Search

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella .

The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro , and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica .

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (Δ prtM 138 - 213 , with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δ lgt 190 - 685 ). Moreover, mucus production was significantly greater in both wild-type-infected and Δ lgt 190 - 685 -infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δ lgt 190 - 685 mutant did still exhibit signs of disease. In contrast, only the Δ prtM 138 - 213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δ lgt 190 - 685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

Transcriptional profiling of Campylobacter jejuni during colonization of the chick cecum identified 59 genes that were differentially expressed in vivo compared with the genes in vitro. The data suggest that C. jejuni regulates electron transport and central metabolic pathways to alter its physiological state during establishment in the chick cecum.

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5 + cells to flagellin induced elevated levels of chicken interleukin-1β (chIL-1β) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1β mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken.

The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148: 473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5 , flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.

To efficiently colonize and persist in the lower respiratory tract, bacteria must survive multiple host immune mechanisms.Bordetella bronchisepticais a gram-negative respiratory pathogen that naturally infects mice and persists in the lower respiratory tract for up to 49 days postinoculation. In this work, we examined the effect of mutation of thepagPgene on the persistence ofB. bronchisepticain the lower respiratory tract of mice. ThepagPgene encodes a palmitoyl transferase that is responsible for the addition of a palmitoyl group to the lipid A region ofB. bronchisepticalipopolysaccharide. Data presented here confirm that aB. bronchisepticaΔpagPmutant demonstrates defective persistence in the lower respiratory tract of wild-type mice. We hypothesized that the defective persistence of theB. bronchisepticaΔpagPmutant was due to an increased susceptibility of this mutant to a host immune response. In vivo data indicate that both B cells and the complement component C3 are required for the reduced bacterial numbers of the ΔpagPmutant on day 14 postinoculation. In addition, an in vitro complement killing assay demonstrated thatB. bronchisepticaexhibitspagP-dependent resistance to antibody-mediated complement killing at low concentrations of immune serum. Taken together, these results suggest thatpagPis required forB. bronchisepticato resist antibody-mediated complement lysis during respiratory infection.

Poultry meat and eggs contaminated withSalmonella entericaserovar Enteritidis orSalmonella entericaserovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective againstSalmonellainfection in chickens has not been characterized at the molecular level. In the present study, bacterial colonization, development of pathological lesions, and proinflammatory cytokine and chemokine gene expression were investigated in the liver, spleen, jejunum, ileum, and cecal tonsils in newly hatched chickens 6, 12, 24, and 48 h after oral infection withSalmonellaserovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereasSalmonellastarted to appear in the liver only from 24 h postinfection. Large numbers of heterophils, equivalent to neutrophils in mammals, and inflammatory edema could be seen in the lamina propria of the intestinal villi and in the liver. Interleukin 8 (IL-8), K60 (a CXC chemokine), macrophage inflammatory protein 1 β, and IL-1β levels were significantly upregulated in the intestinal tissues and in the livers of the infected birds. However, the spleens of the infected birds show little or no change in the expression levels of these cytokines and chemokines. Increased expression of the proinflammatory cytokines and chemokines (up to several hundred-fold) correlated with the presence of inflammatory signs in those tissues. This is the first description of in vivo expression of chemokines and proinflammatory cytokines in response to oral infection withSalmonellain newly hatched chickens.

A major Salmonella component involved in cellular activation is the lipopolysaccharide (LPS) molecule which can act as a dendritic cell (DC) stimulator. The structure of the lipid A domain of the LPS molecule dictates its immunostimulatory capacity with various cell types. In this study, the role of lipid A as an integral component of Salmonella in stimulating murine DCs was studied by using a Salmonella enterica serovar Typhimurium lpxM mutant with defective lipid A. This study revealed that a mutation in lpxM did not significantly affect the ability of bacteria to activate DCs. Although the lpxM mutant less tumor necrosis factor alpha, interleukin-1β, and inducible nitric oxide synthase than the parental strain, this was only seen at lower multiplicities of infection (MOIs). Both strains upregulated surface molecule expression on DCs and augmented the T-cell-stimulating capacity of these cells in an MOI-independent manner. Thus, the lpxM mutation did not appear to affect the stimulatory capacity of the Salmonella mutant.

Campylobacter jejuni can cause an enteritis that is associated with an acute inflammatory response at the gut epithelial surface. The signals inducing inflammation are unknown. C. jejuni can penetrate the intestinal epithelial barrier and may then interact with leucocytes, potentially inducing proinflammatory responses. To investigate this, we studied the interaction of C. jejuni with the human monocytic cell line THP-1 and show that a range of proinflammatory cytokines and chemokines is induced. These include interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and tumor necrosis factor alpha. Responses can be induced by killed Campylobacter as well as live bacteria and do not depend on the cytolethal distending toxin. C. jejuni infection of THP-1 cells triggers both nuclear translocation of functional NF-κB and stimulation of IL-1α, indicating that NF-κB-dependent and -independent stimulation is occurring. The extent of proinflammatory cytokine stimulation suggests that monocytes might significantly contribute to intestinal inflammation and disease pathology.

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection.

Sublethal infection of C57BL/6 mice with Salmonella enterica serovar Typhimurium M525P initiates a strong inflammatory response. We measured organ expression of mRNA for Toll-like receptors and their associated signaling molecules during S. enterica serovar Typhimurium infection. During infection, the Toll-lie receptor 1 (TLR1), TLR2, and TLR9 mRNA levels increased, while TLR6 mRNA expression decreased.

Bordetella avium causes an upper respiratory tract disease (bordetellosis) in avian species. Commercially raised turkeys are particularly susceptible. Like other pathogenic members of the genus Bordetella ( B. pertussis and B. bronchiseptica ) that infect mammals, B. avium binds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease. These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused by B. pertussis . One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting that B. avium does not express a number of the factors known to be associated with virulence in the other two Bordetella species. Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence in B. pertussis and B. bronchiseptica ( bvgS , fhaB , fhaC , and fimC ) were identified. None of the four B. avium genes had been previously identified as encoding factors associated with virulence, and three of the insertions (in fhaB , bvgS , and fimC ) were in genes or gene clusters inferred as being absent or incomplete in B. avium , based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products. We further found that the genotypic arrangements of most of the B. avium orthologues were very similar in all three Bordetella species. In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations. Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species.

Bordetella pertussis,Bordetella parapertussis, andBordetella bronchisepticaare closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of thewlblocus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have usedwlbdeletion (Δwlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Δwlbmutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5−/−mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects.B. pertussisandB. parapertussis Δwlbmutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, theB. bronchiseptica Δwlbstrain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting thatwlb-dependent LPS modifications inB. bronchisepticamodulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.

Salmonella typhimurium strains harboring independent, defined mutations in aroA , invA , ssrA , or msbB were assessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ileal loops. Three wild-type strains of S. typhimurium , TML, HWSH, and SL1344, and two mutant strains, S. typhimurium SL1344 ssrA and S. typhimurium SL1344 msbB , consistently induced fluid accumulation in the lumen of loops and inflammation of loop-associated tissues. In contrast, three different S. typhimurium aroA strains and an invA mutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops. However, the S. typhimurium aroA strains did induce an inflammatory infiltrate and some local villus-associated damage, but the invA mutant did not. Histologically, wild-type S. typhimurium , S. typhimurium SL1344 ssrA , and S. typhimurium SL1344 msbB demonstrated more severe effects on villus architecture than S. typhimurium aroA strains, whereas S. typhimurium invA -infected loops showed no detectable damage. This suggests that villus damage most likely contributes to fluid accumulation within the loop.

The attenuated S. typhimurium SL3261 ( aroA ) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A d /I-E d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80 + and CD11b + spleen cells.