confluence, macrophages were infected at an MOI of 1 with either single bacterial species or combinations. Plates were then immediately centrifuged for 10 min at 930 ⫻ g to synchronize bacterial invasion. After 4 h of incubation, 100 ␮g ml⫺1 gentamicin was added to wells for 1 h to kill extracellular bacteria. Each coverslip was then washed three times with PBS without Ca2⫹ and Mg2⫹, and fresh RPMI was then added. Coverslips were harvested at 4, 24, and 48 h postinfection and fixed at room temperature for 30 min with 10% formalin buffered with 1⫻ PBS containing Ca2⫹ and Mg2⫹. After being washed 3 times with PBS, coverslips were incubated with PBS containing 0.2% Triton X-100 for 15 min. Coverslips were then washed 3 times with PBS and mounted with ProLong Gold (Invitrogen, Carlsbad, CA). Images were acquired with a Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, Thornwood, NY) using the eGFP settings for detection of mClover or mWasabi, the tdTomato settings for tdTomato, and the mPlum settings for mCardinal or mNeptune2. Area measurements were determined using the microscope’s ZEN blue or lite software (Carl Zeiss). In these experiments, coinfected J774A.1 cells were chosen randomly, and the area (square nanometers) of the LCV and BCV was calculated based on the contours of respective reporter signal in confocal images. For experiments investigating colocalization of B. neotomae with LAMP-1 and calreticulin, we similarly infected J774A.1 LAMP-1::mTurquoise2 or J774A.1 mTurquoise2::calreticulin transfectants at an MOI of 1. At the indicated time points, coverslips were fixed as described above. Images were evaluated for colocalization using the Zeiss LSM 880 tdTomato and eCFP confocal settings for detection of bacteria and mTurquoise2 fusion protein signal, respectively. Statistical analysis. Statistical significance was determined in Prism 7 (Graphpad, Inc., La Jolla, CA) using the two-tailed, Mann-Whitney U test for continuous data and Fisher’s exact test for categorical data. A P value of ⬍0.01 was considered statistically significant.

SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/ IAI.00991-16. SUPPLEMENTAL FILE 1, PDF file, 1.8 MB. May 2017 Volume 85 Issue 5 e00991-16

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ACKNOWLEDGMENTS This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R01AI099122, R21AI112694, and R21AI076691 to J.E.K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Gireesh Rajashekara (Department of Veterinary Preventive Medicine, The Ohio State University) for generously providing the pUWGR4 plasmid, the E. coli Genetic Stock Center (New Haven, CT) for providing the BW25113 E. coli strain, and the Harvard-ICCB Longwood Screening facility for use of the Envision instrument. We also thank Lucius Chiaraviglio, Jennifer Tsang, and Kenneth P. Smith for critical reading of the manuscript.

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