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Despite the maintenance of YopP/J alleles throughout the human-pathogenic Yersinia lineage, the benefit of YopP/J-induced phagocyte death for Yersinia pathogenesis in animals is not obvious. To determine how the sequence divergence of YopP/J has impacted Yersinia virulence, we examined protein polymorphisms in this type III secreted effector protein across 17 Yersinia species and tested the consequences of polymorphism in a murine model of subacute systemic yersiniosis. Our evolutionary analysis revealed that codon 177 has been subjected to positive selection; the Yersinia enterocolitica residue had been altered from a leucine to a phenylalanine in nearly all Yersinia pseudotuberculosis and Yersinia pestis strains examined. Despite this change being minor, as both leucine and phenylalanine have hydrophobic side chains, reversion of YopJF177 to the ancestral YopJL177 variant yielded a Y. pseudotuberculosis strain with enhanced cytotoxicity toward macrophages, consistent with previous findings. Surprisingly, expression of YopJF177L in the mildly attenuated ksgA− background rendered the strain completely avirulent in mice. Consistent with this hypothesis that YopJ activity relates indirectly to Yersinia pathogenesis in vivo, ksgA− strains lacking functional YopJ failed to kill macrophages but actually regained virulence in animals. Also, treatment with the antiapoptosis drug suramin prevented YopJ-mediated macrophage cytotoxicity and enhanced Y. pseudotuberculosis virulence in vivo. Our results demonstrate that Yersinia-induced cell death is detrimental for bacterial pathogenesis in this animal model of illness and indicate that positive selection has driven YopJ/P and Yersinia evolution toward diminished cytotoxicity and increased virulence, respectively.

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues., To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein YtfE contributes to the survival of Yersinia pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than that of the wild type (WT), consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and ΔytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across ΔytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro . Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii , demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the Δ lbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted.

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system.

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10 3 -fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

Legionella pneumophila replicates within alveolar macrophages, causing a severe pneumonia termed Legionnaires' disease. The bacterium resides within a vacuole that escapes immediate transport to the host lysosome. Instead, the vacuole interacts with the early secretory pathway to establish an environment suitable for rapid multiplication. A type IV secretion system is central to the pathogenicity of the bacterium, and many protein substrates that are translocated by this system to the host cell have been identified. One of these, VipD, was found to interrupt the late secretory pathway when overproduced in Saccharomyces cerevisiae . We independently identified VipD in a previous study and have further characterized this protein as well as its three paralogs. The vipD gene belongs to a family of L. pneumophila open reading frames that are predicted to contain a phospholipase A domain with sequence similarity to the type III-secreted toxin ExoU from Pseudomonas aeruginosa . Similarly to other known translocated proteins of L. pneumophila , VipD is strongly induced in early stationary phase, a time when the bacterium is most virulent. Detergent extraction studies of infected macrophages confirm that VipD is translocated into host cells via the type IV secretion system. A second assay for translocation revealed that two paralogs of VipD, VpdA and VpdB, also have translocation signals recognized by the type IV system. A strain lacking VipD and its three paralogs grew at wild-type rates in murine macrophages, although secondary mutations that cause growth defects in strains lacking VipD accumulate. The quadruple mutant displayed a growth advantage in the amoebal host Dictyostelium discoideum , indicating that the protein family may modulate intracellular growth in a complex fashion. VipD is mildly toxic when overproduced in eukaryotic cells, and the toxicity is partially dependent on the putative phospholipase active site. VipD and its paralogs therefore define a family of translocated proteins that may assist in the establishment of a vacuole suitable for bacterial replication through functioning as a phospholipase.

Legionella pneumophila uses a type IV secretion system to deliver effector molecules into the host cell and establish its replication vacuole. In this study, we investigated the role of LidA, a translocated substrate associated with the surface of the L. pneumophila -containing vacuole. LidA is secreted into the host cell throughout the replication cycle of the bacteria and associates with compartments of the early secretory pathway. When overexpressed in mammalian cells or yeast, LidA interferes with the early secretory pathway, probably via a domain predicted to be rich in coiled-coil structure. Finally, during intracellular replication, the replication vacuoles are in close contact with the endoplasmic reticulum-Golgi intermediate compartment and the Golgi apparatus, suggesting a positive correlation between intracellular growth and association of the vacuole with compartments of the early secretory pathway. We propose that LidA is involved in the recruitment of early secretory vesicles to the L. pneumophila -containing vacuole and that the vacuole associates with the secretory pathway to facilitate this process.

Although Legionella pneumophila can multiply in diverse cell types from a variety of species, macrophages from most inbred mouse strains are nonpermissive for intracellular replication and allow little or no growth of the bacteria. This phenomenon is likely genetically controlled by the mouse naip5 ( birc1e ) gene located within the Lgn1 locus. In this study, we have investigated the resistance of C57BL/6J macrophages to L. pneumophila infection by examining the fate of both the bacterium and the infected cells compared to that in macrophages from the permissive A/J strain. Our results indicate that although the trafficking of the L. pneumophila -containing vacuole is partially disrupted in C57BL/6J macrophages, this cannot account for the severity of the defect in intracellular growth observed in this strain. Infected macrophages are lost shortly after infection, and at later times a larger fraction of the C57BL/6J macrophages in which L. pneumophila undergoes replication are apoptotic compared to those derived from A/J mice. Finally, a loss of bacterial counts occurs after the first round of growth. Therefore, the resistance mechanism of C57BL/6J macrophages to L. pneumophila infection appears to be multifactorial, and we discuss how early and late responses result in clearing the infection.

Legionella pneumophila vacuole biogenesis was analyzed by using a cell-free system. We show that calnexin, Sec22b, and Rab1 are recruited to the vacuole very shortly after bacterial uptake, and we have identified Rab1 as a potential host factor involved in the endoplasmic reticulum recruitment process.

A promoter-probe strategy was devised to identify genes specifically expressed byLegionella pneumophiladuring growth within the macrophage. Random fragments from theL. pneumophilachromosome were inserted upstream of a promoterless phage T4tdgene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes theL. pneumophilaortholog ofahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis ofahpCgene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate ofL. pneumophilain cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.

The binding of the Yersinia pseudotuberculosis and Yersinia enterocolitica invasin proteins to β 1 integrin receptors allows internalization of these organisms by cultured cells. The C-terminal 192-residue superdomain of the Y. pseudotuberculosis invasin is necessary and sufficient for integrin recognition, while a region located outside, and N-terminal to, this superdomain strongly enhances the efficiency of bacterial uptake. Within the enhancer region is a domain called D2 that allows invasin-invasin interaction. To investigate the role of the enhancer region, bacterial cell binding and entry mediated by the Y. pseudotuberculosis invasin protein (invasin pstb ) was compared to that of Y. enterocolitica invasin (invasin ent ), which lacks the D2 self-association domain. Invasin ent was shown to be unable to promote self-interaction, using the DNA binding domain of λ repressor as a reporter. Furthermore, two genetically engineered in-frame deletion mutations that removed D2 from invasin pstb were significantly less proficient than wild-type invasin pstb at promoting uptake, although the amount of surface-exposed invasin as well as the cell binding capacity of the recombinant Escherichia coli strains remained similar. Competitive uptake assays showed that E. coli cells expressing invasin pstb had a significant advantage in the internalization process versus either E. coli cells expressing invasin ent or the invasin pstb derivatives deleted for D2, further demonstrating the importance of invasin self-interaction for the efficiency of invasin-mediated uptake.

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 and 73 amino acids and a carboxyl-terminal cytoplasmic domain of 122 amino acids. Protein fractionation studies were consistent with DotA residing in the inner membrane. An alkaline phosphatase fusion located 9 amino acids upstream from the C terminus of DotA still retained function and was able to restore intracellular growth when harbored by two L. pneumophila dotA mutants. A hybrid protein from which the carboxyl-terminal 48 amino acids of DotA were deleted was unable to complement the intracellular growth defect in the dotA mutants, indicating that this cytoplasmic region is required for function.

Yersinia pseudotuberculosis inv mutant strains cured of the virulence plasmid exhibit thermoinducible adhesion to cultured mammalian cells. To identify the genes responsible for this phenotype, Y. pseudotuberculosis homologs of the Y. enterocolitica ail and the Y. pestis psa loci were identified. Mutations in the Y. pseudotuberculosis ail and psa loci were constructed and tested for thermoinducible binding. Results of cellular binding assays indicated that only mutations in psa, not in ail, resulted in defects for thermoinducible binding, with inv yadA psa strains showing no detectable cell adhesion. In addition, an inv psa strain was defective for hemagglutination of sheep erythrocytes, in contrast to an inv psa+ strain which was fully competent for hemagglutination. The introduction of a plasmid containing a 6.7-kb KpnI-ClaI fragment of Y. pseudotuberculosis encompassing the psa locus was sufficient to complement both the cell adhesion and hemagglutination defects of the psa mutant. Results from subcloning and transposon mutagenesis indicated that the complete 6.7-kb region was required for thermoinducible binding and hemagglutination.

The Yersinia pseudotuberculosis surface protein invasin binds to multiple β1 integrins with high affinity, leading to misregulation of Rac1 activity. Upon host cell binding, alteration of Rho GTPase activity results from the action of several Yersinia outer proteins (Yops) that are translocated into the cytoplasm. We report here that three virulence determinants encoded by Y. pseudotuberculosis manipulate the Rho GTPase RhoG. Y. pseudotuberculosis binding to cells caused robust recruitment of RhoG to the site of attachment, which required high-affinity invasin-β1 integrin association. Furthermore, inactivation of RhoG significantly reduced the efficiency of invasin-mediated bacterial internalization. To investigate the activation state of RhoG, a fluorescence resonance energy transfer-based activation biosensor was developed and used to show distinct spatial activation of RhoG at the site of bacterial attachment. The biosensor was also used to show efficient RhoG inactivation by Y. pseudotuberculosis YopE, a potent Rho GTPase activating protein. Additionally, RhoG mislocalization by the prenylcysteine endoprotease YopT was demonstrated by two independent assays. Functional bacterial uptake experiments demonstrated that RhoG activation can bypass a deficit in Rac1 activity. Interestingly, increasing the size of the particle gave results more consistent with a linear pathway, in which RhoG acts as an upstream activator of Rac1, indicating that increased surface area introduces constraints on the signaling pathways required for efficient internalization. Taken together, these data demonstrate the misregulation of RhoG by multiple Y. pseudotuberculosis virulence determinants. Since RhoG is imperative for proper neutrophil function, this misregulation may represent a unique mechanism by which Yersinia species dampen the immune response.

The Yersinia pseudotuberculosis invasin protein is a 986-amino-acid outer membrane protein that promotes bacterial penetration into mammalian cells by binding to beta 1-chain integrin receptors. We previously showed that the integrin binding domain is encoded by the carboxyl-terminal 192 amino acids. To further investigate the structure of this protein, we characterized a set of 32 monoclonal antibodies (MAbs) directed against invasin. Invasin deletion derivatives and fusion proteins carrying different segments of invasin were used to map the epitopes of this set of MAbs into 10 overlapping but distinct intervals. Indirect immunofluorescence of intact bacteria expressing invasin demonstrated that two large regions of invasin contain epitopes exposed on the bacterial surface. To assess the role of these surface-exposed regions in the binding and invasion of mammalian cells, each of the MAbs was tested for its ability to inhibit these processes. All of the MAbs that recognized bacterial surface-exposed epitopes in the cell binding domain of invasin inhibited both cell attachment and cell penetration, and no other MAbs inhibited either activity.

The contribution of Toll-like receptors (TLRs) to phagocytosis ofBorrelia burgdorferihas not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88−/−mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalizeB. burgdorferi.Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis ofB. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88−/−BMDM but only partially affected in TLR2−/−BMDM or knockdown of TLR5 and unaffected in TLR9−/−BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88−/−BMDM. Addition of cytochalasin D to TLR2−/−BMDM inhibited inflammatory responses toB. burgdorferito the level of MyD88−/−BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake ofB. burgdorferiand that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.

The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU , are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans , one mutant by a gene termed dotH and two additional mutants by a gene termed dotO . A deletion mutation was created in a third gene, dotI , which is located directly upstream of dotH . The Δ dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans . Based on sequence analysis and structural predictions, possible roles of dotH , dotI , and dotO in intracellular growth are discussed.

After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.

Yersinia pseudotuberculosis is able to enter normally nonphagocytic host cells by multiple pathways, the most efficient of which is mediated by invasin, a 986-amino-acid bacterial outer membrane protein. It has previously been shown that the C-terminal 192 amino acids of invasin are sufficient to bind mammalian cells. To determine if additional regions of the invasin protein are necessary to promote entry, we developed a novel assay that tests the ability of various invasin derivatives to confer on Staphylococcus aureus the ability to enter animal cells. We determined that the 192-amino-acid cell-binding region of invasin, when used to coat the bacterial cell surface, was also sufficient to promote cellular penetration. These results suggest that the simple binding of invasin to its receptors is sufficient to mediate entry and that the bacterium plays a largely passive role in the entry process.

Salmonella typhimurium, enteroinvasive Escherichia coli, Yersinia pseudotuberculosis, and Yersinia enterocolitica possess the ability to enter intestinal epithelial cells. We used a quantitative tissue culture model employing HEp-2 cells to compare the abilities of these bacteria to enter epithelial cells. S. typhimurium and Yersinia species were highly infective for HEp-2 cells but were unable to replicate extensively intracellularly. Enteroinvasive E. coli exhibited low infectivity but replicated extensively intracellularly. The growth of enteroinvasive E. coli led to destruction of the HEp-2 monolayer, whereas Yersinia spp. and S. typhimurium were maintained intracellularly for prolonged periods without damage to the monolayer. The ability of enteroinvasive E. coli to enter HEp-2 cells required prior growth at 37 degrees C; neither S. typhimurium nor Yersinia spp. exhibited this temperature dependence for cell entry. An E. coli K-12 derivative containing a 230-kilobase plasmid from enteroinvasive E. coli was constructed. This derivative shared all the invasive characteristics of the parental enteroinvasive strain, suggesting that determinants required for cell entry and intracellular multiplication were at least partially plasmid encoded. An HB101 derivative containing a cloned invasion determinant from Y. pseudotuberculosis was constructed in our laboratory. HEp-2 monolayers were coinfected with these two K-12 derivatives to compare invasion determinants from enteroinvasive E. coli with those of Y. pseudotuberculosis in a common genetic background. Results from these experiments suggest that these organisms reside within separate intracellular compartments.

A series of translational fusions between the Yersinia pseudotuberculosis inv locus and lacZ was constructed. Each Lac+ fusion strain expressed a hybrid protein containing invasin, the product of the inv locus, at its amino-terminal end. Analysis of these gene fusions allowed determination of the direction of translation of the inv gene. Previous studies of Y. pseudotuberculosis invasion have shown that entry into animal cells is temperature regulated. It is shown here that control of expression of the inv gene is also temperature regulated. phoA gene fusions to inv, when present in Y. pseudotuberculosis, were expressed at lower levels when bacteria were grown at 37 degrees C rather than at 28 degrees C. Similar fusions, in contrast, were regulated in a temperature-independent fashion in Escherichia coli, as was the wild-type inv gene. This implies that Y. pseudotuberculosis has chromosomally encoded trans-acting functions that normally thermoregulate expression of inv.