PsaF Is a Membrane-Localized pH Sensor That Regulates psaA Expression in Yersinia pestis.

The Yersinia pestis pH 6 antigen (PsaA) forms fimbria-like structures and is required for full virulence during bubonic plague. High temperature and low pH regulate PsaA production, and while recent work has uncovered the molecular aspects of temperature control, the mechanisms underlying this unusual regulation by pH are poorly understood. Using defined growth conditions, we recently showed that high levels of PsaE and PsaF (two regulatory proteins required for expression of psaA) are present at mildly acidic pH, but these levels are greatly reduced at neutral pH, resulting in low psaA expression. In prior work, the use of translational reporters suggested that pH had no impact on translation of psaE and psaF, but rather affected protein stability of PsaE and/or PsaF. Here, we investigated the pH-dependent posttranslational mechanisms predicted to regulate PsaE and PsaF stability. Using antibodies that recognize the endogenous proteins, we showed that the amount of PsaE and PsaF is defined by a distinct pH threshold. Analysis of histidine residues in the periplasmic domain of PsaF suggested that it functions as a pH sensor and indicated that the presence of PsaF is important for PsaE stability. At neutral pH, when PsaF is absent, PsaE appears to be targeted for proteolytic degradation by regulated intramembrane proteolysis. Together, our work shows that Y. pestis utilizes PsaF as a pH sensor to control psaA expression by enhancing the stability of PsaE, an essential psaA regulatory protein. IMPORTANCE Yersinia pestis is a bacterial pathogen that causes bubonic plague in humans. As Y. pestis cycles between fleas and mammals, it senses the environment within each host to appropriately control gene expression. PsaA is a protein that forms fimbria-like structures and is required for virulence. High temperature and low pH together stimulate psaA transcription by increasing the levels of two essential integral membrane regulators, PsaE and PsaF. Histidine residues in the PsaF periplasmic domain enable it to function as a pH sensor. In the absence of PsaF, PsaE (a DNA-binding protein) appears to be targeted for proteolytic degradation, thus preventing expression of psaA. This work offers insight into the mechanisms that bacteria use to sense pH and control virulence gene expression.

Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis.

PsaA, the subunit of the fimbria originally referred to as the "pH 6 antigen," is required for full virulence of Yersinia pestis during bubonic plague. The expression of psaA is dependent upon specific environmental signals, and while the signals (high temperature and acidic pH) are defined, the mechanisms underlying this regulation remain unclear. In the closely related species Yersinia pseudotuberculosis, psaA transcription requires two regulatory genes, psaE and psaF, and it is speculated that posttranscriptional regulation of PsaE and/or PsaF contributes to the regulation of psaA transcription. Few studies have examined the regulation of psaA expression in Y. pestis, and prior to this work, the roles of psaE and psaF in Y. pestis had not been defined. The data presented here show that both psaE and psaF are required for psaA transcription in Y. pestis and that the impact of temperature and pH is mediated through discrete posttranscriptional effects on PsaE and PsaF. By generating antibodies that recognize endogenous PsaE and PsaF, we determined that the levels of both proteins are impacted by temperature and pH. High temperature is required for psaE and psaF translation via discrete mechanisms mediated by the mRNA 5' untranslated region (UTR) upstream of each gene. Additionally, levels of PsaE and PsaF are impacted by pH. We show that PsaF enhances the stability of PsaE, and thus, both PsaE and PsaF are required for psaA transcription. Our data indicate that the environmental signals (temperature and pH) impact the expression of psaA by affecting the translation of psaE and psaF and the stability of PsaE and PsaF.IMPORTANCEY. pestis is a Gram-negative bacterial pathogen that causes bubonic plague. As a vector-borne pathogen, Y. pestis fluctuates between an arthropod vector (flea) and mammalian host. As such, Y. pestis must recognize environmental signals encountered within each host environment and respond by appropriately regulating gene expression. PsaA is a key Y. pestis mammalian virulence determinant that forms fimbriae. Our work provides evidence that Y. pestis utilizes multiple posttranscriptional mechanisms to regulate the levels of two PsaA regulatory proteins in response to both temperature and pH. This study offers insight into mechanisms that bacteria utilize to sense environmental cues and regulate the expression of determinants required for mammalian disease.

Comparison of Models for Bubonic Plague Reveals Unique Pathogen Adaptations to the Dermis.

UNLABELLED: Vector-borne pathogens are inoculated in the skin of mammals, most likely in the dermis. Despite this, subcutaneous (s.c.) models of infection are broadly used in many fields, including Yersinia pestis pathogenesis. We expand on a previous report where we implemented intradermal (i.d.) inoculations to study bacterial dissemination during bubonic plague and compare this model with an s.c. MODEL: We found that i.d. inoculations result in faster kinetics of infection and that bacterial dose influenced mouse survival after i.d. but not s.c. inoculation. Moreover, a deletion mutant of rovA, previously shown to be moderately attenuated in the s.c. model, was severely attenuated in the i.d. MODEL: Lastly, based on previous observations where a population bottleneck from the skin to lymph nodes was observed after i.d., but not after s.c., inoculations, we used the latter model as a strategy to identify an additional bottleneck in bacterial dissemination from lymph nodes to the bloodstream. Our data indicate that the more biologically relevant i.d. model of bubonic plague differs significantly from the s.c. model in multiple aspects of infection. These findings reveal adaptations of Y. pestis to the dermis and how these adaptations can define the progression of disease. They also emphasize the importance of using a relevant route of infection when addressing host-pathogen interactions.

The BBA33 lipoprotein binds collagen and impacts Borrelia burgdorferi pathogenesis.

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2-RpoN-RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS-dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2-RpoN-RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33-dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance.

Dissemination of a highly virulent pathogen: tracking the early events that define infection.

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.

Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules.

Systemic dissemination of microbial pathogens permits microbes to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to bacterial infections. Dissemination is a critical stage of disease progression by the Lyme spirochaete, Borrelia burgdorferi. However, many mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular surfaces in the face of the shear forces present in flowing blood. Using real-time microscopic imaging of the Lyme spirochaete in living mice we previously identified the first bacterial protein (B. burgdorferi BBK32) shown to mediate vascular adhesion in vivo. Vascular adhesion is also dependent on host fibronectin (Fn) and glycosaminoglycans (GAGs). In the present study, we investigated the mechanisms of BBK32-dependent vascular adhesion in vivo. We determined that BBK32-Fn interactions (tethering) function as a molecular braking mechanism that permits the formation of more stable BBK32-GAG interactions (dragging) between circulating bacteria and vascular surfaces. Since BBK32-like proteins are expressed in a variety of pathogens we believe that the vascular adhesion mechanisms we have deciphered here may be critical for understanding the dissemination mechanisms of other bacterial pathogens.

Bioluminescence imaging to track bacterial dissemination of Yersinia pestis using different routes of infection in mice.

BACKGROUND: Plague is caused by Yersinia pestis, a bacterium that disseminates inside of the host at remarkably high rates. Plague bacilli disrupt normal immune responses in the host allowing for systematic spread that is fatal if left untreated. How Y. pestis disseminates from the site of infection to deeper tissues is unknown. Dissemination studies for plague are typically performed in mice by determining the bacterial burden in specific organs at various time points. To follow bacterial dissemination during plague infections in mice we tested the possibility of using bioluminescence imaging (BLI), an alternative non-invasive approach. Fully virulent Y. pestis was transformed with a plasmid containing the luxCDABE genes, making it able to produce light; this lux-expressing strain was used to infect mice by subcutaneous, intradermal or intranasal inoculation. RESULTS: We successfully obtained images from infected animals and were able to follow bacterial dissemination over time for each of the three different routes of inoculation. We also compared the radiance signal from animals infected with a wild type strain and a Δcaf1ΔpsaA mutant that we previously showed to be attenuated in colonization of the lymph node and systemic dissemination. Radiance signals from mice infected with the wild type strain were larger than values obtained from mice infected with the mutant strain (linear regression of normalized values, P<0.05). CONCLUSIONS: We demonstrate that BLI is useful for monitoring dissemination from multiple inoculation sites, and for characterization of mutants with defects in colonization or dissemination.

Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity.

The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.

Genetic transformation of Borrelia burgdorferi.

The development of robust genetic tools to manipulate Borrelia burgdorferi, the etiologic agent of Lyme disease, now allows investigators to assess the role(s) of individual genes in the context of experimental Lyme borreliosis. This unit is devoted to the description of experimental approaches that are available for the molecular genetic analysis of B. burgdorferi with an emphasis on cultivation, electrotransformation, selection of desired mutants, and genetic complementation of acquired mutants. The intent is to provide a consensus protocol that encapsulates the methodologies currently employed by the B. burgdorferi research community and describe pertinent issues that must be accounted for when working with these pathogenic spirochetal bacteria.

The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background.

Yersinia pestis is a highly pathogenic Gram-negative organism and the causative agent of bubonic and pneumonic plague. Y. pestis is capable of causing major epidemics; thus, there is a need for vaccine targets and a greater understanding of the role of these targets in pathogenesis. Two prime Y. pestis vaccine candidates are the usher-chaperone fimbriae Psa and Caf. Herein we report that Y. pestis requires, in a nonredundant manner, both PsaA and Caf1 to achieve its full pathogenic ability in both pneumonic and bubonic plague in C57BL/6J mice. Deletion of psaA leads to a decrease in the organ bacterial burden and to a significant increase in the 50% lethal dose (LD50) after subcutaneous infection. Deletion of caf1 also leads to a significant decrease in the organ bacterial burden but more importantly leads to a significantly greater increase in the LD50 than was observed for the ΔpsaA mutant strain after subcutaneous infection of C57BL/6J mice. Furthermore, the degree of attenuation of the Δcaf1 mutant strain is mouse background dependent, as the Δcaf1 mutant strain was attenuated to a lesser degree in BALB/cJ mice by the subcutaneous route than in C57BL/6J mice. This observation that the degree of requirement for Caf1 is dependent on the mouse background indicates that the virulence of Y. pestis is dependent on the genetic makeup of its host and provides further support for the hypothesis that PsaA and Caf1 have different targets.

Invasion of eukaryotic cells by Borrelia burgdorferi requires ß(1) integrins and Src kinase activity.

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most widespread tick-borne infection in the northern hemisphere that results in a multistage disorder with concomitant pathology, including arthritis. During late-stage experimental infection in mice, B. burgdorferi evades the adaptive immune response despite the presence of borrelia-specific bactericidal antibodies. In this study we asked whether B. burgdorferi could invade fibroblasts or endothelial cells as a mechanism to model the avoidance from humorally based clearance. A variation of the gentamicin protection assay, coupled with the detection of borrelial transcripts following gentamicin treatment, indicated that a portion of B. burgdorferi cells were protected in the short term from antibiotic killing due to their ability to invade cultured mammalian cells. Long-term coculture of B. burgdorferi with primary human fibroblasts provided additional support for intracellular protection. Furthermore, decreased invasion of B. burgdorferi in murine fibroblasts that do not synthesize the ß(1) integrin subunit was observed, indicating that ß(1)-containing integrins are required for optimal borrelial invasion. However, ß(1)-dependent invasion did not require either the α(5)ß(1) integrin or the borrelial fibronectin-binding protein BBK32. The internalization of B. burgdorferi was inhibited by cytochalasin D and PP2, suggesting that B. burgdorferi invasion required the reorganization of actin filaments and Src family kinases (SFK), respectively. Taken together, these results suggest that B. burgdorferi can invade and retain viability in nonphagocytic cells in a process that may, in part, help to explain the phenotype observed in untreated experimental infection.

Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection.

Several Borrelia burgdorferi genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the dbpBA locus, whose products bind host decorin and glycosoaminoglycans. Recently, the dbpBA genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a Delta dbpBA::Gent(r) derivative of B. burgdorferi strain B31. The results indicate that the Delta dbpBA::Gent(r) mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of B. burgdorferi, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the dbpBA mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by B. burgdorferi and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.

SIMPLE approach for isolating mutants expressing fimbriae.

Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.

Salmonella enterica serotype Typhimurium MisL is an intestinal colonization factor that binds fibronectin.

MisL is an autotransporter protein encoded by Salmonella pathogenicity island 3 (SPI3). To investigate the role of MisL in Salmonella enterica serotype Typhimurium (S. Typhimurium) pathogenesis, we characterized its function during infection of mice and identified a host receptor for this adhesin. In a mouse model of S. Typhimurium intestinal persistence, a misL mutant was shed with the faeces in significantly lower numbers than the wild type and was impaired in its ability to colonize the cecum. Previous studies have implicated binding of extracellular matrix proteins as a possible mechanism for S. Typhimurium intestinal persistence. A gluthathione-S-transferase (GST) fusion protein to the MisL passenger domain (GST-MisL(29-281)) was constructed to investigate binding to extracellular matrix proteins. In a solid-phase binding assay the purified GST-MisL(29-281) fusion protein bound to fibronectin and collagen IV, but not to collagen I. MisL expression was not detected by Western blot in S. Typhimurium grown under standard laboratory conditions. However, when expression of the cloned misL gene was driven by the Escherichia coli arabinose promoter, MisL could be detected in the S. Typhimurium outer membrane by Western blot and on the bacterial cell surface by flow cytometry. Expression of MisL enabled S. Typhimurium to bind fibronectin to its cell surface, resulting in attachment to fibronectin-coated glass slides and in increased invasiveness for human epithelial cells derived from colonic carcinoma (T84 cells). These data identify MisL as an extracellular matrix adhesin involved in intestinal colonization.

The Salmonella enterica serotype Typhimurium lpf, bcf, stb, stc, std, and sth fimbrial operons are required for intestinal persistence in mice.

Salmonella enterica serotype Typhimurium causes human infections that can frequently be traced back through the food chain to healthy livestock whose intestine is colonized by the pathogen. Little is known about the genes important for intestinal carriage of S. enterica serotype Typhimurium in vertebrate animals. Here we characterized the role of 10 fimbrial operons, agf, fim, lpf, pef, bcf, stb, stc, std, stf, and sth, using competitive infection experiments performed in genetically susceptible (BALB/c) and resistant (CBA) mice. Deletion of agfAB, fimAICDHF, lpfABCDE, pefABCDI, bcfABCDEFG, stbABCD, stcABCD, stdAB, stfACDEFG, or sthABCDE did not reduce the ability of S. enterica serotype Typhimurium to colonize the spleen and cecum of BALB/c mice 5 days after infection. Similarly, deletion of agfAB, fimAICDHF, pefABCDI, and stfACDEFG did not result in reduced recovery of S. enterica serotype Typhimurium from fecal samples collected from infected CBA mice over a 30-day time period. However, S. enterica serotype Typhimurium strains carrying deletions in lpfABCDE, bcfABCDEFG, stbABCD, stcABCD, stdAB, or sthABCDE were recovered at significantly reduced numbers from the feces of CBA mice. There was a good correlation (R(2) = 0.9626) between competitive indices in the cecum and fecal samples of CBA mice at 30 days after infection, suggesting that the recovery of S. enterica serotype Typhimurium from fecal samples closely reflected its ability to colonize the cecum. Collectively, these data show that six fimbrial operons (lpf, bcf, stb, stc, std, and sth) contribute to long-term intestinal carriage of S. enterica serotype Typhimurium in genetically resistant mice.

The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons.

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB, pefBACDI, lpfABCDE, bcfABCDEFG, stbABCD, stcABC, stdAB, stfACDEFG, sthABCDE or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria-Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in the expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA and StiA. These data show that in vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.

Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants.

The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus Salmonella. Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.

Population heterogeneity of Salmonella enterica serotype Typhimurium resulting from phase variation of the lpf operon in vitro and in vivo.

The lpf fimbrial operon oscillates between phase ON and phase OFF expression states, thereby generating heterogeneity within S. enterica serotype Typhimurium populations with regard to expression of long polar fimbrial antigens. To determine whether the proportion of lpf phase variants changes with growth conditions, the lpf phase ON content of cultures was determined after in vitro and in vivo passage. After passage in Luria-Bertani (LB) broth for 120 generations, 96% of cells in a serotype Typhimurium culture carried the lpf operon in the phase ON expression state, regardless of the phase ON/OFF ratio in the inoculum. In contrast, a culture passaged on LB agar plates for 500 generations contained approximately 2% lpf phase ON cells. Differences in the lpf phase ON content of cultures passaged in broth and on plates were not caused by an outgrowth of lpf phase ON or lpf phase OFF cells, since deletion of lpf biosynthesis genes did not alter the phase ON/OFF ratio attained after passage. Instead, growth in LB broth resulted in a eightfold increase in the phase OFF-to-ON transition frequency and a decrease of the lpf phase ON-to-OFF transition frequency by a factor of 150 compared to growth on LB agar plates. After infection of naïve CBA/J mice with an lpf phase ON culture of serotype Typhimurium, the proportion of lpf phase ON cells continuously decreased over time, regardless of whether the strain carried intact fimbrial biosynthesis genes. These data suggest that elaboration of fimbriae does not have a major influence on the population heterogeneity produced by phase variation of the lpf operon in naïve mice.