Salmonella-containing Vacuole Research Articles

Dynamic Behavior of Salmonella‐Induced Membrane Tubules in Epithelial Cells

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct ‘leader’ domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

Maintenance of the Salmonella-containing vacuole in the juxtanuclear area: A role for intermediate filaments

Until recently, intermediate filaments (IF) were thought to be only involved in resistance to physical stress and mechanical integrity of cells and tissues. Recent data indicate that IF play a much more important role in cellular physiology including organelle structure and positioning within the cell. Here, we show that Salmonella enterica serovar Typhimurium ( S. typhimurium) induces in epithelial cells and macrophages the formation of an aggresome-like structure with a dramatic remodelling of cytoplasmic IF (vimentin and cytokeratin) networks and the adaptor proteins 14-3-3 which are recruited around intracellular S. typhimurium microcolonies. These rearrangements are not necessary for bacterial replication. Depletion of vimentin and cytokeratin by siRNA indicates that IF remodelling is required to maintain Salmonella microcolonies in the juxtanuclear area.

Cytolocalization of the PhoP response regulator in Salmonella enterica: modulation by extracellular Mg2+ and by the SCV environment

The PhoP/PhoQ two-component system plays an essential role regulating numerous virulence phenotypes in Salmonella enterica. Previous work showed that PhoQ, the sensor protein, switches between the kinase- and the phosphatase-dominant state in response to environmental Mg2+ availability. This switch defines the PhoP phosphorylation status and, as a result, the transcriptional activity of this regulator. In this work, using the FlAsH labelling technique, we examine PhoP cytolocalization in response to extracellular Mg2+ limitation in vitro and to the Salmonella-containing vacuole (SCV) environment in macrophage cells. We demonstrate that in these PhoP/PhoQ-inducing environments PhoP displays preferential localization to one cell pole, while being homogeneously distributed in the bacterial cytoplasm in repressing conditions. Polar localization is lost in the absence of PhoQ or when a non-phosphorylatable PhoP(D52A) mutant is expressed. However, when PhoP transcriptional activation is achieved in a Mg2+- and PhoQ-independent manner, PhoP regains asymmetric polar localization. In addition, we show that, in the analysed conditions, PhoQ cellular distribution does not parallel PhoP location pattern. These findings reveal that PhoP cellular location is dynamic and conditioned by its environmentally defined transcriptional status, showing a new insight in the PhoP/PhoQ system mechanism.

The Salmonella SPI-2 effector SseJ exhibits eukaryotic activator-dependent phospholipase A and glycerophospholipid : cholesterol acyltransferase activity

Intracellular replication of Salmonella enterica serovar Typhimurium within membrane-bound compartments, called Salmonella-containing vacuoles, depends on the activities of several effector proteins translocated by the Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system. The SPI-2 effector protein SseJ shows similarity at the amino acid level to several GDSL lipases with glycerophospholipid : cholesterol acyltransferase (GCAT) activity. In this study, we show that catalytic serine-dependent phospholipase A (PLA) and GCAT activity of recombinant SseJ is potentiated by factor(s) present in HeLa cells, RAW macrophages and Saccharomyces cerevisiae. SseJ activity was enhanced with increasing amounts of, or preincubation with, eukaryotic cell extracts. Analysis of the activating factor(s) shows that it is soluble and heat- and protease-sensitive. We conclude that PLA and GCAT activities of SseJ are potentiated by proteinaceous eukaryotic factor(s).

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.

Sorting nexin-1 defines an early phase of Salmonella-containing vacuole-remodeling during Salmonella infection

Salmonella enterica serovar Typhimurium replicate within host cells in a specialized membrane-bound compartment, the Salmonella-containing vacuole (SCV). Interaction of SCVs with the host endocytic network is modulated by bacterial effectors, some of which, such as SigD/SopB, manipulate the level of endosomal phosphoinositides. Here, we establish that at early stages of Salmonella infection, sorting nexin-1 (SNX1) - a host phosphoinositide-binding protein that normally associates with early endosomes and regulates transport to the trans-Golgi network (TGN) - undergoes a rapid and transient translocation to bacterial entry sites, an event promoted by SigD/SopB. Recruitment of SNX1 to SCVs results in the formation of extensive, long-range tubules that we have termed ;spacious vacuole-associated tubules'. Formation of these tubules is coupled with size reduction of vacuoles and the removal of TGN-resident cargo. SNX1 suppression perturbs intracellular progress of bacteria, resulting in a delayed replication. We propose that SNX1 is important in tubular-based re-modeling of nascent SCVs and, in doing so, regulates intracellular bacterial progression and replication.

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella-containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro, in cells and during infection. Intracellular infections with wild-type as compared with DeltasseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in DeltasseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.

Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells

Intramacrophage survival appears to be a pathogenic trait common to Salmonellae and definition of the metabolic requirements of Salmonella within macrophages might provide opportunities for novel therapeutic interventions. We show that loss of PurG function in Salmonella enterica serovar Typhimurium SL1344 leads to death of the bacterium in RAW264.7 cells, which was due to unavailability of purine nucleotides but not thiamine in the phagosome of RAW264.7 cells. Phagosomal escape of purG mutant restored growth, suggesting that the phagosomal environment, but not the cytosol, is toxic to Salmonella purine auxotrophs. NADPH oxidase inhibition restored the growth of purG mutant in RAW264.7 cells, implying that the Salmonella-containing vacuole acquires reactive oxygen species (ROS) that are lethal to purine auxotrophs. Under purine limiting conditions, purG mutant was unable to repair the damage caused by hydrogen peroxide or UV irradiation, suggesting that ROS-mediated DNA damage may have been responsible for the attenuated phenotype of purG mutant in RAW264.7 cells and in mice. These studies highlight the possibility of utilizing the Salmonella purine nucleotide biosynthetic pathway as a prospective therapeutic target and also underline the importance of metabolic pathways in assembling a comprehensive understanding of the host-pathogen interactions inside phagocytic cells.

During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems

The biogenesis of the Salmonella-containing vacuole within mammalian cells has been intensively studied over recent years. However, the ability of Salmonella to sense and adapt to the intracellular environment of different types of host cells has received much less attention. To address this issue, we report the transcriptome of Salmonella enterica serovar Typhimurium SL1344 within epithelial cells and show comparisons with Salmonella gene expression inside macrophages. We report that S. Typhimurium expresses a characteristic intracellular transcriptomic signature in response to the environments it encounters within different cell types. The signature involves the upregulation of the mgtBC, pstACS and iro genes for magnesium, phosphate and iron uptake, and Salmonella pathogenicity island 2 (SPI2). Surprisingly, in addition to SPI2, the invasion-associated SPI1 pathogenicity island and the genes involved in flagellar biosynthesis were expressed inside epithelial cells at later stages of the infection, while they were constantly downregulated in macrophage-like cells. To our knowledge, this is the first report of the simultaneous transcription of all three Type Three Secretion Systems (T3SS) within an intracellular Salmonella population. We discovered that S. Typhimurium strain SL1344 was strongly cytotoxic to epithelial cells after 6 h of infection and hypothesize that the time-dependent changes in Salmonella gene expression within epithelial cells reflects the bacterial response to host cells that have been injured by the infection process.

Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.

Protective role of Hemidesmus indicus R. Br. root extract against Salmonella typhimurium‐induced cytotoxicity in Int 407 cell line

The present investigation deals with the effect of the chloroform fraction composed of sterols and fatty acids isolated from Hemidesmus indicus root extract (CHI) on Salmonella enterica serovar Typhimurium (S. typhimurium)-induced cytotoxicity in a human intestinal epithelial cell line (Int 407). The optimum dose was fixed as 100 microg/mL for CHI against S. typhimurium, which was quite safe for Int 407 cells as the CD(50) concentration (50% cell death) of CHI was determined to be 500 microg/mL in the Int 407 cell line. CHI-treated S. typhimurium were 10-fold less cytotoxic and 40% less adherent to host cells than wild-type. Treatment of CHI significantly abrogated the invasion ability to 10- to 15-fold in S. typhimurium. The cells infected with CHI-treated S. typhimurium had a comparable viability to uninfected cells in the epithelial cell detachment assay. Immunofluorescence showed the CHI-treated bacteria were unhealthy and shrunken rods in comparison with the wild-type bacteria; those were firmly attached and invaded to deceased and hypertrophoid Int 407 cells. Transmission electron micrographs of Int 407 cells infected with wild bacteria showed a coat of adherent and invaded bacteria completely occupying the cytoplasm with characteristic Salmonella-containing vacuoles (SCV). Both necrotic and apoptotic type of cell death were observed in cells infected with wild-type bacteria, whereas most of the cells infected with treated bacteria were normal in morphology and a few had invaded bacteria, but the typical proliferated SCV was not observed in cells infected with CHI-treated S. typhimurium. In summary, the sterols and fatty acids present in CHI may be capable of taming S. typhimurium by suppressing its cytotoxic activity in an intestinal epithelial cell line.

SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling

Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates inside mammalian cells within membrane-bound compartments called Salmonella-containing vacuoles. Intracellular replication is dependent on the activities of several effector proteins translocated across the vacuolar membrane by the Salmonella pathogenicity island 2 (SPI-2)-type III secretion system (T3SS). This is accompanied by the formation in the vicinity of bacterial vacuoles of an F-actin meshwork, thought to be involved in maintaining the integrity of vacuolar membranes. In this study, we investigated the function of the SPI-2 T3SS effector SteC. An steC mutant strain was not defective for intracellular replication or attenuated for virulence in mice. However, the steC mutant was defective for SPI-2-dependent F-actin meshwork formation in host cells, although the vacuolar membranes surrounding mutant bacteria appeared to be normal. Expression of SteC in fibroblast cells following transfection caused extensive rearrangements of the F-actin cytoskeleton. Sequence analysis identified amino acid similarity between SteC and the human kinase Raf-1. A His-tagged SteC fusion protein had kinase activity in vitro and a point mutant lacking kinase activity was unable to induce F-actin rearrangements in vivo. We conclude that SPI-2-dependent F-actin meshwork formation depends on the kinase activity of SteC, which resembles more closely eukaryotic than prokaryotic kinases.

The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles

Intracellular replication of Salmonella enterica occurs in membrane-bound compartments, called Salmonella-containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella-pathogenicity island-2 (SPI-2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 μm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule-dependent. An intact Golgi, SseG and SPI-2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live-cell imaging between 4 and 8 h post invasion revealed that the majority of wild-type SCVs displaced less than 2 μm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 μm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi-associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.

Complement Receptor 3 and Toll-Like Receptor 4 Act Sequentially in Uptake and Intracellular Killing of UnopsonizedSalmonella entericaSerovar Typhimurium by Human Neutrophils

The uptake and subsequent killing of Salmonella enterica serovar Typhimurium by human neutrophils was studied. In particular, two pattern recognition receptors, complement receptor 3 (CR3) and Toll-like receptor 4 (TLR4), were found to be essential for the efficient uptake and activation, respectively, of the NADPH oxidase. The uptake of Salmonella was almost completely inhibited by various monoclonal antibodies against CR3, and neutrophils from a patient with leukocyte adhesion deficiency type 1, which lack CR3, showed almost no uptake of Salmonella. A lipopolysaccharide (LPS) mutant strain of Salmonella was used to show that the expression of full-length, wild-type, or so-called smooth LPS is important for the efficient killing of intracellular Salmonella. Infection with wild-type-LPS-expressing Salmonella resulted in the generation of reactive oxygen species (ROS) in TLR4-decorated, Salmonella-containing vacuoles, whereas ROS were not induced by an LPS mutant strain. In addition, the recognition of Salmonella by neutrophils, leading to ROS production, was shown to be intracellular, as determined by priming experiments with intact bacteria under conditions where the bacterium is not taken up. Finally, the generation of ROS in the wild-type-Salmonella-infected neutrophils was largely inhibited by the action of a TLR4-blocking, cell-permeable peptide, showing that signaling by this receptor from the Salmonella-containing vacuole is essential for the activation of the NADPH oxidase. In sum, our data identify the sequential recognition of unopsonized Salmonella strains by CR3 and TLR4 as essential events in the efficient uptake and killing of this intracellular pathogen.

Salmonella SPI1 Effector SipA Persists after Entry and Cooperates with a SPI2 Effector to Regulate Phagosome Maturation and Intracellular Replication

SummarySalmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

Salmonella Trafficking is Defined by Continuous Dynamic Interactions with the Endolysosomal System

Following invasion of non-phagocytic host cells, Salmonella enterica survives and replicates within a phagosome-like compartment known as the Salmonella-containing vacuole (SCV). It is now well established that SCV biogenesis, like phagosome biogenesis, involves sequential interactions with the endocytic pathway. However, Salmonella is believed to limit these interactions and, in particular, to avoid fusion of terminal lysosomes with the SCV. In this study, we reassessed this process using a high-resolution live-cell imaging approach and found an unanticipated level of interaction between the SCV and the endocytic pathway. Direct interactions, in which late endosomal/lysosomal content was transferred to SCVs, were detected within 30 min of invasion and continued for several hours. Mechanistically, these interactions were very similar to phagosome–lysosome fusion because they were accompanied by rapid acidification of the SCV, could be blocked by chemical perturbation of microtubules or vacuolar acidification and involved the small GTPase Rab7. In comparison with vacuoles containing internalized Escherichia coli or heat-killed Salmonella, SCVs did show some delay of fusion and acidification, although, this appeared to be independent of either type III secretion system. These results provide compelling evidence that inhibition of SCV–lysosome fusion is not the major determinant in establishment of the Salmonella replicative niche in epithelial cells.

Murine osteoblasts respond to LPS and IFN-γ similarly to macrophages

Osteoblasts are bone-forming mesenchymal cells, while macrophages are cells of hematopoietic origin responsible for innate immunity. Lipopolysaccharide (LPS) can induce tolerance in macrophages, whereas interferon (IFN)-gamma can activate macrophages to produce cytokines, exert bactericidal effects, and present antigens. In this study, we examined such macrophagic phenotypes regulated by LPS and IFN-gamma in murine osteoblasts. In both primary calvarial osteoblasts and osteoblastic MC3T3-E1 cells, LPS pretreatment resulted in reduced production of IL-6 in response to a subsequent LPS stimulation or to Salmonella infection, indicating the existence of LPS-induced tolerance in osteoblasts. Furthermore, IFN-gamma treatment of MC3T3-E1 cells resulted in both enhanced IL-6 production in response to LPS and upregulation of major histocompatibility complex class II (MHC II). Following infection, Salmonella-containing vacuoles (SCVs) were formed in MC3T3-E1 cells, and IFN-gamma pretreatment enhanced bactericidal effects on intracellular Salmonella. Taken together, these observations indicate that osteoblasts can exhibit a subset of phenotypes reminiscent of macrophages in the course of bacterial infection.

Regulation of Salmonella pathogenicity island 2 genes by independent environmental signals

The function of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) is essential for the intracellular lifestyle of Salmonella enterica serovar Typhimurium. Expression of SPI2 genes is induced within the Salmonella-containing vacuole (SCV) inside host cells and is controlled by the SsrAB two-component system. However, the nature of the signals leading to expression of SPI2 genes is controversial. Here we report that expression of SPI2 genes can be induced independently by two different environmental stimuli. Exposure of bacteria to slightly acidic pH was sufficient to induce a rapid up-regulation of SPI2 genes. In contrast, limitation of inorganic phosphate (P i) in the growth media led to activation of SPI2 genes in the late exponential growth phase and was independent of the media pH. Limitation of P i induced an over-expression of sensor protein SsrB. Response to both environmental stimuli required a functional SsrAB system. In vivo analyses indicated that limitation of P i is also encountered by intracellular Salmonella. Our observations allow the integration of previous disparate reports on the induction of SPI2 genes.

Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.