The role of host cell death in Salmonella infections.

Salmonella enterica is an important enteric pathogen of humans and a variety of domestic and wild animals. Infection is initiated in the intestinal tract, and severe disease produces widespread destruction of the intestinal mucosa. Salmonella strains can also disseminate from the intestine and produce serious, sometimes fatal infections with considerable cytopathology in a number of systemic organs. A combination of bacterial genetic and cell biology studies have shown that Salmonella uses specific virulence mechanisms to induce host cell death during infection. Salmonella produces one set of virulence proteins to promote invasion of the intestine and a different set to mediate systemic disease. Significantly, each set of virulence factors mediates a distinct mechanism of host cell death. The Salmonella pathogenicity island-1 (SPI-1) locus encodes a type III protein secretion system (TTSS) that delivers effector proteins required for intestinal invasion and the production of enteritis. The SPI-1 effector SipB activates caspase-1 in macrophages, releasing IL-1beta and IL-18 and inducing rapid cell death by a mechanism that has features of both apoptosis and necrosis. Caspase-1 is required for Salmonella to infect Peyer's patches and disseminate to systemic tissues in mice. Progressive Salmonella infection in mice requires the SPI-2 TTSS and associated effector proteins as well as the SpvB cytotoxin. Apoptosis of macrophages in the liver is found during systemic infection. In cell culture, Salmonella strains induce delayed apoptosis dependent on SPI-2 function in macrophages from a variety of sources. This delayed apoptosis also requires activation of TLR4 on macrophages by the bacterial LPS. Downstream activation of kinase pathways leads to balanced pro- and antiapoptotic regulatory factors in the cell. NF-kappaB and p38 mitogen-activated protein kinase (MAPK) are particularly important for the induction of antiapoptotic factors, whereas the kinase PKR is required for bacterial-induced apoptosis. The Salmonella SPI-2 TTSS is essential for altering the balance in favor of apoptosis during intracellular infection, but the effectors involved remain poorly characterized. The SpvB cytotoxin has been shown to play a role in apoptosis in human macrophages by depolymerizing the actin cytoskeleton. A model for the role of bacteria-induced host cell death in Salmonella pathogenesis is proposed. In the intestine, the Salmonella SPI-1 TTSS and SipB mediate macrophage death by caspase-1 activation, which also releases IL-1beta and IL-18, promoting inflammation and subsequent phagocytosis by incoming macrophages and leading to dissemination to systemic tissues. Intracellular secretion of virulence effector proteins by the SPI-2 TTSS facilitates growth of Salmonella in these macrophages and the delayed onset of apoptosis in extraintestinal tissues. These infected, apoptotic cells are targeted for engulfment by incoming macrophages, thus perpetuating the cycle of cell-to-cell spread that is the hallmark of systemic Salmonella infection.

Diverse virulence traits underlying different clinical outcomes of Salmonella infection.

Salmonella strains have evolved to infect a wide variety of reptiles, birds, and mammals resulting in many different syndromes ranging from colonization and chronic carriage to acute fatal disease. Adaptation to a large number of different evolutionary niches has undoubtedly driven the high degree of phenotypic and genotypic diversity in Salmonella strains. Differences in LPS and flagellar structure generate the antigenic variation that is reflected in the more than 2,000 known serotypes. Moreover, variations of LPS structure affect the virulence of the strain. The differential expression of various fimbriae by Salmonella is likely to be due to the wide variety of mucosal surfaces that are encountered by various strains, and the host immune response may select for a different expression pattern. As with these surface structures, a variety of other important virulence determinants show a variable distribution in Salmonella strains and also serve to delineate the divergence of the Salmonella lineage from E. coli. The acquisition of the SPI-1 region may have represented the defining genetic event in the separation of the Salmonella and E. coli lineages. The SPI-1 cell invasion function allowed Salmonella to establish a separate niche in epithelial cells. The mgtC locus on SPI-3 is also present in all lineages and facilitates the adaptation of the bacteria to the low Mg2+, low pH environment of the endosome that results from SPI-1-mediated invasion. Subsequent acquisition of SPI-2 allowed Salmonella to manipulate the sorting of the endosome or phagosome, altering the intracellular environment and facilitating bacterial growth within infected cells. The ability to disseminate from the bowel and establish extraintestinal niches is promoted by the spv locus. Since Salmonella proliferates within macrophages and must avoid phagocytosis by neutrophils to establish a systemic infection, the spv genes appear to promote the macrophage phase of the disease process. Here the polymorphism of the spv locus is clearly demonstrated, since the serovars that cause most cases of nontyphoid bacteremia contain the spv genes. The absence of the spv genes from S. typhi is particularly puzzling and is a strong indication that the pathogenesis of typhoid fever is fundamentally different from that of bacteremia due to nontyphoid Salmonella. There is currently no genetic explanation for the phenotype of host adaptation or for the finding that only a few serovars cause the majority of human infections. Based on recent findings that multiple individual virulence genes have a variable distribution in Salmonella, it is unlikely that a single locus will be found to be responsible for these complex biological traits. Instead, a complicated combination of genes are likely to contribute to the overall virulence phenotype.

The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells.

ADP-ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP-ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site-directed mutagenesis demonstrates that the ADP-ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP-ribosylating toxin critical for the pathogenesis of Salmonella infections.

The best defense is a good offense--Salmonella deploys an ADP-ribosylating toxin.

The dramatic clinical manifestations of toxigenic infections such as cholera and diphtheria occur without substantial bacterial invasion. Disease is mediated by the secretion of potent toxins that use ADP-ribosylation as the catalytic mechanism underlying their action. ADP-ribosylating toxins comprise a large family, including the cholera, diphtheria, pertussis and Escherichia coli heat-labile (LT) toxins, and all produce disease by altering key metabolic processes after transfer of an ADP-ribose moiety from NAD to specific host-cell target proteins. A new paradigm implicating ADP-ribosylation during intracellular pathogenesis is beginning to emerge from recent research in Salmonella.

The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages.

The pathogenesis of serious systemic Salmonella infections is characterized by survival and proliferation of bacteria inside macrophages. Infection of human monocyte-derived macrophages in vitro with S. typhimurium or S. dublin produces cytopathology characterized by detachment of cells that contain large numbers of proliferating bacteria. This cytopathology is dependent on the expression of the bacterial spv genes, a virulence locus previously shown to markedly enhance the ability of Salmonella to produce systemic disease. After 24 h of infection, macrophage cultures contain two populations of bacteria: (i) proliferating organisms present in a detached cell fraction; and (ii) a static bacterial population in macrophages remaining attached to the culture well. Mutations in either the essential transcriptional activator SpvR or the key SpvB protein markedly reduce the cytopathic effect of Salmonella infection. The spv-dependent cytopathology in macrophages exhibits characteristics of apoptosis, with release of nucleosomes into the cytoplasm, nuclear condensation and DNA fragmentation. The current findings suggest that the mechanism of the spv effect is through induction of increased cytopathology in host macrophages.

Effect of nitric oxide on Helicobacter pylori morphology.

Helicobacter pylori causes chronic gastritis punctuated with fluctuating episodes of acute distress that can lead to peptic ulcer disease. Several factors produced by the bacterium have been shown to initiate the inflammatory response, but mechanisms potentially involved in the down-regulation of inflammation have not been described. We show that nitric oxide (NO) released from synthetic NO generators causes a rapid and dose-dependent morphologic conversion of H. pylori from the replicating spiral form to the nonreplicating, but viable, coccoid form. Because only spiral organisms-and not coccoid forms-are capable of inducing interleukin-8 secretion by epithelial cells, this conversion could result in down-regulation of the inflammatory response. These data suggest that the increase in NO synthase activity observed during gastritis results in morphologic conversion to a potentially dormant but viable H. pylori.

Mutational characterization of promoter regions recognized by the Salmonella dublin virulence plasmid regulatory protein SpvR.

The virulence plasmid-encoded spv regulon is essential for virulence of Salmonella dublin in mice. The spvR gene product belongs to the LysR family of transcriptional regulator proteins. SpvR induces the expression of the spvABCD operon and positively regulates its own expression. DNase I protection analysis with purified SpvR fusion proteins identified SpvR binding sites within the spvA and spvR promoters (P. Grob and D. G. Guiney, J. Bacteriol. 178:1813-1820, 1996). We have used PCR mutagenesis, combined with functional selection for reduced SpvR affinity, to define the DNA elements essential for SpvR binding. For the spvR promoter fragment, a screen for reduced expression was also applied. Sequence analysis of the resulting mutant fragments reveals that the base pair changes are clustered in distinct regions. Determination of the apparent dissociation constants of SpvR for the mutant promoters showed that the spvA LysR-type motif and the upstream palindromic sequences of both promoters play an important role in SpvR recognition.

SlyA, a transcriptional regulator of Salmonella typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages.

Appropriate regulation of genes enables Salmonella typhimurium to adapt to the intracellular environment of the host. The Salmonella slyA gene is in a family of transcriptional regulators that may play an important role in this adaptation. We have previously shown that slyA mutant Salmonella strains are profoundly attenuated for virulence and do not survive in macrophages. In this study, we demonstrate that the expression of multiple Salmonella proteins is regulated by SlyA during stationary phase and during infection of macrophages. Both of these conditions also induced the expression of a slyA::lacZ transcriptional fusion. Expression of the slyA::lacZ transcriptional fusion increased 15-fold in stationary phase and was not dependent on the stationary-phase sigma factor, RpoS. slyA mutant Salmonella strains were sensitive to oxidative products of the respiratory burst, including hydrogen peroxide and the products of the redox cycling compound paraquat, but not to nitric oxide donors. These results suggest that the SlyA regulon is activated during infection of the host and is required for resistance to toxic oxidative products of the reticuloendothelial system.

The spv genes on the Salmonella dublin virulence plasmid are required for severe enteritis and systemic infection in the natural host.

The pathogenic role of the spv (Salmonella plasmid virulence) genes of Salmonella dublin was determined in the natural, bovine host. Since the lack of overt signs of enteritis or enterocolitis due to Salmonella infections in mice has limited the development of a convenient experimental system to study enteric disease, we used calves to study the contribution of the spv genes to S. dublin-induced salmonellosis. Since the SpvR transcriptional regulator is required for expression of the spvABCD operon, we constructed an spvR knockout mutation in a calf-virulent strain of S. dublin. Calves were infected with the wild-type strain, an spvR mutant, and an spvR mutant containing a complementing plasmid. Calves that were infected with the wild type or the complemented spvR mutant rapidly developed severe diarrhea and became moribund. Calves that were infected with the spvR mutant showed little or no clinical signs of systemic salmonellosis and developed only mild diarrhea. The survival and growth of the wild-type strain and the spvR mutant were determined by using blood-derived bovine monocytes. Wild-type S. dublin survived and grew inside cells, while the spvR mutant did not proliferate. These results suggest that the spv genes of S. dublin promote enhanced intracellular proliferation in intestinal tissues and at extraintestinal sites in the natural host.

Plasmid virulence gene expression induced by short-chain fatty acids in Salmonella dublin: identification of rpoS-dependent and rpo-S-independent mechanisms.

The Salmonella plasmid virulence spvABCD genes are growth phase regulated and require RpoS for maximal expression in stationary phase. We identified a growth phase-independent expression of spv which is mediated by short-chain fatty acids. During this fatty acid-mediated expression of spv, RpoS is required for induction only during exponential phase. In stationary phase, an rpoS-independent mechanism is responsible for expression of spv.

Coccoid and spiral Helicobacter pylori differ in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion.

Helicobacter pylori exists as an actively dividing spiral form and a nonculturable, but viable, metabolizing coccoid form. Both forms are present in the stomach, but their relative pathophysiologic significances are unknown. Here we show that the coccoid form of H. pylori, in contrast to the spiral form, binds poorly to gastric epithelial cells and induces little, if any, interleukin-8 secretion by these cells.

Expression of Salmonella typhimurium rpoS and rpoS-dependent genes in the intracellular environment of eukaryotic cells.

Adaptation to the intracellular environment of host cells is crucial for the pathogenesis of Salmonella infections. The alternative sigma factor RpoS is a global regulator of gene expression during starvation and stress conditions and is required for virulence in Salmonella spp. We have used lacZ reporter fusions to rpoS and rpoS-dependent genes to study rpoS regulation after entry of Salmonella typhimurium into macrophages and epithelial cells. The results demonstrate that expression of an rpoS::lacZ translational fusion increases rapidly in S. typhimurium after phagocytosis. Activity of RpoS also increases after bacterial entry into both macrophages and epithelial cells, as demonstrated by the induction of the rpoS-regulated genes katE and spvB. A control rpoS-independent promoter for neomycin resistance does not show significant induction after cell entry. These results demonstrate that the regulatory system mediated by RpoS in S. typhimurium is activated by the intracellular environment of eukaryotic cells.

Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein.

sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.

Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion.

Rck is encoded on the Salmonella typhimurium virulence plasmid and is a member of a family of related 17- to 19-kDa outer membrane proteins of Enterobacteriaceae, including Ail (Yersinia enterocolitica) and PagC (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and outer loops. When expressed in Escherichia coli, Rck and Ail, but not PagC, confer high-level resistance to the bactericidal activity of complement as well as the ability to adhere to and invade mammalian cell lines. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single amino acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryotic cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresponding PagC fragment containing both the third and the fourth outer loops abolishes the Rck-mediated serum resistance and invasion phenotypes. Substitution of Rck with a smaller C-terminal portion of PagC containing the fourth outer loop did not affect the invasive phenotype or serum resistance. These data reveal that the third putative outer membrane loop region is important for the virulence-associated properties of the Rck protein and suggest a similarity between the mechanism of serum resistance and epithelial cell invasion involving the same domain of Rck.

In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR.

The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125-and 147-bp DNA fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.

Site-specific mutations in the traI relaxase and upstream region of plasmid RP4.

The relaxase of RP4 nicks the double-stranded plasmid at the oriT site and binds covalently to DNA at the 5' end of the nick. The 80-kDa relaxase (TraI) is encoded on an operon with several overlapping open reading frames (ORFs). The importance in conjugation of a short ORF (traX) with a start site overlapping the 5' terminus of traI was investigated, as well as the effects of specific mutations in the relaxase. Elimination of TraX reduced the transfer efficiency by approximately 50% in several intergeneric matings, especially when Escherichia coli was the donor. While TraI was essential for transfer to occur, deletion of the C-terminus of TraI decreased, but did not eliminate plasmid transfer. Mutation of the active site tyrosine resulted in residual transfer associated with amino acid misincorporation.

Biology and clinical significance of virulence plasmids in Salmonella serovars.

Non-typhoid Salmonella strains containing virulence plasmids are highly associated with bacteria and disseminated infection in humans. These plasmids are found in Salmonella serovars adapted to domestic animals, such as Salmonella dublin and Salmonella choleraesuis, as well as in the widely distributed pathogens Salmonella typhimurium and Salmonella enteritidis. Although virulence plasmids differ between serovars, all contain a highly conserved 8-kb region containing the spv locus that encodes the spvR regulatory gene and four structural spvABCD genes. Studies in mice suggest that the spv genes enhance the ability of Salmonella strains to grow within cells of the reticuloendothelial system. The spv genes are not expressed during exponential growth in vitro but are rapidly induced following entry of Salmonella strains into mammalian cells, including macrophages. Transcription of the spv genes is controlled by the stationary-phase sigma factor RpoS, and mutations in RpoS abolish virulence. These studies suggest that the ability of Salmonella strains to respond to starvation stress in the host tissues is an essential component of virulence.