Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches

Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection. It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer's patches, the M cell. In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions of Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells by using a murine ligated-loop model. Our results indicate that S. typhimurium possesses a highly efficient mechanism for M cell entry that targets and destroys these cells, while L. monocytogenes and S. flexneri appear to be internalized into M cells in a less disruptive fashion. Early uptake of Listeria or Shigella into M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium. At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction. These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.     

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.     

Mycoplasma pulmonis infection in gnotobiotic and conventional mice: aspects of pathogenicity including microbial enumeration and studies of tracheal involvement

Intranasal inoculation of Mycoplasma pulmonis into gnotobiotic mice resulted in positive cultures from the anterior nares, nasopharynx, trachea, and lungs persisting for 21 da. Similar inoculation of conventional, mycoplasma-free mice resulted in an increase in colony-forming units reaching a maximum by the 8th post-inoculation day and remaining constant in trachea and lungs through the 22nd day by an organ homogenate serial dilution method. All mice appeared ill with loss of total body weight, an increase in lung weight, and a peak of mortality between the 6th and 8th days. Between the 8th and 22nd, day, the number of deaths declined and survivors improved in appearance despite persistence of large numbers of colony-forming units in the trachea and lungs. M pulmonis was demonstrated in the trachea by immunofluorescence and electron microscopy.     

Salmonella typhimurium infection in mice induces nitric oxide-mediated immunosuppression through a natural killer cell-dependent pathway

Splenocytes isolated from C57BL/6J female mice 3 to 7 days after inoculation with an attenuated strain of Salmonella typhimurium produced high levels of nitric oxide (39 to 77 microM) and gamma interferon (IFN-gamma). Additionally, spleen cell cultures from Salmonella-inoculated mice were markedly suppressed in their ability to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes. Depletion of natural killer (NK) cells from the immune splenocyte population markedly reduced nitric oxide production, prevented suppression of PFC responses, and completely abrogated IFN-gamma release. Treatment of NK cell-depleted immune cells with IFN-gamma restored nitric oxide production to levels comparable to those of intact immune cells and also restored the immunosuppression. These results suggest that NK cells regulate the induction of nitric oxide-mediated immunosuppression following infection with S. typhimurium through the production of IFN-gamma.     

At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.     

Antibody and cytokine responses in a mouse pulmonary model of Shigella flexneri serotype 2a infection

A murine pulmonary model was used to study the mucosal immune response to Shigella flexneri serotype 2a infection. Inoculation of BALB/cJ mice with shigellae via the intranasal route resulted in bacterial invasion of bronchial and alveolar epithelia with concomitant development of acute suppurative bronchiolitis and subsequent development of lethal pneumonia. The pathology of pulmonary lesions resembled the colitis that characterizes shigellosis in humans and primates. Significant protection against a lethal dose of S. flexneri 2a was observed in mice previously infected with two sublethal doses of the homologous strain. Immunity against lethal challenge was associated with decreased bacterial invasion of the mucosal epithelium. Over the course of two sublethal challenges, which constituted primary and secondary immunizations, mice developed pulmonary and serum immunoglobulin G and A antibody recognizing both lipopolysaccharide and invasion plasmid antigens IpaB and IpaC. Immune mice and naive control mice differed in lung lavage cytokine levels following lethal challenge. Immune mice developed significantly elevated levels of pulmonary gamma interferon within 6 h of challenge, while naive control mice developed elevated levels of this cytokine later during the initial 24-h period. Both groups had elevated levels of gamma interferon during the 24- to 48-h period of infection. Both groups also had elevated levels of tumor necrosis factor alpha within 6 h of challenge, but the control mice had significantly higher levels at the 48- and 72-h time points. Elevated levels of interleukin-4 were observed only in immunized mice. This cytokine appeared within 24 h and receded between 48 and 72 h. Fluorescence-activated cell sorter analysis of lung parenchymal cells showed that both groups experienced an initial influx of monocytes, but the proportion of this cell type began to recede in immunized mice after 48 h of infection, while peak levels were maintained in the control animals. These studies suggest that elements of local B lymphocyte activity, as well as Th1 and Th2 lymphocyte activity, may contribute to the survival of immune mice after intranasal challenge with shigellae.     

Priming of T-cell responses in mice by porins of Salmonella typhimurium

We have investigated systematically the effects of short-term exposure to main stream cigarette smoke condensates (CSC-MS) on basal and inducible functional capacities of murine peritoneal exudate macrophages. Macrophages treated with CSC-MS form granules that fluoresce orange under blue excitation, consistent with the speculation that they are polycyclic aromatic hydrocarbons (PAH). CSC-MS selectively suppressed interferon gamma (IFN gamma) induction of four macrophage functional capacities: enhanced phagocytosis of immunoglobulin-opsonized sheep red blood cells, TPA-induced H2O2 production, class II major histocompatibility complex expression, and nitric oxide synthesis. In contrast, two macrophage functions that are not induced by IFN gamma, basal electron transport and LPS-induced TNF alpha production, were enhanced by treatment with CSC-MS. These results suggest that the suppressive effects of CSC-MS on macrophage responsiveness were selective and were not due to nonspecific inhibition of general functions such as RNA or protein synthesis. Since macrophage responsiveness to IFN gamma can result in induction of functional capacities that are fundamental to immunity, the data suggest that CSC-MS maybe deleterious to the general health of the smoker.     

Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma

A live attenuated AroA- auxotrophic mutant of Salmonella typhimurium (SL7207) has been used as carrier for the pCMVbeta vector that contains the beta-galactosidase (beta-gal) gene under the control of the immediate early promoter of Cytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether beta-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the beta-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to beta-gal. Mice vaccinated with the Salmonella harboring pCMVbeta, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80(+) macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.     

Serological and bacteriological observations on experimental infection with Salmonella hadar in chickens

Over the past 3 years the frequency of Salmonella hadar infections has increased in Belgium in both poultry and humans. Therefore, the course of infection with S. hadar in poultry was investigated. One day-old and 4 week-old specific pathogen-free chickens were orally infected with one of two S. hadar strains, SH1 or SH2. Mortality was 6% (SH1) and 17% (SH2) in birds infected at 1 day-old. Chickens infected at 1 day-old with SH2 showed a mild diarrhoea. The S. hadar faecal excretion in birds infected at 1 day-old remained high throughout the experiment until 12 weeks post-inoculation (pi). Faecal excretion was lower in older birds. Antibodies to S. hadar were observed from 2 weeks pi (SH2, infected at 1 day-old) or 4 weeks pi (SH1, both groups; SH2, chickens infected at 4 weeks of age). The percentage of chickens with antibodies was higher after infection at 1 day-old than after infection at 4 weeks of age. In a second experiment 1 day-old chicks were infected with SH1 and autopsied at regular intervals until 42 days pi. SH1 was isolated from the caeca from 3 h pi onwards and from the liver and spleen from 18 h until 14 days pi. Serous typhlitis and omphalitis were the main lesions. The number of macrophages in the lamina propria of the caecal tonsils was slightly increased from 18 h until 2 weeks pi. In the liver, inflammation was observed in the portal triads and in the sinusoids. This study indicates that infections with S. hadar lead to intense colonisation of the gut and extensive faecal shedding. It may also cause invasive infections in 1 day-old chickens.     

Multiple fimbrial adhesins are required for full virulence of Salmonella typhimurium in mice

Adhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the Salmonella typhimurium fimbrial operons lpf, pef, or fim only moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or S. typhimurium may encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of S. typhimurium: fim, lpf, pef, and agf. A mutation in the agfB gene resulted in a threefold increase in the oral 50% lethal dose (LD50) of S. typhimurium for mice. In contrast, an S. typhimurium strain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD50. The quadruple mutant, but not the agfB mutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever.     

Characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection

This study characterised the primary immune response in gnotobiotic lambs after infection with a lamb rotavirus (RV). Lambs were infected and killed over a 7 week period together with controls. RV-ELISA and neutralising antibodies were determined in serum, nasal secretions, and intestinal scrapings. RV-antibody secreting cells (ASC) were enumerated in blood. Lymphocyte proliferations were determined in blood and gut-associated lymphoid tissues and cytokine expression was analysed in jejunal Peyer's patches (JPPs) and mesenteric lymph nodes (MLNs). Infected lambs cleared the virus by 8-9 days after infection without showing any clinical signs. The first indication of a specific immune response to RV was an increased expression of IL-4 mRNA in the JPPs in the infected group compared to the control group 3 days after infection. Rotavirus-specific IgA ASC in blood and IgA antibodies in serum and nasal secretions were detected from 7 days after infection followed at 10 days after infection by RV-specific IgG ASC and antibodies. Rotavirus-specific IgA antibodies were not detected in intestinal scrapings in the first 10 days after infection, but were detected by 52 days after infection. No RV-specific neutralising antibodies were seen in the intestine during the course of the experiment.     

Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis

An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy. Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S. typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age. A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S. typhimurium or S. enteritidis showed that delta cya delta crp S. typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella. The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated. S. enteritidis and S. typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella. S. typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S. enteritidis showed no pathological lesion in the visceral and reproductive organs. Vaccination with chi 3985 prevented transmission of S. typhimurium or S. enteritidis into eggs laid by vaccinated layers with no effect on egg production. To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers.     

Effect of vaccination of hens with an avirulent strain of Salmonella typhimurium on immunity of progeny challenged with wild-Type Salmonella strains

The avirulent Salmonella typhimurium chi3985 was used to vaccinate white leghorn chickens at 16 and 18 weeks of age, and the effect of maternal antibody on Salmonella colonization of progeny of vaccinated hens was assessed with S. typhimurium F98 or chi3985. Progeny of hens that had been vaccinated at 1 and 3 or 2 and 4 weeks of age with chi3985 were used to determine the effect of maternal immunity on vaccine efficacy. Vaccination of hens induced long-lasting Salmonella-specific antibodies which were transferred into eggs and were detected as immunoglobulin G (IgG) in the egg yolk. Maternal antibody was detected in the progeny of vaccinated birds as IgG and IgA in serum and intestinal fluid, respectively. The titer of maternally transmitted IgG or IgA was highest in the first week of life of the progeny and declined with age. Maternal antibodies prevented colonization of the chicks by S. typhimurium chi3985 and reduced colonization by S. typhimurium F98. Overall, chicks from vaccinated hens had significantly higher antibody responses than did the progeny of nonvaccinated hens after oral infection with Salmonella strains. Maternal antibody reduced the efficacy of vaccination of progeny with chi3985 at 1 and 3 weeks of age. But vaccination at 2 and 4 weeks of age induced excellent protection against challenge with S. typhimurium F98 or S. enteritidis 27A PT 8 in birds from vaccinated hens and in specific-pathogen-free chickens. Vaccination of chickens at 2 and 4 weeks of age has been shown to protect the birds against challenge with homologous and heterologous Salmonella serotypes. A combination of vaccination of adult animals and use of the progeny of vaccinated birds will enhance effective control of Salmonella infections in the poultry industry. This will complement the present control of Salmonella-associated food poisoning caused by Salmonella enteritidis in eggs because the avirulent S. typhimurium vaccine strain chi3985 induced excellent protection against S. enteritidis in chickens.     

Experimental transmission of an apparent viral pneumonia in conventional and gnotobiotic pigs

Endemic pneumonia in five- to eight-week-old pigs induced microscopic lesions of proliferative interstitial pneumonia which were compatible with a viral aetiology. The disease was transmitted experimentally to conventional and gnotobiotic pigs by means of a lung homogenate filtered through a 0.22 micron filter. No common viral respiratory pathogens of pigs were isolated. Two types of virus particles were observed in cell culture by electron microscopy; one was about 70 nm in diameter and had an envelope and short surface spicules, the other also had an envelope, was elongated, pleomorphic, measured 80 x 320 nm and was coated by antibodies.     

Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium

Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.     

Directed formation of chromosomal deletions in Salmonella typhimurium: targeting of specific genes induced during infection

In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection. Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands). It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region. We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region. Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible.     

The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046-4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.     

Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF)

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a commercially expensive respiratory disease of swine. Salmonella typhimurium SL3261 was used as a live carrier of plasmid pKF1, which encodes a 15-kDa recombinant M. hyopneumoniae protein. This expressed recombinant protein consists of the carboxy-terminal 11 kDa of a 42-kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Rabbit anti-15-kDa serum was able to inhibit the growth of viable M. hyopneumoniae J in vitro. When used as a live oral vaccine, S. typhimurium SL3261(pKF1) induced a significant secretory immunoglobulin A immune response in the lungs of mice orally immunized against the M. hyopneumoniae antigen. Utilization of live oral vaccines expressing potentially protective M. hyopneumoniae proteins, such as the NrdF antigen, which can stimulate a lung mucosal response against surface-accessible proteins may provide a cost-effective alternative to the present control strategies used for porcine enzootic pneumonia.