Natural and recombinant interferons inhibit epithelial cell invasion by Shigella spp.

The effect of natural and recombinant interferons (IFNs) on the abilities of Shigella flexneri, S. sonnei, and Salmonella typhimurium to invade different human and murine cells was examined. Pretreatment of cell monolayers with natural and recombinant IFNs reduced the number of Shigella-infected cells in a dose-dependent manner. Establishment of an anti-invasive cellular state was time dependent, requiring 10 h for 50% inhibition of bacterial invasion. The inhibitory effect of IFN was species specific, with human or murine IFN effective against homologous but not heterologous cells. Gamma IFN was slightly more potent than alpha IFN at inhibiting bacterial invasion. Inhibition of Shigella invasion was dependent on the challenge dose of bacteria. Little inhibition of invasion was seen when cells were pretreated with low concentrations of IFN and challenged with high multiplicities of infection of Shigella sp. In contrast to Shigella invasion, the maximum inhibitory effect of IFN on Salmonella invasion of cells was observed at low levels (5 to 50 U) of IFN. These results suggest that Shigella and Salmonella invasions occur at unique sites on eucaryotic cells or by different penetration mechanisms. More importantly, these data suggest that IFN may play a significant role in host defense against Shigella and Salmonella infections.     

Natural killer cell activation and interferon production by peripheral blood lymphocytes after exposure to bacteria.

We have previously shown that peripheral blood natural killer (NK) cells have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. In this report, we show that NK cell activity against K562 tumor cells and Shigella flexneri-infected HeLa cells can be greatly enhanced by preincubating peripheral blood lymphocytes (PBL) for 18 h with kanamycin-treated Shigella flexneri or Salmonella typhimurium. Cell-free supernatants obtained from PBL-bacteria cultures contained high levels of interferon (IFN) activity, which was characterized as a mixture of IFN-gamma and IFN-alpha. Cytotoxic activity associated with PBL precultured with shigellae was associated with predominantly CD16+ (Leu-11+) and CD2+ (OKT-11+) cells. Further, IFN production was dependent upon the presence of CD16+ and CD2+ cells at culture initiation. Enhancement of cytotoxic activity associated with PBL-bacteria cultures did not, however, appear to be dependent upon IFN production, since low numbers of bacteria which failed to stimulate IFN production induced high levels of NK cell activity. Lipopolysaccharide appeared not to be involved in bacteria-induced IFN production or enhanced NK cell activity, since Salmonella lipopolysaccharide failed to induce IFN production or enhance NK cell activity. These results suggest that IFN production by NK cells and the killing of bacteria-infected cells play an important role in host defense against facultative intracellular bacterial infections.     

Genetic control of in vitro natural cell-mediated activity against Salmonella typhimurium by intestinal and splenic lymphoid cells in mice.

In vitro natural anti-bacterial activity against Salmonella typhimurium by lymphocytes from Peyer's patches and spleens was assessed in several mouse strains. C3H/HeN and CBA/J mice, which are resistant to S. typhimurium infections, showed a natural anti-bacterial activity significantly higher than BALB/c, C57BL/10, C57BL/6 and C3H/HeJ mice, i.e. strains susceptible to the in vivo bacterial infection. In these susceptible strains and also in A/J mice, a significantly higher natural activity was observed in females compared to males. The sex control of natural anti-bacterial activity was further stressed by the fact that orchidectomy could induce a strong activity in low responder C57BL/10 male mice. With the exception of Beige mice, a low natural killer (NK) strain also with no natural activity against S. typhimurium in both sexes, the genetic distribution of natural anti-bacterial activity was extremely different from that of the NK activity. Thus, these results further stress the difference between natural anti-bacterial activity and NK cytotoxicity. Furthermore, our data establish a possible link, although with some exceptions, between in vivo susceptibility to S. typhimurium infections and in vitro natural activity against these bacteria.     

Immune response to Salmonella: location, location, location?

Successful immunity against Salmonella infections is dependent on the generation of CD4(+) T helper cells and to a lesser extent on antibody production and CD8(+) T cells. The cells within the lymphatic tissue of the gut are likely to be central for the orchestration of a proper and rapid response. The anatomical restriction of the pathogen may also determine the distribution of effector cells. In this issue of Immunity, McSorley et al. address both of these processes using identifiable CD4 T cells that are specific for Salmonella typhimurium. Such cells localize to the Peyer's patches of the small intestine when the bacteria are delivered orally.     

Enhancement of host resistance against Salmonella typhimurium infection by a diet supplemented with yogurt

The effect of a diet supplemented with yogurt containing live lactobacilli (LAB) - Lactobacillus bulgaricus and Streptococcus thermophilus - on the response of inbred mice to infection with Salmonella typhimurium was elaborated. The results of our experiments were consistent with the hypothesis that modifications of the microflora influence the adherence of S. typhimurium to intestinal mucosa, the natural antibacterial activity of the Peyer's patches lymphocytes, the accumulation of the macrophages in the liver, the proliferative responses of the splenocytes. The relationship between modifications of the immune response following ingestion of yogurt with live LAB and increased defense mechanisms was confirmed by the bacterial counts in livers and spleens and by the reduced mortality to S. typhimurium infection.     

The induction of interferon production in fibroblasts by invasive bacteria: a comparison of Salmonella and Shigella species

As the role of interferon (IFN) in host defense against facultative intracellular bacterial infections continues to expand, it has become increasingly important to understand what cell types can produce IFN following infection and/or interaction with these invasive bacteria. We have demonstrated previously that Shigella flexneri was able to induce high levels of IFN in primary cultures of human and murine fibroblasts following bacterial invasion. In this study, we examined the ability of Salmonella typhimurium to induce IFN production in different cell lines. S. typhimurium-infected primary cell cultures of mouse embryo-fibroblasts (MEF) were shown to produce high levels of IFN following bacterial challenge. In contrast to Shigella, Salmonella required a much lower multiplicity of infection for optimal IFN induction. Examination at the RNA level of IFN production by MEF following challenge with either bacteria revealed that the IFN produced was a mixture of IFN alpha and IFN beta (IFN alpha/beta), with IFN beta 1 as the predominant species. As previously demonstrated for Shigella, bacterial invasion of cells appeared to be required for the induction of IFN production by S. typhimurium. Salmonella rendered non-invasive by UV-treatment failed to induce IFN production in MEF. Furthermore, Salmonella LPS, when tested over a wide range of concentrations, was unable to induce IFN production in these cells. In contrast to MEF, human and murine continuous cell lines did not produce IFN following Salmonella challenge. These results taken together suggest that IFN may be a common factor involved in Salmonella and Shigella infections. Furthermore, IFN may play an important role in the front line host defense against these types of infections.     

In Vivo and In Vitro Antibacterial Activity of Conglutinin, a Mammalian Plasma Lectin

Conglutinin is a mammalian C-type lectin which agglutinates iC3b-coated erythrocytes. Ingram [13] found that euglobulin from bovine serum may confer partial protection against experimental infections in mice. We now present evidence that the protective activity in euglobulin against infections of BALB/c mice with Salmonella typhimurium is mediated by conglutinin. Conglutinin also demonstrated antibacterial activity against E. coli and S. typhimurium in vitro. The expression of this activity required the presence of heat-labile serum factors and peritoneal exudate or spleen cells, but not antibodies to the bacteria. Antibacterial activity was also demonstrated when the bacteria were pretreated with serum at 37 degrees C before incubation with conglutinin and cells. The activity of conglutinin was not observed when factor I-deficient or EDTA-treated serum was used instead of normal serum. The active peritoneal exudate or spleen cells showed adherence to plastic.     

Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium.

Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.     

Electrophoretic analysis of Salmonella typhi and other bacteria

A comparative study involving SDS-PAGE of Salmonella typhi and other Bacteria was conducted. Protoplasmic antigens of Salmonella typhi. Salmonella paratyphi A, Salmonella typhimurium, Proteus sp, Klebsiellas sp. Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus were separated and compared on SDS-PAGE followed by checking of their cross reactivity by gel diffusion using antisera raised against whole cell and lysates of Salmonella typhi. Lines of identity between Salmonella typhi, Salmonella paratyphi A and Salmonella typhimurium were observed. No lines of identity were seen among Salmonella typhi, Pseudomonas aeruginosa and Sfaphylococcus aureus.     

Antibacterial activity and mutagenicity studies of water-soluble phosphazene high polymers.

Eight water-soluble phosphazene high polymers, [NPR2]n (R, organic, water-solubilizing side-group; n, approx: 15,000) and the small-molecule counterparts of the polymers were examined for antibacterial activity against six different strains of bacteria (Escherichia coli, Salmonella typhimurium (TA 100), Salmonella pullorum, Streptococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa). Antibacterial testing was carried out by measuring zones of inhibition and changes in solution turbidity over time. In addition, the antibacterial activity of the surfaces of cross-linked poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels were investigated. A number of the high polymers, as well as the MEEP hydrogels, impeded bacterial growth. Only E. coli was unaffected by the phosphazenes. A possible explanation for the antibacterial character of the polymers is presented. The same compounds were monitored for potential mutagenic activity using the Salmonella typhimurium tester strains TA 100 and TA 98. None of the high polymers or their small-molecule analogues showed mutagenic activity in either strain of Salmonella at the concentrations tested. The use of these materials as coatings for artificial implants is discussed.     

Entry route of Salmonella typhimurium directs the type of induced immune response

Secretory immunoglobulin A (SIgA) represents a first line of defense against mucosal pathogens by limiting their entrance. By using different strains of Salmonella typhimurium that target the two mechanisms of bacterial entry (microfold cell [M cell]- or dendritic cell-mediated), we demonstrated here that the distribution of bacteria after oral infection directed the type of induced immune response. M cell-penetrating invasive, but not noninvasive, S. typhimurium was found in large numbers in Peyer's patches (PPs), leading to the activation of immune cells and the release of fecal IgA. In contrast, both strains of bacteria were equally capable of reaching the mesenteric lymph node and the spleen and inducing IgG responses. These data suggest that PPs are absolutely required for the initiation of an IgA response to Salmonella, whereas they are dispensable for a systemic response. This compartmentalization could allow the fast generation of both mucosal and systemic acquired immunity to pathogens.     

Screening of in vitro antibacterial activity of Terminalia chebula, Eclapta alba and Ocimum sanctum

Study of in vitro antibacterial activity of extracts from the plants T. chebula, E. alba and O. sanctum was carried out by the disk diffusion technique. All showed such activity against human pathogenic Gram positive and Gram negative bacteria. The activity against Salmonella organisms was shown only by T. chebula; against Shigella organisms by T. chebula and E. alha; but not by O. sanctum. The widest spectrum of antibacterial activity was shown by T. chebula. It was also most potent. The antibacterial spectrum of E. alba was in between that of T. chebula and O. sanctum. The narrowest spectrum of antibacterial activity was also most potent. The antibacterial spectrum of E. alba was in between that of T. chebula and O. sanctum. The narrowest spectrum of antibacterial activity was observed in O. sanctum.     

Host restriction phenotypes of Salmonella typhi and Salmonella gallinarum.

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.     

Invasiveness of enterobacteria related to the presence of high molecular weight plasmids

A study of 41 strains of Shigella, Escherichia coli, and Salmonella was performed. The presence of high-molecular-weight plasmids, invasiveness in HEp-2 cells and the ability to produce a positive Serény test were tested. Five of the seven strains of Salmonella typhimurium harboured a 62 Md plasmid and invaded HEp-2 cells. Two strains of S. typhimurium and a S. paratyphi-B strain lacked plasmids but were still invasive. Among the 27 strains of Shigella and enteroinvasive E. coli (EIEC) 25 strains harboured a high molecular weight plasmid. 27 of the Shigella/EIEC strains invaded HEp-2 cells and 25 produced a positive Serény test. One strain of Shigella sonnei was invasive in HEp-2 cells and gave a positive Serény test, but plasmids were not demonstrated. Of the eight non-enteropathogenic E. coli 5 strains harboured plasmids of 100 to 140 Md size; only one of the strains invaded HEp-2 cells; none of the strains produced a positive Serény test. The study shows that tests for pathogenicity (Serény test, HEp-2 cell test) are usually positive in the Shigella and EIEC group of bacteria; these bacteria also usually carry a high molecular weight plasmid. However, among non-enteropathogenic bacteria plasmids of 100 to 140 Md size can be observed without any correlation to invasive properties. Genetic information from gene loci located to plasmids and chromosomes is required to give a positive Serény test. The presence of high molecular weight plasmids does not seem to be necessary for expression of in vitro invasiveness of S. typhimurium.     

Effective antibacterial protection induced by a Listeria monocytogenes-specific T cell clone and its lymphokines.

The capacity of the murine Listeria monocytogenes-specific T cell clone 9-36-1 and of lymphokines derived therefrom to induce antibacterial protection in vivo was studied. Clone 9-36-1 was stimulated to proliferate and to produce lymphokines by in vitro culture with syngeneic accessory cells and heat-killed L. monocytogenes. Although 9-36-1 cells were highly active in vitro, intravenous transfer of the cells resulted in marginal protection against a systemic infection with L. monocytogenes. In contrast, 9-36-1 cells injected subcutaneously together with L. monocytogenes into the footpad induced marked protection in syngeneic, but not in allogeneic, mice. Multiplication of Salmonella typhimurium was not reduced by the T cell clone. Studies with 51Cr-labeled T cells indicated that the low activity of intravenously transferred cells was due to an altered migration pattern. Lymphokines produced by 9-36-1 cells in vitro induced protection against L. monocytogenes in syngeneic recipient mice. Lymphokine-induced protection was also demonstrable in allogeneic recipients and against S. typhimurium. These findings suggest that the L. monocytogenes-specific T cell clone 9-36-1, although unable to immigrate into sites of bacterial deposition, had retained its ability to mobilize antibacterial defense mechanisms once present at the site of reaction.     

Resistance and susceptibility of mice to bacterial infection: course of listeriosis in resistant or susceptible mice.

Within 3 h after oral challenge of mice with Salmonella typhimurium, foci of infection developed in the Peyer's patches of the small intestine. The numbers of organisms in the cecum, although in excess of those found in the small intestine, were not firmly associated with the cecal wall but were present largely in the cecum's contents. The Peyer's patches at first were remarkably incapable of eliminating even small numbers of Salmonella, but at about 7 days after infection developed the ability to eliminate a less virulent strain of S. typhimurium. Selected strains of Salmonella of varied virulence, and hybrid Escherichia coli/Salmonella typhimurium with varied O-antigens, revealed that those of low virulence could multiply within the intestinal Peyer's patches at nearly the same rate as a virulent strain, and the ability to multiply within the Peyer's patches was not dependent upon O-antigen type or smooth lipopolysaccharide. The ability of these strains to adhere to intestinal mucosa in vitro did not reflect on their ability to colonize the Peyer's patches, although strains of high in vitro adhesive ability appeared in greater numbers initially after oral challenge. Anti-O serum, ineffective in reducing the in vitro adhesive ability of virulent S. typhimurium, when given with the oral challenge prevented Peyer's patch colonization but was unable to prevent the appearance of a systemic infection. Anti-H serum, although effective in vitro in preventing adherence, had no effect in vivo. These experiments suggest that adhesiveness is neither essential nor sufficient for the virulence of Salmonella and that the usual development of a systemic infection after colonization of the small intestinal Peyer's patches may be subverted by the presence of O-antibody.     

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.     

Effect of human leukocyte interferon on invasiveness of Salmonella species in HEp-2 cell cultures.

The effect of human leukocyte interferon on the invasiveness of Salmonella and Shigella species in HEp-2 cell cultures was examined. The intracellular and extracellular bacteria were identified by a combination of Nomarski differential interference contrast microscopy and UV incident light microscopy applied on the same microscope. Pretreatment of HEp-2 cells with human leukocyte interferon reduced the number of Salmonella typhimurium and Salmonella paratyphi-B bacteria per cell and the proportion of cells containing bacteria in a dose-dependent manner. Maximum inhibitory effect was observed with ca. 100 U of interferon per ml. The inhibitory effect was neutralized with anti-human interferon globulin. Murine fibroblast interferon did not influence the invasiveness of Salmonella species. Invasiveness of Shigella flexneri was not influenced by treatment of cells with human interferon.     

Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage.

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.