Biomedical subjects
Publications and source records attributed to T S Wallis.
Mutation of slyA, which reduces Salmonella typhimurium virulence in mice, caused only minor attenuation of S. typhimurium virulence in orally inoculated calves. This correlated with modest reductions in intestinal invasion and enteropathogenic responses in bovine ligated ileal loops. slyA appears to regulate virulence genes involved in systemic, but not enteric, salmonellosis.
Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5, 6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456-14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.
Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella-specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA1ser (serT) on one side and copS/copR on the other. Thus, this Salmonella-specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA, pipB, pipC, pipD (pathogenicity island-encoded proteins) and orf, in addition to sopB. The effect of mutations in pipA, pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.
The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in Salmonella-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of Salmonella spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the sopB gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of Salmonella dublin. Using the cya reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of sopD has an additive effect in relation to the sopB mutation.
The induction of secretory and inflammatory responses in calves by Salmonella typhimurium and Salmonella dublin strains was compared, and the effects of mutations in the invH and stn genes were assessed. S. typhimurium induced greater secretory and inflammatory responses than S. dublin in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P. R. Watson, S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis, Infect. Immun. 63:2743-2754, 1995). These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis. The secretory and inflammatory responses induced by S. typhimurium and S. dublin in bovine ligated ileal loops were not significantly altered by mutation of stn, which suggests that stn does not have a major role in Salmonella-induced enteritis. The invH mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves. The attenuation associated with the invH mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of S. typhimurium to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered. However, lysis of macrophages following infection with S. typhimurium was significantly reduced by the invH mutation. The invH mutation prevented the normal secretion of several proteins, including SipC, by S. typhimurium, indicating that the function of the inv-spa-encoded type III protein secretion system was disrupted. Taken together, these observations implicate inv-spa-dependent effectors in mediation of Salmonella-induced enteritis in cattle. Clearly, however, other undefined serotype-specific virulence factors are also involved in Salmonella-induced enteritis.
Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC, encoding [Cu,Zn]-SOD, from Salmonella typhimurium. The predicted protein sequence shows only 58% identity to Escherichia coil SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC, in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S. typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S. typhimurium, S. choleraesuis and S. dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro, perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella, the most efficient overwhelming the proposed protective effect of periplasmic SOD.
Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.
Ileal loops including Peyer's patch were prepared in five 28-day-old calves and infused Salmonella typhimurium strain ST4/74. Loops were fixed 5 minutes to 2 hours after inoculation, and the mucosa was examined by light and electron microscopy. Within 5 minutes, the bacteria were interacting with the follicle-associated epithelium (FAE); the surface of M cells changed to lamellipodia, engulfing many bacteria. This process proceeded rapidly to 30 minutes, involving most M cells above crypt level. Most cells were exfoliated, and many were packed with bacteria, and the domed villi became stunted. There was a rapid migration of neutrophils through the FAE into the lumen by 15 minutes. By 60 minutes, there was no further interaction between the bacteria and the FAE; at this time bacteria were present in macrophages in the lamina propria. Restitution of the FAE was complete by 2 hours in spite of the many bacteria in the cell debris overlying the epithelium. Interaction of bacteria with the absorptive villi was delayed compared with interaction with the FAE. After 15 minutes, bacteria were seen adhering to some enterocytes of the upper third of the villi; many bacteria were adhering to the surface of the enterocytes at 20 and 30 minutes, but few were seen thereafter. Adherence was patchy and largely confined to cells whose surfaces were depressed relative to others. The microvillous surface of these enterocytes was extensively remodelled. Tissue response, with uptake of bacteria into vacuoles, exfoliation of enterocytes containing bacteria, and subsequent stunting of the villi, began at 30 minutes and was severe and progressive to 2 hours. Following the initial attachment and uptake of the bacteria loss of enterocytes progressed from these initial sites; bacteria were associated with the lateral cell membrane of cells adjacent to cells being extruded and not with the microvilli of cells at new sites. In a calf 4 hours after dosing orally with the same strain, M cells were engulfing bacteria and their cell surface was changed as seen in the inoculated loops; absorptive enterocytes were also taking up bacteria as seen in the ileal loops, indicating the process seen in the loops and after oral dosage was similar. For this strain of S typhimurium, there was an initial concentration of bacilli around the domed villus epithelium. This distribution was not random but may have resulted from a specific attraction to the FAE.
The virulence plasmid in Salmonella dublin mediates systemic infection in mice and cattle. The role of gamma delta T cells or hepatic extrathymic T cells has recently been reported to be important in the control of the early stage of Salmonella choleraesuis infections of mice. Here, we report on T-cell responses in conventional mice after challenge with a virulent strain of S. dublin carrying a virulence plasmid or with a strain cured of the plasmid. Over a period of 4 days postinfection, when both strains could be compared, similar changes in alpha beta and gamma delta T-cell subsets in peritoneal cavities, livers, and spleens were recorded, demonstrating no clear role of the virulence plasmid in modulation of early T-cell responses. To investigate further the role of the virulence plasmid in pathogenesis, the growth of the plasmid-cured strain was assessed in SCID, SCID bg, and irradiated mice. During the first 6 days after infection, there was no statistically difference in the net growth of Salmonella cells in the livers and spleens of SCID and SCID bg mice compared with conventional BALB/mice. This observation excludes a key role for a T- or B-cell-mediated immune response in controlling the initial growth of the plasmid-cured S. dublin strain. Thereafter, the immunocompromised mice were no longer able to control infection, although SCID mice were more efficient at controlling net bacterial multiplication than SCID bg mice, potentially implicating NK cells in the control of infection in SCID mice. The early control of net bacterial multiplication in the spleens and livers of BALB/c mice was ablated by whole-body X-irradiation. Both wild-type and plasmid-cured strains multiplied significantly more rapidly in irradiated than in conventional BALB/c mice. However, the numbers of wild-type bacterial still increased more rapidly than in the numbers of the cured strains. These results are consistent with a role of the S. dublin virulence plasmid in promoting in vivo growth of Salmonella cells.
The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle. Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo. The intracellular recovery of S. dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage. However, the virulence plasmid increased the lytic activity of S. dublin, Salmonella typhimurium, and Salmonella choleraesuis for resident or activated mouse peritoneal macrophages. Lysis was not mediated by spv genes and was abolished by cytochalasin D treatment. Peritoneal and splenic macrophages were isolated from mice 4 days after intraperitoneal infection with wild-type or plasmid-cured S. dublin strains. The wild-type strain was recovered in significantly higher numbers than the plasmid-cured strain. However, the intracellular killing rates of such cells cultured in vitro for both S. dublin strains were not significantly different. Four days after infection, there was a lower increase of phagocyte numbers in the peritoneal cavities and spleens of mice infected with the wild-type strain compared with the plasmid-cured strain. The virulence plasmid influenced the survival of macrophages in vitro following infection in vivo as assessed by microscopy. Cells from mice infected with the plasmid-cured strain survived better than those from mice infected with the wild-type strain. This is the first report demonstrating an effect of the virulence plasmid on the interaction of Salmonella strains with macrophages. Plasmid-mediated macrophage dysfunction could influence the recruitment and/or the activation of phagocytic cells and consequently the net growth of Salmonella strains during infection.
The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S. typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops. S. dublin was found to be significantly less invasive in cultured cells than S. typhimurium, but this difference was not observed in bovine intestines. The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines. The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy. The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer. The degree of damage in the bovine ileum was related to the magnitude of the invasion. There was no difference in the amount of S. typhimurium or S. dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop.
Plasmid-bearing and plasmid-free isolates and a plasmid-cured strain of Salmonella dublin were compared for virulence in calves. The plasmid-bearing strains were highly virulent, causing severe enteric and systemic disease with high mortality. In contrast, the plasmid-free strains caused diarrhea but only low mortality. The infection kinetics of a wild-type and a derivative plasmid-cured strain were compared. Both strains were isolated in high numbers from intestinal sites at 3 and 6 days after oral challenge and were isolated at comparable frequencies from systemic sites at 3 days, but not at 6 days, when the wild-type strain was predominant. The strains were equally invasive in intestinal epithelia with and without Peyer's patch and elicited comparable secretory and inflammatory responses and intestinal pathology in ligated ileal loops. The effect of the virulence plasmid on growth kinetics and on the outer membrane protein profile was assessed in an in vivo growth chamber. The virulence plasmid did not influence either extracellular growth or the expression of major outer membrane proteins. These observations demonstrate that the virulence plasmid is not involved in either the enteric phase of infection or the systemic dissemination of S. dublin but probably mediates the persistence of S. dublin at systemic sites.
Nitrogen mustard (N2M) treatment of rabbits induced neutropenia, and, in ligated ileal loops, it inhibited fluid secretion induced by salmonella or by cholera toxin (CT). Pretreatment of rabbits with indomethacin almost abolished salmonella-induced fluid secretion and significantly reduced that induced by CT. Similar effects of N2M and indomethacin on fluid secretion induced by salmonella, but not by CT, have been reported by other workers and used to implicate prostaglandins, from the salmonella-induced inflammation, as mediators of fluid secretion. In contrast, we show that N2M treatment, in addition to reducing CT-induced secretion, caused severe morphological alterations to ileal mucosa. Irradiation techniques were developed for inducing neutropenia, but they did not totally inhibit salmonella-induced leucocyte influx into ileal mucosa. We propose an alternative mechanism for the inhibitory effect of N2M on salmonella- and CT-induced secretion, based on the known anti-mitotic activity of N2M. Also, the anti-secretory effect of indomethacin cannot be attributed uniquely to its anti-inflammatory activity because it depressed CT-induced secretion as well as salmonella-induced secretion. These results support the concept of pathophysiological secretion in infectious diarrhoea, developed previously for rotavirus and extended to bacterial infections.
The abilities of six strains of Salmonella typhimurium to associate with rabbit ileal mucosa have been measured in vitro. Two were "virulent" strains (TML and W118 which are invasive and inducers of fluid secretion in rabbit ileal loops); four were "avirulent" (LT7, M206 and SL1027 which are invasive but induce negligible fluid secretion, and Thax-1 which is neither invasive nor an inducer of fluid secretion). A special organ-culture apparatus was designed to expose only the luminal surface of the mucosa to organisms. Viable counts of washed homogenised tissue taken 30 min after challenge showed that virulent strains TML and W118 and avirulent strains LT7 and M206 could not be distinguished from each other. Avirulent strain SL1027 associated less well than the other four strains, and Thax-1 associated less well than SL1027; both these strains were non-motile whereas the other four were motile. Thus, early association with gut mucosa did not discriminate all avirulent strains from the virulent strains. Qualitative examination of tissues by scanning electronmicroscopy did not detect strains LT7 and M206 on the mucosal surface whereas strains TML and W118 were readily seen, suggesting that the nature of association of virulent and avirulent strains was different. Qualitative examination by transmission electronmicroscopy of tissues challenged in vivo for 120 min showed virulent strains TML and W118 invading epithelial cells; similar events were reproduced after 120-min challenge in vitro. In contrast, invasion by avirulent strains was observed only very rarely.
Leucocyte influx into rabbit ileal loops, induced by strains of Salmonella typhimurium of different virulence, was assessed with 111Indium-labelled leucocytes. Strains fell into two groups on the basis of their leucotactic potential: "virulent" strains (which induced fluid secretion) caused a dose-dependent leucocyte influx; strains which did not induce fluid secretion failed to induce a significant leucocyte influx. Fluid secretion was never observed in the absence of leucocyte influx, but leucocyte influx per se did not induce fluid secretion. The phenotype of the challenge inoculum influenced fluid secretion; young log-phase organisms induced fluid secretion with a higher frequency than overnight cultures. These findings support earlier evidence implicating leucocytes in an interactive but not exclusive role in the genesis of salmonella-induced fluid secretion. They suggest, though do not prove, that interaction of leucocytes with the appropriate phenotype of organisms results in the release of a host-derived or bacterial secretagogue, or both. The bacterial factor may or may not be the antigen related to cholera toxin, described previously.
The choice of strain, culture conditions, composition of medium and size of inoculum all affected the expression of a cholera-toxin-related antigen (CTRA) in Salmonella typhimurium. A previous study had shown that the number of organisms expressing CTRA in Casamino acid Yeast Extract (CYE) medium decreased between 4 h and 6 h in uninterrupted culture. In the present experiments, organisms harvested at 4-5 h were subcultured into fresh CYE medium and incubated for a further 2 h; the total number of organisms increased, and the decrease in the proportion of organisms expressing CTRA was reduced. Use of Hartley Digest Broth in place of CYE medium increased the proportion of organisms expressing CTRA in all strains tested, in both the uninterrupted and the subculture procedures. The higher the initial inoculum, the lower was the proportion of organisms expressing CTRA. The presence of the antigen in cells remained constant for about 18 h after transfer from 37 degrees C to 4 degrees C. These data have important implications for the production and purification of CTRA: they show that it was expressed during log-phase of growth, and they suggest that expression was regulated by a non-growth-limiting factor. Moreover, some avirulent strains were better producers of the antigen than virulent ones. The significance of the data is discussed in relation to the in-vivo situation.
Six strains of Salmonella typhimurium (W118, TML, SL1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1-4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.
Mice that did not contain antibodies to rotavirus were orally infected with murine rotavirus (EDIM strain) and observed over 7 days. As judged by ELISA, only the small intestine was infected, not the colon. The infection was biphasic, viral antigen peaks being observed at 48 h and approximately 120 h post-infection. Clinically evident diarrhoea was maximal at 72 h. Virus in the upper, middle and lower regions of the small intestine was mainly tissue-associated; most virus was found in the middle small intestine. Two peaks (48 h and 120 h post-infection) of virus antigen were observed in the colon, but these corresponded to luminal, not tissue-associated viral antigen. Only enterocytes in the upper two-thirds of villus epithelia were infected as judged by fluorescent-antibody analysis and transmission electron microscopy. Scanning electron microscopy revealed morphological appearances not hitherto correlated with the progress of the infection: villus tips were convoluted, corresponding to the shedding of virus-infected cells but the lower regions of infected villi were shrunken and considerably narrowed compared to tips.