Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice, cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

Exposure of Salmonella senftenberg 4969 to sublethal heating in phosphate buffer, pH 7·0, at 52· produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37· in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favourable environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.     

Cellular routes of invasion by enteropathogens

The cellular pathways of infection utilized by pathogenic enteric bacteria have important implications for their clinical manifestations. Yersinia reaches Peyer's patches via M cells and uses plasmid-encoded factors to resist phagocytic cells. Shigella also translocates via M cells and incapacitates phagocytes, but subsequently re-enters the epithelium basolaterally to elicit an acute inflammatory response. Salmonella has recently been shown to both colonize Peyer's patches via M cells and independently disseminate to extraintestinal sites via CD18-expressing phagocytes. M cell-mediated entry can lead to gastroenteritis and mucosal antibody production, while systemic dissemination can result in septicemia and elicitation of systemic immune responses.     

A Salmonella SipB-derived polypeptide blocks the 'trigger' mechanism of bacterial entry into eukaryotic cells

Entry into non-phagocytic mammalian cells by the invasive pathogens Salmonella and Shigella is triggered by the delivery of bacterial virulence effector proteins into the host cell. This is dependent upon Salmonella SipB or its Shigella homologue IpaB, which insert into the eukaryotic cell plasma membrane. Here we show that a SipB-derived 166 residue alpha-helical polypeptide is a potent inhibitor of SipB-directed liposome fusion in vitro, preventing the membrane-associated form of SipB from inserting deeply into the bilayer. This polypeptide blocks Salmonella entry into cultured mammalian cells at 10(-10) M, and is a heterologous inhibitor of analogous IpaB activity and Shigella cell entry. These findings reveal a potential strategy to identify inhibitors of the 'trigger' mechanism underlying cell entry by these major invasive pathogens.     

Effect of short-chain fatty acids on the size of enteric bacteria

J.L. THOMPSON AND M. HINTON. 1996. The size of Escherichia coli and Salmonella enteritidis PT4 cells was measured using both transmission electron micrographs and image analysis. Incubation in the presence of formic and propionic acids resulted in larger cells, possibly as a result of DNA inhibition, with no apparent damage to the cell membranes. Bacteria incubated with propionic acid were more resistant to shrinkage after fixation, possibly as a result of altered phospholipid and fatty acid composition.     

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.     

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.     

Variation in Salmonella core lipopolysaccharide as detected by the monoclonal antibody M105

L.P. MANSFIELD, E. BILLETT, E. OLSEN AND S.J. FORSYTHE. 1996. The lipopolysaccharide antigenicity of 22 Salmonella strains (representing nine serogroups) and four non-salmonellae Enterobacteriaceae to the Salmonella genus specific monoclonal antibody M105 was analysed. The monoclonal antibody M105 reacted with all 22 Salmonella strains. Probing SDS-PAGE separated LPS molecules with MAb M105 revealed that the antibody reacted with the core region of all Salmonella serovars. However, no reaction was obtained to the long-chain LPS of serovars O ( Salm. adelaide and Salm. ealing ), C₁ ( Salm. infantis, Salm. livingstone and Salm. virchow ) or Salm. arizonae . It is plausible that the presence of a second core antigenic type results in the lack of reaction between long-chain LPS and the Salmonella genus specific monoclonal antibody M105.     

Interaction of pathogenic bacteria with rabbit appendix M cells: bacterial motility is a key feature in vivo

Rabbit appendix consists mainly of lymphoid follicles (LF) covered by M cells, the specialized antigen-sampling cells of the mucosal immune system, and surrounded by glandular epithelium. Until now, these M cells have been characterized morphologically and histologically by using cellular markers. Here, the adhesion and transport of pathogenic bacteria were investigated to assess the function of M cells of the appendix. We used the enteroinvasive motile Salmonella typhimurium and the rabbit enteropathogenic non-motile Escherichia coli RDEC-1, which are known to target specifically rabbit M cells of Peyer's patches (PPs). We found that S. typhimurium efficiently attached and was transported through appendix M cells in vivo. In contrast to S. typhimurium, RDEC-1 targeted M cells only ex vivo, when bacteria were allowed to have direct contact with the surface of the follicle. The difference in interaction of the two bacteria with appendix M cells led us to investigate whether this could be correlated with the lack of motility of RDEC-1. We used an aflagellate mutant of S. typhimurium and found that it had the same infection phenotype as RDEC-1. Gene complementation restored the efficiency of infection to that of S. typhimurium wild-type strain. In conclusion, we show that M cells of the appendix display features of the canonical M cells of PP, since they efficiently sample luminal pathogenic bacteria. However, due to the morphology of the appendix, motile bacteria appear to be more potent in their interactions with appendix M cells.     

Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed decreased expressions of MMP-2 and MMP-9 at the mRNA and protein levels. Taken together, the present findings suggest that Neu3 is a promising molecular target for the prevention of prostate cancer metastasis.     

A parallel intraphagosomal survival strategy shared by mycobacterium tuberculosis and Salmonella enterica

Mycobacterium tuberculosis and Salmonella enterica cause very different diseases and are only distantly related. However, growth within macrophages is crucial for virulence in both of these intracellular pathogens. Here, we demonstrate that in spite of the phylogenetic distance, M. tuberculosis and Salmonella employ a parallel survival strategy for growth within macrophage phagosomes. Previous studies established that the Salmonella mgtC gene is required for growth within macrophages and for virulence in vivo. M. tuberculosis contains an open reading frame exhibiting 38% amino acid identity with the Salmonella MgtC protein. Upon inactivation of mgtC, the resulting M. tuberculosis mutant was attenuated for virulence in cultured human macrophages and impaired for growth in the lungs and spleens of mice. Replication of the mgtC mutant was inhibited in vitro by a combination of low magnesium and mildly acidic pH suggesting that the M. tuberculosis-containing phagosome has these characteristics. The similar phenotypes displayed by the mgtC mutants of M. tuberculosis and Salmonella suggest that the ability to acquire magnesium is essential for virulence in intracellular pathogens that proliferate within macrophage phagosomes.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.     

Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications for immunogenicity

Abstract Two different expression systems were investigated for the production of an 80 amino acid polypeptide, M3, from the C-terminus of the Plasmodium falciparum blood stage antigen Pf155/RESA in an attenuated Salmonella typhimurium vaccine strain. Upon expression, the malarial polypeptide was targeted either to the periplasm as a soluble fusion protein containing two IgG-binding domains (ZZ) from the staphylococcal protein A or, to the bacterial surface as an insert within a chimeric outer membrane protein A (OmpA) derived from Escherichia coli and Shigella dysenteriae . Both the ZZM3 and the OmpAM3 proteins were stably expressed in the periplasm or on the surface of Salmonella , respectively. The ZZ expression system yielded 10–100 times more malarial immunogen than did the OmpA system. Live recombinant Salmonella expressing ZZM3 or OmpAM3 were used to immunize mice intraperitoneally. Both the ZZM3 and OmpAM3 genes persisted for up to three weeks in bacteria isolated from different lymphoid organs. Bacteria expressing ZZM3 induced antibodies to M3, ZZ and to the Pf155/RESA antigen whereas, bacteria producing OmpAM3 induced similar levels of antibodies reactive with M3 but not with Pf155/RESA. Both recombinants induced a memory response of antibodies reactive with both M3 and Pf155/RESA. The high levels of M3 produced by the ZZ expression system make it suitable for the expression of heterologous antigens in Salmonella . Nevertheless, in spite of the quantitative difference in M3 expression, the ZZ and OmpA constructs elicited comparable immune responses to M3.     

Vasoactive intestinal peptide (VIP) prevents killing of virulent and phoP mutant Salmonella typhimurium by inhibiting IFN-gamma stimulated NADPH oxidative pathways in murine macrophages

Vasoactive intestinal peptide is an immunomodulator with great potential in the treatment of inflammatory pathology. In this study, we have examined the effect of VIP on the growth dynamics of virulent Salmonella enterica. Serovar typhimurium (S. typhimurium) 14028 and 4/74 and an avirulent mutant (14028 phoP) in a murine, macrophage cell line (J774.2). In contrast to standard growth dynamics, in which phoP mutants do not survive in macrophages, we show that VIP (10(-10) M) significantly enhances phoP growth over a 24 h post-infection period even when the cells are co-cultured with IFN-gamma. We examined the effect of VIP on the generation of NADPH-induced reactive oxygen species (ROS) in Salmonella-infected/IFN-gamma cultured J774 cells. VIP inhibited gp91 mRNA levels, gp91 protein and subsequent ROS. The importance of ROS in killing of Salmonella by J774 cells was highlighted by experiments in which ROS production by J774 cells was inhibited using a conventional inhibitor, N-acetyl-L-cysteine captopril (ACC) and in which Salmonella growth significantly increased. Our findings suggest that although VIP inhibits inflammatory pathways in myeloid cells it also promotes the growth of avirulent (phoP) mutants.     

The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).     

Isolation of salmonellas by immunomagnetic separation

A.E.M. VERMUNT, A.A.J.M. FRANKEN AND R.R. BEUMER. 1992. Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10⁷) were generally incubated with 10⁴ Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.9±7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9±12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salim. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.