https://orcid.org/0000-0002-0770-7784
Figures
Abstract
Listeria monocytogenes is a Gram-positive pathogen that causes the severe foodborne disease listeriosis. Following oral infection of the host, L. monocytogenes disseminates from the gastrointestinal tract to peripheral organs, including the gallbladder, where it replicates to high densities, establishing the gallbladder as the primary bacterial reservoir. Despite its importance in pathogenesis, little is known about how L. monocytogenes survives and replicates in the gallbladder. In this study, we assessed the L. monocytogenes genes required for growth and survival in ex vivo non-human primate gallbladders using a transposon sequencing approach. The screen identified 43 genes required for replication in the gallbladder, some of which were known to be important for virulence, and others had not been previously studied in the context of infection. We evaluated the roles of 19 genes identified in our screen both in vitro and in vivo, and demonstrate that most were required for replication in bile in vitro, for intracellular infection of murine cells in tissue culture, and for virulence in an oral murine model of listeriosis. Interestingly, strains lacking the mannose and glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS) permeases Mpt and Mpo exhibited no defects in intracellular growth or intercellular spread, but were significantly attenuated during murine infection. While the roles of PTS systems in vivo were not previously appreciated, these results suggest that PTS permeases are necessary for extracellular replication during infection. Overall, this study demonstrates that L. monocytogenes genes required for replication in the gallbladder also play broader roles in disease.
Author summary
Listeria monocytogenes is a Gram-positive foodborne pathogen capable of spreading from the gastrointestinal tract to internal organs, including the spleen, liver, and gallbladder. The gallbladder is proposed to serve as a primary bacterial reservoir during infection, but the mechanisms by which L. monocytogenes survives and replicates extracellularly in this environment remain poorly understood. In this study, a genome-wide screen in ex vivo gallbladders identified 43 genes as essential for L. monocytogenes growth and survival in this niche, of which 19 were validated. Notably, most of these genes are required for full virulence in a murine model of oral infection. Interestingly, not all identified genes are necessary for intracellular replication or intercellular spread, processes generally regarded as critical for pathogenesis. For example, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) permeases Mpt and Mpo, which mediate glucose and mannose import, play important roles in murine gallbladder colonization, but are dispensable in tissue culture infection models. These findings advance our understanding of L. monocytogenes pathogenesis by highlighting genes essential for survival in the gallbladder environment.
Citation: Schwardt NH, Halsey CR, Sanchez ME, Ngo BM, Reniere ML (2025) A genome-wide screen in ex vivo gallbladders identifies Listeria monocytogenes factors required for virulence in vivo . PLoS Pathog 21(3): e1012491. https://doi.org/10.1371/journal.ppat.1012491
Editor: Mary X. O'Riordan, University of Michigan Medical School, UNITED STATES OF AMERICA
Received: August 9, 2024; Accepted: January 29, 2025; Published: March 3, 2025
Copyright: © 2025 Schwardt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by NIH research grant AI132356 to M.L.R. and NIH training grant T32GM007270 to M.E.S.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that no competing interests exist.
Introduction
Listeria monocytogenes is a Gram-positive bacterium known to survive and replicate in a variety of environments, including soil, sludge, and in mammalian hosts, where it is the etiologic agent of the severe foodborne illness listeriosis [1]. Humans are exposed to L. monocytogenes multiple times per year, as it contaminates ready-to-eat foods due to its frequent occurrence in food processing facilities [2]. While the incidence of disease is relatively low compared to other foodborne illnesses, the case fatality rate of invasive listeriosis is 15-22%, making L. monocytogenes one of the most deadly bacterial pathogens [2,3]. In addition, it is estimated that 10-12% of healthy adults exhibit asymptomatic fecal shedding of L. monocytogenes, potentially contributing to the spread of this deadly pathogen [1].
After ingestion of L. monocytogenes-contaminated food, the bacteria first colonize the lumen of the gastrointestinal (GI) tract where they replicate extracellularly, primarily in the cecum and colon [4]. Small mammal models of infection have revealed that a small portion of the L. monocytogenes in the GI tract invade intestinal epithelial cells, replicate intracellularly, and spread to neighboring enterocytes via actin-mediated motility [5]. L. monocytogenes then cross the mucosal barrier and gain access to the lamina propria, Peyer’s patches, and draining mesenteric lymph nodes (MLN). Bacteria disseminate from the GI tract indirectly to the spleen through the MLN and lymphatics, and directly to the liver via the portal vein [5,6]. Subsequently, 1-5 bacteria migrate from the liver through the hepatic ducts to seed the murine gallbladder, where they replicate extracellularly in the lumen to very high densities [7]. After a meal, the gallbladder contracts and delivers a bolus of bile and bacteria into the small intestines, reseeding the GI tract [8]. Thus, the gallbladder becomes the main reservoir of L. monocytogenes during infection and the primary source of bacteria excreted in the feces [7]. These observations suggest that replication in the gallbladder is important for infection outcomes and potentially pathogen transmission, and yet little is known about the requirements for L. monocytogenes colonization and proliferation in this organ.
The gallbladder is a sac-like organ in which bile is stored and concentrated. Bile is composed of bile salts, cholesterol, phospholipids, and the heme degradation products biliverdin and bilirubin, which give bile its characteristic color. Bile acts as an emulsifier, aiding in the digestion of lipids in food and exhibiting antimicrobial activity by damaging microbial membranes, nucleic acids, and proteins [9]. Despite this, some bacteria have evolved methods of bile detoxification that render them tolerant to bile and capable of colonizing the gallbladder, including L. monocytogenes, Salmonella enterica, and Campylobacter jejuni [10–12]. Following foodborne infection, S. enterica replicates in the gallbladder both extracellularly in biofilms on gallstones and intracellularly within epithelial cells [13]. Additionally, fecal shedding from chronic asymptomatic carriers is important for the pathogenesis and transmission of S. enterica, the most famous example of which being Typhoid Mary [11]. In contrast, C. jejuni, a pathogen that infects both livestock and humans, replicates extracellularly and localizes to the mucosal folds of the gallbladder, although the role of gallbladder colonization in disease remains unclear [14]. These infection strategies are distinct from that of L. monocytogenes, which replicates extracellularly in the lumen of the gallbladder [10].
Animal models of infection have been crucial for understanding disease pathogenesis and gallbladder colonization of bacterial pathogens in vivo. Murine infection models are most commonly used, but they pose two major limitations. First, there is a severely restrictive bottleneck in which fewer than five L. monocytogenes initially seed the gallbladder following either oral or intravenous inoculation [7]. The undefined bottlenecks in vivo render the mouse model unsuitable for genetic screening approaches, such as Tn-seq or competitive mixed infections. Second, the murine gallbladder is extremely small, containing only 5 - 15 µL of biofluid [15], limiting its utility for biochemical studies aimed at identifying the requirements for L. monocytogenes colonization. Guinea pig models of oral infection have been used to assess L. monocytogenes dissemination from the GI tract to peripheral organs [5], but gallbladder colonization was not assessed in this model. Sheep have been used to study C. jejuni infection of gallbladders in vivo, but these studies are low-throughput and require veterinary surgical expertise to complete [12,14]. Purified bile salts and reconstituted powdered bile are frequently used to mimic the gallbladder environment in vitro, but it is not clear what concentration and diluent accurately represent gallbladder biofluid.
In this study, we sought to identify L. monocytogenes genes required for replication in the mammalian gallbladder using a transposon sequencing (Tn-seq) approach. This technique combines saturating transposon mutagenesis with next-generation sequencing to assess the contribution of every genetic locus in a high-throughput manner [16,17]. Tn-seq has been used to identify essential genes and genes conditionally essential for survival in a host for several pathogenic bacteria, including Staphylococcus aureus [18], Vibrio cholerae [19], Streptococcus pneumoniae [17], and recently L. monocytogenes [20]. However, the restrictive bottlenecks in mouse models of listeriosis make the use of global genetic approaches to study gallbladder colonization in vivo unfeasible. Here, we performed Tn-seq on L. monocytogenes in ex vivo non-human primate gallbladders and identified 43 genes necessary for survival and replication in this environment and more broadly in the context of a murine model of listeriosis.
Results
An unbiased approach identifies L. monocytogenes genes required for growth and survival in the gallbladder lumen
To identify Listeria monocytogenes genes required for gallbladder colonization, we developed a novel model using non-human primate (NHP) gallbladders obtained from the Washington National Primate Research Center Tissue Distribution Program. There are several advantages to NHP organs over conventional murine models. First, NHP gallbladders can be inoculated with bacteria via syringe, eliminating the bottlenecks to colonization encountered during murine infections. Second, NHP organs are larger and contain ~1,000-fold more biofluid than murine gallbladders, which can support more bacterial biomass or be harvested for in vitro assays. Finally, organs were obtained from NHPs at the endpoint of other non-infectious experiments, and therefore no additional animals were sacrificed for these studies.
In the development of the gallbladder colonization model, we first assessed whether ex vivo gallbladder biofluid (bile) supports growth of L. monocytogenes. Bile harvested from three independent NHP gallbladders was determined to be sterile and supported exponential growth of L. monocytogenes in vitro. We next injected mid-log L. monocytogenes into the lumen of intact ex vivo gallbladders and monitored bacterial survival over time by removing luminal contents with a syringe and plating to enumerate colony forming units (CFU). Interestingly, we observed consistent reductions in CFU shortly after inoculation, followed by exponential growth that plateaued between 6 and 12 hours post-injection. Additionally, we observed that immersing the organ in medium, such as DMEM, accelerated tissue deterioration. To minimize potential host cell death and preserve organ integrity, we limited the incubation time to 6 hours and conducted subsequent experiments by incubating the organs on dry, sterile petri dishes.
After establishing growth conditions for L. monocytogenes in NHP gallbladders, we used this ex vivo model to investigate the L. monocytogenes genes required for survival and growth in the gallbladder lumen using the unbiased global genetic approach of transposon sequencing (Tn-seq). Four NHP gallbladders were inoculated via syringe with a saturated transposon mutant library of L. monocytogenes containing transposon insertion sequences approximately every 25 base pairs [20]. To monitor growth of the mutant library in the organs, samples of luminal contents were collected 30 minutes and 6 hours post-injection. As observed previously with wild type (WT) L. monocytogenes, an initial reduction in CFU at 30 minutes post-injection was followed by exponential growth through 6 hours, with an average doubling time of 46 minutes (Fig 1A). This doubling time is similar to that observed for L. monocytogenes growing in rich medium, demonstrating robust growth in this environment. After 6 hours of incubation in the gallbladders, the entire luminal contents were harvested, diluted in brain heart infusion (BHI) broth, and incubated for 2 hours to increase biomass. Bacterial genomic DNA was then isolated and libraries were prepared for Illumina sequencing of the transposon insertion sites (Fig 1B and S1 Table). Using the parameters of a log2 fold-change less than -1.50 and an adjusted p-value of less than 0.05, mutants in 43 genes were significantly depleted after incubation in the gallbladders compared to the input libraries, indicating that these genes are required for growth or survival in the NHP gallbladder lumen.
(A) Growth of the L. monocytogenes transposon library in NHP gallbladders over 6 hours. Gallbladders were inoculated via syringe and bacteria were collected for CFU enumeration 30 minutes and 6 hours post-inoculation. Gallbladders from three Macaca mulatta and one M. nemestrina were used. (B) Average read counts of L. monocytogenes genes in the gallbladder experimental condition (Exp) compared to the input library (Ctrl). (C) Model depicting the PTSs and their regulators identified by Tn-seq, color-coded to match panel B. Only Mpo-IIA (gray) was not significantly depleted after incubation in NHP gallbladders.
Genes significantly depleted after incubation in the gallbladder are listed in S2 Table, categorized by the biological pathways associated with the proteins they encode. Based on the known anti-microbial properties of bile, we expected to identify genes involved in combatting membrane, DNA, and protein stress, as well as genes involved in redox homeostasis. We also expected to identify genes in metabolic pathways essential for L. monocytogenes replication in the gallbladder. In fact, the Tn-seq screen identified genes involved in protein homeostasis (clpX, encoding a Clp protease ATPase and prsA2, encoding the PrsA2 chaperone), redox homeostasis (trxA and yjbH, encoding thioredoxins), and DNA recombination and repair (xerD and recR, encoding recombinases). We also identified four genes encoding nucleotide transport and metabolism proteins, including the adenylosuccinate synthesis genes encoded by purA and purB, consistent with the importance of purine biosynthesis in surviving bile stress [21].
Ten genes encoding proteins involved in carbohydrate transport and metabolism were identified as depleted, including two phosphoenolpyruvate-dependent phosphotransferase system (PTS) permeases, mptACD and mpoBCD, which are known to import both glucose and mannose [22]. PTSs are multi-protein complexes utilized by many bacteria to import and phosphorylate defined carbohydrates, with the sugar specificity determined by the Enzyme II (EII) complex proteins [23]. While the L. monocytogenes genome encodes 29 complete PTSs [22], our screen identified Mpt and Mpo as the only EIIs required for growth in the gallbladder (Fig 1C). In addition to the PTS permeases, genes encoding regulators that activate transcription of the mpt and mpo operons (manR and sigL), and for phosphorylation of the PTS sugars (ptsI and ptsH, encoding EI and HPr, respectively) were also significantly depleted in the gallbladder condition. Identification of multiple PTS-related operons and their regulators, which lie at distinct genetic loci, suggests that L. monocytogenes imports glucose and/or mannose via Mpt and Mpo for growth in the gallbladder (Fig 1C). The Tn-seq also identified genes encoding proteins involved in coenzyme metabolism (panCD), amino acid metabolism (ansB, glynA), and energy production. In fact, 8 of the 9 genes encoding the F-type ATP synthase were depleted after growth in the gallbladders. Overall, analysis of our screen identified multiple operons as depleted after growth in the NHP gallbladder, indicating that the screen was robust. Importantly, many genes identified here have not been previously implicated in the context of infection.
Genes identified by Tn-seq in the NHP gallbladder contribute to replication in bile in vitro
To investigate the roles of the identified genes in L. monocytogenes physiology and pathogenesis, we used allelic exchange techniques to generate nine deletion mutants, representative of 19 genes identified in our screen (Table 1). These genes were chosen to represent the biological categories most depleted after growth in the gallbladder, including: protein and redox homeostasis, nucleotide transport and metabolism, carbohydrate transport and metabolism, energy production, and regulation (S2 Table). To evaluate the PTS permeases, the entire mptACD (∆mpt) or mpoABCD (∆mpo) operons were deleted and a double mutant lacking both operons (∆mpt∆mpo) was generated. To evaluate the F-type ATP synthase, the atpB open reading frame was deleted, which eliminates functionality of the entire complex [25].
The mutants were then grown individually either in BHI broth or NHP bile in 96-well plates, and CFU were enumerated after 0.5 and 6 hours of static incubation. Because the oxygen status of the gallbladder lumen is not known, L. monocytogenes growth was evaluated in both aerobic and anaerobic conditions. Several mutants exhibited general growth defects and replicated significantly less than WT in rich medium, including ∆ccpA, ∆ptsI, and ∆trxA (Figs 2A and 2B). The ∆atpB strain had the most striking phenotype in BHI, displaying a slight ~4-fold reduction in CFU in the presence of oxygen and a complete lack of growth in anaerobic conditions (Fig 2B). This is consistent with a previous report documenting that the F-type ATPase is essential for L. monocytogenes anaerobic growth [25]. We additionally measured growth in BHI in shaking flasks to assess growth in maximally aerated conditions in rich medium. Under these growth conditions, the ∆atpB mutant exhibited significantly decreased CFU at the earliest time point, while ∆trxB was attenuated for growth at 6 and 8 hours post-inoculation (Figs 2C and 2D). Although the relevant oxygen levels during infection are unknown, these data provide a broad view of mutant growth in vitro under varying oxygen levels and reveal that ∆atpB and ∆trxA are generally impaired for growth in rich medium.
(A-B) BHI or NHP bile was inoculated with 106 CFU/mL of each L. monocytogenes strain, incubated for 6 hours statically in an aerobic incubator (A) or in an anaerobic chamber (B), and then CFU were enumerated. Each circle is an individual data point, while the bars indicate the means and the error bars represent the standard error of the mean (SEM) of at least 3 biological replicates. (C-D) Flask growth curves in BHI, with shaking. Data in (C) represent the means and SEM of at least 3 biological replicates. Data in (D) represent the means and SEM of at least 2 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by Dunnett’s multiple comparisons test compared to WT in each medium condition and at each time point.
When incubated in bile under anaerobic conditions, all mutants were significantly impaired, with the exception of ∆ccpA (Fig 2B). This decrease in CFU at 6 hours was not driven by differences in killing early after inoculation, as all strains exhibited similar reductions in CFU at 30 minutes (S1 Fig). Interestingly, some mutants exhibited an oxygen-dependent phenotype. For example, ∆atpB, ∆mpt, and ∆mpt∆mpo grew similarly to WT in bile under aerobic conditions (Fig 2A), but displayed reduced growth under anaerobic conditions (Fig 2B). Together, these results demonstrated that the genes identified by Tn-seq as important during growth in the NHP gallbladder lumen are also required for growth in bile in vitro, even in the absence of competing strains.
Genes identified by Tn-seq as critical for survival in the gallbladder lumen also contribute to intracellular fitness
Tn-seq identified L. monocytogenes genes required for growth in the gallbladder lumen, which represents one of the extracellular environments that L. monocytogenes encounters during infection. While extracellular niches of infection remain largely uncharacterized, the determinants of intracellular infection and their roles in systemic disease have been thoroughly described. The intracellular lifecycle begins with L. monocytogenes entering host cells via phagocytosis or receptor-mediated endocytosis, vacuolar escape, followed by cytosolic replication and intercellular spread to neighboring cells via actin-dependent motility [26]. To determine if the genes we identified also have important roles in the intracellular lifecycle, we assessed cell-to-cell spread and cytosolic replication of each of the L. monocytogenes mutants in cell culture.
The intracellular lifecycle was first evaluated via plaque assay in which a monolayer of cells is infected with L. monocytogenes and then immobilized in agarose containing gentamicin to prevent extracellular growth. Three days post-infection, the live cells are stained and the zones of clearance formed by L. monocytogenes are measured as an indicator of intracellular growth and intercellular spread. In this assay, most mutants formed significantly smaller plaques than those formed by WT, while mutants lacking the PTS operons mpt and mpo formed plaques similar in size to WT (Fig 3A). Notably, infections with ∆atpB resulted in no visible plaque formation. We hypothesize this is due to the ∆atpB requirement for oxygen, which may be limiting in cells with the agarose overlay. Plaque areas were also measured after infection with the complemented strains, in which each deleted gene was expressed from its native promoter at an ectopic site on the chromosome. With one exception, complementation restored plaque areas to WT levels (Fig 3A). The ptsI complemented strain produced even smaller plaque areas than ∆ptsI, although this strain did restore other ∆ptsI growth defects, as discussed below.
(A) Plaque areas formed in L2 fibroblasts, measured as a percentage of those formed by WT L. monocytogenes. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by one-way ANOVA with multiple comparisons to the mean of WT. ND, not detected. (B-D) Intracellular growth of mutants in BMDMs, normalized to CFU at 30 minutes post-infection (p.i.) for each strain. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by Dunnett’s multiple comparisons test compared to WT in each condition. Data in panels A, B, and D are the means and SEMs of at least 3 independent experiments, and data in panel C are the means and SEMs of 2 independent experiments.
The plaque assay measures both cytosolic replication and cell-to-cell spread. To identify the role of each gene in intracellular growth, we measured replication kinetics in primary bone marrow-derived macrophages (BMDMs) over 8 hours. The ∆purB mutant exhibited the most dramatic phenotype as it did not replicate in the host cytosol (Fig 3B). Several additional mutants displayed attenuated intracellular growth, including ∆ccpA, ∆ptsI, and ∆trxA (Fig 3B). Intracellular growth was fully restored in the complemented strains, including ∆ptsI + ptsI (Fig 3C). Despite defects in plaque formation, both clpX::Tn and ∆atpB grew similarly to WT in BMDMs, indicating that these mutants are defective specifically in the cell-to-cell spread stage of the intracellular lifecycle. Finally, strains lacking the PTS operons (∆mpt, ∆mpo, ∆mpt∆mpo) displayed no defects in cytosolic growth, consistent with these strains forming WT-sized plaques (Fig 3D). Together, these results indicated that although we identified these genes using the selective pressure of extracellular growth in a mammalian organ, many also contribute to intracellular infection. However, the PTS operons were found to be dispensable during intracellular infection, consistent with prior work [22].
L. monocytogenes genes important in the NHP gallbladder are required for oral infection of mice.
The Tn-seq screen identified many genes important for extracellular replication in NHP gallbladders as well as intracellular growth and intercellular spread in murine cells. Thus, we hypothesized that these genes would be important for virulence in a mouse model of oral listeriosis. For these infections, 6-7 week old female BALB/c mice were given streptomycin in their drinking water for 2 days and fasted for 16 hours prior to infection to increase susceptibility to oral infection [4,27,28]. Mice were then fed 108 CFU of each L. monocytogenes strain via pipette. Body weights were recorded daily as a measurement of global disease severity. Mice infected with WT lost nearly 20% of their initial body weight throughout the 4 day infection, whereas mice infected with most of the mutant strains exhibited significantly less weight loss (Figs 4A and 4B). Notably, mice infected with ∆ccpA, ∆mpo, or ∆mpt∆mpo lost approximately the same amount of weight as mice infected with WT, suggesting that that these genes may not be required for L. monocytogenes pathogenesis in vivo. Conversely, mice infected with ∆ptsI, ∆trxA, and ∆purB lost very little weight over the 4 day infection, suggesting that these strains were severely attenuated in their pathogenicity.
Mice were orally infected with 108 of each L. monocytogenes strain. (A-B) Body weights of infected mice over time, reported as a percentage of initial weight before streptomycin treatment. Data are means and SEM of n=4-33. (C-F) CFU were enumerated from tissues at 1 or 4 days post-infection. Each data point represents a single mouse (n=15-18 for WT, n=4-10 for mutants), horizontal solid lines represent geometric means, and the dotted lines represent the limit of detection. The data are combined from 4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by Dunnett’s multiple comparisons test compared to WT in each condition.
To assess bacterial burdens throughout infection, mice were euthanized and CFU were enumerated from organs at both 1 and 4 days post-infection (dpi). After ingestion, L. monocytogenes in the GI tract disseminates via the portal vein to the liver and subsequently the gallbladder. At 1 dpi, bacterial burdens in the livers and gallbladders were similar between WT and all mutant strains, indicating that these genes are not required for dissemination from the GI tract to the liver or gallbladder (Figs 4C and 4E). By 4 dpi, 7 of the 9 mutants displayed significantly decreased bacterial burdens in the gallbladder compared to WT (Fig 4D). Bacterial burdens in mice infected with ∆ptsI, ∆purB, and clpX::Tn were decreased by more than 300,000-fold compared to mice infected with WT. In contrast, bacterial loads of mice infected with ∆trxA, ∆atpB, ∆mpt, ∆mpo, and ∆mpt∆mpo displayed more variability in CFU between animals and were decreased by 174- to over 38,000-fold compared to mice infected with WT. Interestingly, all mutants were significantly attenuated in the livers at 4 dpi, with the exception of ∆ccpA (Fig 4F). These data demonstrate that the majority of the L. monocytogenes genes identified by Tn-seq as important for colonization of NHP gallbladders ex vivo were also required for infection of murine gallbladders and livers in vivo.
In addition to disseminating directly to the liver via the portal vein, L. monocytogenes disseminates from the GI tract via the lymphatics through the MLN and to the spleen [5]. Most mutants colonized the MLN and displayed similar bacterial burdens as WT at both 1 and 4 dpi, with the exception of ∆ptsI, ∆purB, and ∆atpB, which were significantly attenuated in the MLN compared to WT (Figs 5A and 5B). In the spleens, only ∆ptsI, ∆purB, ∆mpt, and clpX::Tn were significantly attenuated at 4 dpi compared to WT (Fig 5D). Bacterial burdens in the feces were also enumerated as a measure of L. monocytogenes colonization in the lower GI tract lumen. Bacterial burdens in the feces were similar between most mutants and WT at 1 dpi, whereas the majority of mutants exhibited significantly decreased bacterial loads compared to WT at 4 dpi (Figs 5E and 5F). The notable exception is ∆purB, which was decreased ~150-fold compared to WT in the feces at 1 dpi, but similar to WT by 4 dpi. These data collectively demonstrate that the genes identified in our ex vivo screen contribute to infection of multiple organs, including the gallbladder, following oral infection of mice.
Mice were orally infected with 108 of each L. monocytogenes strain and CFU from tissues or feces were enumerated 1 (A, C, E) or 4 (B, D, F) days post-infection. Each data point represents a single mouse (n=15-18 for WT, n=4-10 for mutants), horizontal solid lines represent geometric means, and the dotted lines represent the limit of detection. The data are combined from 4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by Dunnett’s multiple comparisons test compared to WT in each condition.
DISCUSSION
In this study we sought to identify L. monocytogenes genes important for infection of the mammalian gallbladder. To this end, we developed an ex vivo bacterial colonization model of NHP gallbladders and performed Tn-seq to determine the genes necessary for growth and survival in this environment. This unbiased global genetic approach identified mutants in 43 genes that were significantly depleted after growth in the gallbladder condition, including some genes known to be important for virulence and others not previously studied in the context of infection. Several mutants identified by Tn-seq had growth defects in rich medium and most were predictably attenuated for growth in NHP bile in vitro. Many mutants also had defects in the intracellular lifecycle, including cytosolic growth and cell-to-cell spread, with the notable exception of the PTS permeases. A murine model of oral L. monocytogenes infection revealed that nearly all identified genes are required for full virulence. Together, these data identified genes that are important for L. monocytogenes infection of a mammal and, interestingly, not all are required for intracellular replication or intercellular spread.
Animal models have shed light on the importance of gallbladder colonization during L. monocytogenes pathogenesis. Murine models of infection demonstrated that L. monocytogenes replicates extracellularly in the gallbladder lumen to high bacterial densities and that this population can become the primary bacterial reservoir and source of fecally shed L. monocytogenes [4,7,10]. Further, they revealed the presence of an uncharacterized severe within-host bottleneck in which the founding population of the gallbladder is limited to approximately 3 bacteria [7]. Dowd et al. demonstrated that L. monocytogenes readily grows in ex vivo porcine gallbladders and the extracted bile [21]. Given the limitations of murine infection models for examining bacterial gallbladder colonization, and inspired by Dowd’s use of ex vivo organs as incubators, we established a new infection model with a less restrictive bottleneck that is also amenable to Tn-seq analysis. While the present study focused on L. monocytogenes luminal growth within the organs, the ex vivo NHP gallbladder model could be utilized to measure mucosal and epithelial colonization of a variety of gallbladder-tropic pathogens.
Our Tn-seq screen identified mutants in dozens of genes that were significantly depleted after incubation in the NHP gallbladders. These genes include those involved in redox regulation (trxA, yjbH, rex), cell wall modifications (walK), and protein stability (clpX), consistent with the known antimicrobial activities of bile which result in cell envelope stress and protein damage [9]. Purine biosynthesis was previously identified as important for growth in porcine bile and here, we identified purB as essential for replication in the gallbladder [21]. Interestingly, the screen did not identify the known bile resistance genes mdrT, bsh, bilE, or sigB as required for growth or survival in the gallbladder [29–31]. MdrT is a multidrug resistance transporter originally identified to secrete c-di-AMP [32,33] and subsequently suggested to be an efflux pump for cholic acid [34], a component of bile. The bsh gene encoding bile salt hydrolase was originally described as required for survival in bile in vitro and for intestinal persistence in a guinea pig model of infection [30]. The bile exclusion system encoded by bilE was proposed to be a transporter that protected L. monocytogenes from toxicity induced by 30% reconstituted bovine bile [31]. SigB is a stress response alternative sigma factor that positively regulates both bsh and bilE [29,31]. It is now appreciated that bsh, bilE, and sigB confer resistance to acidified bile acids, as may be found in the small intestine, but are not necessary to detoxify bile at neutral pH, as is found in the gallbladder lumen [21,35]. Furthermore, BilE was recently renamed EgtU when it was conclusively demonstrated that it specifically binds and transports the low molecular weight thiol ergothioneine [36].
Some genes we identified by Tn-seq had been previously described as necessary for virulence in vivo, while many others had not been studied in the context of infection. The purB, trxA, yjbH, clpX, and rex genes are known to be required for full virulence, though some of the studies used different mouse strains and different inoculation methods [37–40]. Moreover, rex is one of the few L. monocytogenes genes specifically required for replication in the murine gallbladder [35]. Conversely, we identified genes encoding all 8 structural components of the F-type ATP synthase, which was not previously examined in vivo [25]. We also identified operons encoding two PTS EII complexes (mpt, mpo), as well as genes encoding their transcriptional (sigL, manR) and post-transcriptional regulators (ptsH, ptsI). Mpt and Mpo were previously designated as dispensable for virulence based on tissue culture assays, although were never tested in vivo [22].
Most mutants under investigation were deficient for growth in NHP bile in vitro, which was unsurprising given the conditions under which the Tn-seq screen was performed. The most attenuated strains after growth in bile were ∆ptsI, ∆trxA, and ∆purB, which also displayed growth defects in rich medium. Additionally, the ∆atpB strain was severely attenuated in BHI, which was expected based on the published requirement for the F-type ATP synthase for anaerobic replication [25]. In the aerobic condition, the cultures were incubated statically and we hypothesize that the lack of aeration led to ∆atpB growing more slowly than WT in BHI. Surprisingly, ∆atpB did replicate in NHP bile under anaerobic conditions. Future research will investigate the role of atpB in L. monocytogenes growth in bile and in vivo. It has been hypothesized that the F-type ATP synthase is required during anaerobic growth to combat acid stress and generate a proton motive force, rather than for ATP synthesis [25,41]. It remains unclear, however, if these mechanisms contribute to the role of the F-type ATP synthase during infection.
Tissue culture models of infection have historically been reliable indicators of L. monocytogenes pathogenesis in vivo, although the correlation was not as strong in this study. Several mutants displayed defects in a plaque assay, which measures both intracellular growth and intercellular spread over three days. Specifically, ∆ccpA, ∆trxA, ∆purB, and clpX::Tn formed significantly smaller plaques than WT. Despite this, ∆ccpA was surprisingly fully virulent in a murine model of listeriosis. Furthermore, mpt and mpo were completely dispensable for intracellular growth and intercellular spread in tissue culture, although they were required for infection of mice. Importantly, studies solely using tissue culture models of infection would not have identified these operons as important for pathogenesis in vivo.
To assess the validity of the ex vivo NHP gallbladder model, we evaluated the roles of genes identified by Tn-seq in an oral murine model of listeriosis. Nearly all mutants tested were significantly attenuated in the gallbladders and livers of infected mice 4 dpi. The notable exception was the strain lacking ccpA, the catabolite control protein A that represses transcription of metabolic genes based on the phosphorylation state of HPr and the overall nutrient status of the cell. In the ∆ccpA mutant, approximately 100 genes are de-repressed [42], resulting in attenuated in vitro growth in rich medium, bile, and BMDMs, yet exhibiting no virulence defect in mice after oral infection. Interestingly, not all strains were attenuated in the spleens and MLNs of infected mice, suggesting that factors required for colonizing the liver and gallbladder are distinct from those needed to colonize other peripheral organs. For example, ∆trxA was attenuated approximately 29,000-fold in the gallbladder, but colonized the spleen and MLN at levels similar to WT, despite a significant growth defect in rich medium in vitro. Relatedly, it was recently reported that L. monocytogenes folate metabolism is specifically required in the livers but not the spleens of infected mice [20,43,44]. Conversely, ∆ptsI and ∆purB were significantly attenuated in the MLN at both 1 and 4 dpi, indicating that these genes are necessary for dissemination beyond the gut and/or replication in the MLN. Taken together, oral infections of mice revealed that the genes identified by Tn-seq in the NHP gallbladder have broader roles in disease pathogenesis than simply conferring resistance to bile stress.
Similarly to the gallbladders, most mutants were significantly attenuated in the feces at 4 dpi, supporting the notion that the gallbladder is the primary source of fecally excreted bacteria [7]. The two mutants not attenuated in the feces were ∆ccpA and ∆purB. While ∆ccpA was fully virulent in mice, the ∆purB mutant was severely attenuated in vitro and in all murine organs after infection. These results indicate that purine biosynthesis is required for intracellular infection and virulence, but not for extracellular survival in the lumen of the lower GI tract or feces. The factors influencing fecal shedding of L. monocytogenes are incompletely understood. Zhang et al. used a barcoded library of L. monocytogenes to demonstrate that the gallbladder is the source of fecally shed bacteria and that neutrophils and monocytes restrict bacterial dissemination to the gallbladder [7]. A subsequent study using a similar approach determined that the bacterial population in the feces was derived from the inoculum after infection with a severely attenuated strain of L. monocytogenes [4]. Thus, multiple factors contribute to fecal shedding during listeriosis, including the level of gallbladder colonization, specific strain virulence capacity, and the host immune response.
Taken together, our Tn-seq approach revealed several novel insights into L. monocytogenes carbon metabolism during infection. It is well-accepted that the primary carbon sources consumed by L. monocytogenes in the cytosol are host-derived glycerol and hexose-phosphates and thus, the permeases that import these sugars are required for intracellular replication and virulence [45–47]. Conversely, L. monocytogenes encodes 84 genes that assemble into 29 complete PTSs, which were previously thought to be dispensable for virulence [22]. Indeed, the main glucose and mannose PTS EII proteins, encoded by the mpt and mpo operons, are not required for intracellular growth or intercellular spread [22,48]. However, we found that strains lacking mpt or mpo are significantly attenuated in a murine model of listeriosis. These results suggest that glucose and mannose are important nutrients for L. monocytogenes replicating in extracellular sites in vivo, such as the gallbladder and, to a lesser extent, the liver. Recent studies established that significant populations of L. monocytogenes are extracellular in the liver, spleen, and MLN [6,49], although the bacterial requirements for surviving extracellularly and the role that extracellular bacteria play in pathogenesis remain unclear. Interestingly, infection with mutants lacking mpt and mpo resulted in similar bacterial loads in the MLN as WT at both 1 and 4 dpi, which suggests that these PTS operons are not required for dissemination beyond the GI tract. Moreover, all PTSs are activated by a phosphorelay between Enzyme I (encoded by ptsI) and HPr (encoded by ptsH). Thus, the ∆ptsI mutant, which functionally lacks all 29 PTSs, is deficient for intracellular replication and dramatically attenuated in vivo. This suggests that PTS-dependent carbohydrates are important nutrients in the host cytosol. Ongoing studies are aimed at characterizing the additional PTSs that are required for full virulence.
Methods
Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were reviewed and approved by the Animal Care and Use Committee at the University of Washington (Protocol 4410–01).
Bacterial strains and conditions
The bacterial strains used in this study are listed in S2 Table. L. monocytogenes was cultured in brain heart infusion (BHI) and E. coli was cultured in Luria-Bertani (LB) broth at 37°C, with shaking (220 rpm), unless otherwise specified. Antibiotics (purchased from Sigma Aldrich) were used at the following concentrations: streptomycin, 200 μg/mL; chloramphenicol, 10 μg/mL (E. coli) and 7.5 μg/mL (L. monocytogenes); and carbenicillin, 100 μg/mL. L. monocytogenes mutants were derived from wild type strain 10403S [50,51]. Plasmids were introduced to E. coli via chemical competence and heat shock and introduced into L. monocytogenes via trans-conjugation from E. coli SM10 [52].
Vector construction and cloning
To construct in-frame, unmarked deletion mutants by allelic exchange in L. monocytogenes, ~700 bp regions up- and downstream of the gene of interest were PCR amplified using L. monocytogenes 10403S genomic DNA as a template. PCR products were digested and ligated into pLIM (gift from Arne Rietsche, Case Western). pLIM plasmids were then transformed into E. coli and sequences confirmed via Sanger sequencing (Azenta). Plasmids harboring mutant alleles were then introduced into L. monocytogenes via trans-conjugation and integrated into the chromosome as previously described [53,54].
Complemented strains of L. monocytogenes were generated using the pPL2 integration plasmid [55]. Genes were PCR amplified with their respective native promoters using L. monocytogenes 10403S genomic DNA as a template, and sequences were confirmed by Sanger sequencing. The constructed pPL2 plasmids were then introduced into L. monocytogenes by trans-conjugation and integration into the L. monocytogenes chromosome was confirmed by antibiotic resistance.
Growth curves
NHP bile aliquots were plated to evaluate sterility and stored at -80°C. Before an experiment, aliquots were thawed overnight at 4°C and warmed to room temperature immediately before the inoculation into a 96-well plate. Overnight L. monocytogenes cultures were washed twice, resuspended in PBS, and 105 CFU were inoculated into either NHP bile or BHI, in a total volume of 100 µL per well. Bacterial growth was measured by collecting samples of the cultures, serially diluting in PBS, and plating for CFU. For experiments performed anaerobically, NHP bile aliquots were thawed overnight in GasPak EZ Anaerobe gas-generating pouches (Becton Dickinson), and BHI and the 96-well plate were degassed overnight in a closed-system anaerobic chamber (Don Whitley Scientific A35 anaerobic work station). After washing and resuspending aerobically-grown overnight L. monocytogenes cultures in PBS, the L. monocytogenes suspensions and bile aliquots were transferred into the anaerobic chamber and the plate was inoculated and incubated within the chamber. Bacterial growth was measured by collecting samples of the cultures, serially diluting in PBS, and plating for CFU.
To evaluate aerobic growth in rich medium, L. monocytogenes overnight cultures were normalized to an OD600 of 0.02 in 25 mL BHI in 250-mL flasks and incubated at 37°C, with shaking. At each time point, bacteria were serially diluted and plated on BHI agar to enumerate CFU. Raw data are included in S6 Table.
L. monocytogenes transposon library in NHP gallbladders.
The NHP gallbladders were obtained from animals at the WaNPRC that had been part of non-infectious experiments. The organs were transported between facilities on ice and used for experiments within 2 hours of excision. The animals included a mix of females and males, ages 8-12 years old, with body weights ranging from 10 – 13.7 kg.
The L. monocytogenes transposon library [20] was inoculated directly from the -80°C stock into BHI broth and incubated at 37°C for 2 hours, with shaking. The library was then washed twice and resuspended in PBS to a density of 108 CFU per 2 kg of NHP body weight. The inoculum size was determined to maintain 1,000-fold coverage of the library. Gallbladders were injected via syringe with 100 µL of inoculum, the injection site was sealed with liquid bandage (3M), and incubated in a dry 15 cm petri dish at 37°C, with 5% CO2. After 30 minutes, 200 µL of bile was removed from the gallbladder via syringe, serially diluted, and plated to enumerate CFU. 6 hours post-injection, bile was extracted from the organ via syringe for CFU enumeration and the remaining luminal contents collected via cell scraper after resection. The gallbladder contents were diluted into 50 mL BHI broth and incubated at 37°C for 2 hours, with shaking. The cultures were pelleted, washed twice with PBS, and stored at -80°C.
Tn-seq library preparation, sequencing, and analysis
Genomic DNA was extracted using a Quick-DNA Fungal/Bacterial MiniPrep Kit (Zymo Research). DNA was diluted to 3 µg/130 µL in microTUBES (Covaris) and sheared in duplicate on a Covaris LE220 Focused-Ultrasonicator using the following settings: duty cycle 10%; peak intensity 450; cycles per burst 100; duration 100 sec. Sheared DNA was then end-repaired with NEBNext End Repair (NEB), and purified with Ampure SPRIselect beads (Beckman Coulter). Poly-C tails were added to 1 µg of end-repaired DNA with Terminal Transferase (Promega), then purified with Ampure SPRIselect beads. Transposon junctions were PCR amplified with primers olj376 and pJZ_RND1 (S5 Table) using 500 ng DNA and KAPA HiFi Hotstart Mix (Kapa Biosystems). PCR reactions were stopped once the inflection point of amplification was reached (6-14 cycles), and amplified transposon junctions were purified with Ampure SPRIselect beads. Barcoded adaptors were added using KAPA HiFi Hotstart Mix, and primers pJZ_RND2 and one TdT_Index per sample. DNA was purified and size-selected with Ampure SPRIselect beads for 250-450 bp fragments. Samples were pooled and sequenced as single end 50 bp reads on a NextSeq MO150 sequencer with a 7% PhiX spike in and primer pJZTnSq_SeqPrimer.
Trimmed reads were mapped to the L. monocytogenes 10403S NC_17544 reference genome in PATRIC (now https://www.bv-brc.org/) and assessed for essentiality using TRANSIT software [56–58]. Genes were considered required for survival or growth in ex vivo NHP gallbladders if they met the following criteria: 5 or more insertion sites in the input libraries, a p value less than 0.05, and a 1.5-fold or greater depletion after incubation in the gallbladder.
Murine cells
L2 fibroblasts were incubated at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) (Cytiva) and supplemented with sodium pyruvate (1 mM) and L-glutamine (1 mM) (L2 Medium). For passaging, cells were maintained in Pen-Strep (100 U/ml) but were plated in antibiotic-free media for infections.
Bone marrow-derived macrophages (BMDMs) were routinely incubated in DMEM supplemented with 20% heat-inactivated FBS, 1 mM sodium pyruvate, 1 mM L-glutamine, 10% supernatant from M-CSF-producing 3T3 cells, and 55 μM β-mercaptoethanol (BMDM medium). BMDMs were isolated as previously described [59]. Briefly, femurs and tibias from C57BL/6 mice bred in-house were crushed with a mortar and pestle in 20 mL BMDM medium and strained through 70-μm cell strainers. Cells were plated in 150-mm untreated culture dishes, supplemented with fresh BMDM medium at day 3, and then harvested by resuspending cells in cold PBS at day 7. BMDMs were aliquoted in 80% BMDM medium, 10% FBS, and 10% DMSO and stored in liquid nitrogen.
Intracellular growth curves
BMDMs were plated in TC-treated 24-well plates at a density of 6 x 105 cells per well in BMDM medium. L. monocytogenes cultures were grown overnight at 30°C, stationary. The next day, L. monocytogenes cultures were washed twice, resuspended in PBS, and added to BMDMs at an MOI = 0.1. After 30 minutes, cells were washed twice with PBS and BMDM medium containing gentamicin (50 µg/mL) was added to kill extracellular bacteria. At various time points post-infection, cells were washed twice with PBS and lysed in 250 μL cold 0.1% Triton-X in PBS. Lysates were then serially diluted and plated to enumerate intracellular CFU.
Plaque assays
Plaque assays were performed as previously described [39,60]. In brief, 1.2 x 106 L2 fibroblasts were plated in tissue-culture treated 6-well plates overnight in L2 medium. L. monocytogenes cultures were grown overnight at 30°C stationary. The next day, L. monocytogenes cultures were diluted 1:10 in PBS and 5 µL of diluted bacteria was added to cell monolayers. After 1 hour of infection, monolayers were washed twice with PBS, then overlaid with 3 mL of molten agarose solution (1:1 mixture of 2X DMEM and 1.4% SuperPure Agarose (U.S. Biotech Sources, LLC), containing 10 µg/mL gentamicin). After 3 days of incubation, 2 mL of molten agarose solution containing Neutral Red was added to wells to visualize plaques. After 12-24 hours, plates were scanned, plaque areas quantified using ImageJ software [61] and normalized to WT.
Oral murine infections
Female BALB/c mice were purchased from The Jackson Laboratory (Strain 000651) at 5 weeks of age and used in experiments when they were 6–7 weeks old. BALB/c mice were used because they are more susceptible to oral listeriosis and gallbladder colonization, in particular [28]. Infections were performed as previously described [4,35]. Streptomycin (5 mg/mL) was added to drinking water 48 hours prior to infection and food and water were removed 16 hours before infection. L. monocytogenes cultures were grown overnight at 30°C, stationary. Overnight cultures were diluted 1:10 in 5 mL fresh BHI and incubated at 37°C for 2 hours, with shaking. Bacteria were then washed twice and diluted in PBS. Mice were fed 108 bacteria in 20 µL of PBS and food and water were returned immediately after infection. Inocula were serially diluted and plated. Body weights were recorded daily and mice were humanely euthanized 1 and 4 days post-infection for tissue collection. Tissues were homogenized in the following volumes of 0.1% Igepal CA-630 (Sigma): MLN, 3 mL; cecum (contents removed and tissues rinsed with PBS), 4 mL; liver, 5 mL; spleen, 3 mL. Feces were homogenized in 1 mL of 0.1% Igepal with a sterile stick, and gallbladders were ruptured and crushed in 500 µL of 0.1% Igepal with a sterile stick. All samples were serially diluted in PBS and plated to enumerate CFU.
Supporting information
S1 Fig. Growth curves of L. monocytogenes in BHI or bile in vitro.
BHI or NHP bile was inoculated with L. monocytogenes, incubated statically in an aerobic incubator (A,B) or in an anaerobic chamber (C,D), and CFU were enumerated, as in Fig 2. Data are the means and SEMs of at least three independent experiments. Statistics omitted for clarity.
https://doi.org/10.1371/journal.ppat.1012491.s001
(TIF)
S2 Table. Summary of significant Tn-seq hits.
https://doi.org/10.1371/journal.ppat.1012491.s003
(DOCX)
S5 Table. Primers used in Tn-seq library preparation.
https://doi.org/10.1371/journal.ppat.1012491.s006
(DOCX)
Acknowledgments
We would like to thank all members of the Reniere and Woodward Labs at the University of Washington (UW) for valuable feedback and discussions; the Mougous Lab at UW for equipment; and Christopher English at the Washington National Primate Research Center for coordinating NHP experiments.
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