Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches

Salmonella typhi GIFU10007-3 which lost a viaB locus on its chromosome became highly invasive in our previous study. To investigate the phenomenon, we controlled Vi expression in wild strain S. typhi GIFU10007, and studied the invasive phenotype both in vitro and in vivo. When the wild strain of S. typhi was cultured in 300 mM NaCl containing Luria-Bertani broth (LBH), the expression of Vi antigen was suppressed, but secretion of invasion proteins (SipC, SipB and SipA) was increased. In this condition, wild strain S. typhi became highly invasive toward both epithelial cells and M cells of rat Peyer's patches. When GIFU10007 was cultured under conditions of high osmolarity, the bacteria disrupted Peyer's patches and induced massive bleeding in these structures only 20 min after inoculation into the ileal loop. In contrast, Vi-encapsulated wild strain GIFU10007 cultured under low osmolarity was not destructive, even after 60 min. To understand the role of the type III secretion system under conditions of high osmolarity, we knocked out the invA and sipC genes of both GIFU10007 and GIFU10007-3. Neither invA nor sipC mutants could invade epithelial cells or M cells in a high osmolarity environment. Our data show that the highly invasive phenotype was only expressed when the wild strain S. typhi was cultured under high osmolarity, which induced a state of Vi suppression, and in the presence of the type III secretion system.     

The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail.

A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five-member family with a distinct virulence-associated phenotype.     

Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins.

Entry into intestinal epithelial cells is an essential step in the pathogenesis of Salmonella infections. Our laboratory has previously identified a genetic locus, inv, that is necessary for efficient entry of Salmonella typhimurium into cultured epithelial cells. We have carried out a molecular and functional analysis of invB and invC, two members of this locus. The nucleotide sequence of these genes indicated that invB and invC encode polypeptides with molecular masses of 15 and 47 kDa, respectively. Polypeptides with the predicted sizes were observed when these genes were expressed under the control of a T7 promoter. Strains carrying nonpolar mutations in these genes were constructed, and their phenotypes were examined in a variety of assays. A mutation in invC rendered S. typhimurium defective in their ability to enter cultured epithelial cells, while mutations in invB did not. Comparison of the predicted sequences of InvB and InvC with translated sequences in GenBank revealed that these polypeptides are similar to the Shigella spp. proteins Spa15 and Spa47, which are involved in the surface presentation of the invasion protein antigens (Ipa) of these organisms. In addition, InvC showed significant similarity to a protein family which shares sequence homology with the catalytic beta subunit of the F0F1 ATPase from a number of microorganisms. Consistent with this finding, purified preparations of InvC showed significant ATPase activity. Site-directed mutagenesis of a residue essential for the catalytical function of this family of proteins resulted in a protein devoid of ATPase activity and unable to complement an invC mutant of S. typhimurium. These results suggest that InvC may energize the protein export apparatus encoded in the inv locus which is required for the surface presentation of determinants needed for the entry of Salmonella species into mammalian cells. The role of InvB in this process remains uncertain.     

Identification of Salmonella typhimurium invasiveness loci.

Salmonella typhimurium is capable of entering into (invading) nonphagocytic host cells. To systematically identify the bacterial genes necessary for this process, 15,000 Tn10dCm random transposon mutants of S. typhimurium were individually screened for invasiveness, using the human colonic epithelial Caco-2 cell line. Four hundred and eighty-eight mutants had decreased levels of invasiveness; most were nonmotile. However, five mutants, representing four loci, were completely motile. Further characterization of these five mutants showed that they were also unable to enter the dog kidney epithelial cell line MDCK and the mouse macrophage line J774.A1. In contrast to the parental strain, they were unable to disrupt the transepithelial resistance of polarized epithelial monolayers, nor were they able to penetrate across these epithelial barriers. Three of the four classes of mutants remained virulent in mice. The results confirm several aspects of S. typhimurium invasiveness: (i) intact motility enhances invasiveness of cultured cells; (ii) S. typhimurium invasiveness is multifactorial, and at least six distinct genetic loci are involved; and (iii) invasion loci involved in uptake into epithelial cells are also needed for uptake into cultured phagocytic cells. The results also emphasize that decreased levels of invasiveness eliminate bacterial penetration of polarized epithelial barriers and invasiveness loci mutants are not necessarily avirulent.     

The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells

Type III secretion systems (TTSS) are used by Gram-negative pathogens to translocate proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) has two of these specialized systems, which are encoded on separate Salmonella pathogenicity islands (SPI-1 and SPI-2) and translocate unique sets of effectors. The specific roles of these systems in Salmonella pathogenesis remain undefined, although SPI-1 is required for bacterial invasion of epithelial cells and SPI-2 for survival/replication in phagocytic cells. However, because SPI-1 TTSS mutants are invasion-incompetent, the role of this TTSS in post-invasion processes has not been investigated. In this study, we have used two distinct methods to internalize a non-invasive SPI-1 TTSS mutant (invA) into cultured epithelial cells: (i) co-internalization with wild-type S. Typhimurium (SPI-1-dependent) and (ii) complementation with the Yersinia pseudotuberculosis invasin (inv) gene (SPI-1-independent). In both cases, internalized invA mutants were unable to replicate intracellularly, indicating that SPI-1 effectors are essential for this process and cannot be complemented by wild-type bacteria in the same cell. Analysis of the biogenesis of SCVs showed that vacuoles containing mutant bacteria displayed abnormal maturation that was dependent on the mechanism of entry. Manipulation of Salmonella-containing vacuole (SCV) biogenesis by pharmacologically perturbing membrane trafficking in the host cell increased intracellular replication of wild-type but not mutant S. Typhimurium This demonstrates a previously unknown role for SPI-1 in vacuole biogenesis and intracellular survival in non-phagocytic cells.     

Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.

Entry into host cells is an essential feature in the pathogenicity of Salmonella spp. The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells. We report here the identification of several proteins whose secretion into the culture supernatant of S. typhimurium is dependent on the function of the inv-encoded translocation apparatus. Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp. invasins IpaB and IpaC, respectively. An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC. Nonpolar mutations in sicA, sipB, and sipC rendered S. typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.     

The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

IpaC from Shigella and SipC from Salmonella possess similar biochemical properties but are functionally distinct

Invasion plasmid antigen C (IpaC) is secreted via the type III secretion system (TTSS) of Shigella flexneri and serves as an essential effector molecule for epithelial cell invasion. The only homologue of IpaC identified thus far is Salmonella invasion protein C (SipC/SspC), which is essential for enterocyte invasion by Salmonella typhimurium. To explore the biochemical and functional relatedness of IpaC and SipC, recombinant derivatives of both proteins were purified so that their in vitro biochemical properties could be compared. Both proteins were found to: (i) enhance the entry of wild-type S. flexneri and S. typhimurium into cultured cells; (ii) interact with phospholipid membranes; and (iii) oligomerize in solution; however, IpaC appeared to be more efficient in carrying out several of the biochemical properties examined. Overall, the data indicate that purified IpaC and SipC are biochemically similar, although not identical with respect to their in vitro activities. To extend these observations, complementation analyses were conducted using S. flexneri SF621 and S. typhimurium SB220, neither of which is capable of invading epithelial cells because of non-polar null mutations in ipaC and sipC respectively. Interestingly, both ipaC and sipC restored invasiveness to SB220 whereas only ipaC restored invasiveness to SF621, suggesting that SipC lacks an activity possessed by IpaC. This functional difference is not at the level of secretion because IpaC and SipC are both secreted by SF621 and it does not appear to be because of SipC dependency on this native chaperone as coexpression of sipC and sicA in SF621 still failed to restore detectable invasiveness. Taken together, the data suggest that IpaC and SipC differ in either their ability to be translocated into host cells or in their function as effectors of host cell invasion. Because IpaB shares significant sequence homology with the YopB translocator of Yersinia species, the ability for IpaC and SipC to associate with this protein was explored as a potential indicator of translocation function. Both proteins were found to bind to purified IpaB with an apparent dissociation constant in the nanomolar range, suggesting that they may differ with respect to effector function. Interestingly, whereas SB220 expressing sipC behaved like wild-type Salmonella, in that it remained within its membrane-bound vacuole following entry into host cells, SB220 expressing ipaC was found in the cytoplasm of host cells. This observation indicates that IpaC and SipC are responsible for a major difference in the invasion strategies of Shigella and Salmonella, that is, they escape into the host cell cytoplasm. The implications of the role of each protein's biochemistry relative to its in vivo function is discussed.     

Identification of two targets of the type III protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella IpaD and IpaA proteins.

An important virulence factor of Salmonella spp. is their ability to gain access to host cells. A type III secretion system encoded in the inv and spa loci of these organisms is essential for this phenotype. We have identified two proteins, SipA and SipD, whose secretion from the bacterial cells is dependent on this system. The genes encoding these proteins are located at centisome 63 on the S. typhimurium chromosome, immediately downstream of the previously identified sipB and sipC genes (K. Kaniga, S. Tucker, D. Trollinger, and J. E. Galán, J. Bacteriol. 177:3965-3971, 1995). Nucleotide sequence analysis of the genes encoding these proteins indicated that SipA and SipD have significant sequence similarity to the Shigella IpaA and IpaD proteins. A nonpolar null mutation in sipD rendered S. typhimurium severely deficient for entry into cultured epithelial cells. In addition, this mutant strain exhibited increased secretion of a selected group of proteins whose export is controlled by the inv- and spa-encoded translocon. In contrast, a nonpolar mutation in sipA did not result in an invasion defect or in a significant decreased in virulence in a mouse model of infection. In addition, we have found an open reading frame immediately downstream of SipA that encodes a predicted protein with significant similarity to a family of acyl carrier proteins.     

Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae.

Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE-containing S. typhimurium expressed a YopE-mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.     

A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide)

Invasion of host cells is essential for the pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis. rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness.     

DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic') plasmid.     

Isolation and characterization of lon mutants in Salmonella typhimurium.

In this paper we report the isolation and characterization of lon mutants in Salmonella typhimurium. The mutants were isolated by using positive selection by chlorpromazine resistance. The physiological and biochemical properties of the lon mutants in S. typhimurium are very similar to those of Escherichia coli lon mutants. Mutants altered at this locus contain little or no activity of the ATP-dependent protease La and show a number of pleiotropic phenotypes, including increased production of capsular polysaccharides, increased sensitivity to UV light and other DNA-damaging agents, and a decreased ability to degrade abnormal proteins.     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

his-Linked hydrogen sulfide locus of Salmonella typhimurium and its expression in Escherichia coli.

A his-linked H2S locus of Salmonella typhimurium has been further defined by direct isolation of H2S mutants. Expression of this locus in Escherichia coli has been demonstrated.