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Urinary tract infection (UTI) is one of the most prevalent bacterial infections, particularly in women, children, and the elderly. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Uropathogens are directly instilled in the urinary bladder, bypassing the lower urogenital tract, in the widely used murine model of UTI. We assessed whether vaginal inoculation of UPEC led to UTI and how stages of the estrous cycle would impact bacterial colonization in mice. Mice in proestrus, estrus, metestrus, and diestrus were identified by vaginal cytology and inoculated with UPEC in the vaginal tract. Mice were euthanized 1 day after infection, and bacterial loads in the urogenital tract, liver, and spleen were enumerated. Mice in estrus exhibited the highest and most consistent UPEC burdens in all organs, except the bladder. Vaginal inoculation resulted in bladder colonization in a UPEC strain-specific manner. In contrast, transurethral inoculation of UPEC led to bladder colonization. Importantly, inoculation by both routes led to vaginal and uterine colonization and concomitant systemic dissemination to the spleen and liver. The kinetics of bacterial colonization over 2 weeks following vaginal inoculation was comparable in the urogenital tract. Tissue sections revealed the induction of vaginitis and cystitis upon the vaginal instillation of UPEC. In summary, vaginal inoculation of UPEC in mice during estrus represents a novel approach to investigate infection of the kidneys and genital tract and systemic dissemination from the urogenital tract. Our findings suggest that estrogen primes the urogenital tract to create a conducive milieu for UPEC colonization.

Stealthy intracellular bacterial pathogens are known to establish persistent and sometimes lifelong infections. Some of these pathogens also have a tropism for the reproductive system, thereby increasing the risk of reproductive disease and infertility. To date, the pathogenic mechanism involved remains poorly understood. Here, we demonstrate that Brucella abortus, a notorious reproductive pathogen, has the ability to infect the nonpregnant uterus, sustain infection, and induce inflammatory changes during both acute and chronic stages of infection. In addition, we demonstrated that chronically infected mice had a significantly reduced number of pregnancies compared to naive controls. To investigate the immunologic mechanism responsible for uterine tropism, we explored the role of regulatory T cells (Tregs) in the pathogenesis of Brucella abortus infection. We show that highly suppressive CD4(+)FOXP3(+)TNFR2(+) Tregs contribute to the persistence of Brucella abortus infection and that inactivation of Tregs with tumor necrosis factor receptor II (TNFR2) antagonistic antibody protected mice by significantly reducing bacterial burden both systemically and within reproductive tissues. These findings support a critical role of Tregs in the pathogenesis of persistence induced by intracellular bacterial pathogens, including B. abortus. Results from this study indicate that adverse reproductive outcomes can occur as sequelae of chronic infection in nonpregnant animals and that fine-tuning Treg activity may provide novel immunotherapeutic and prevention strategies against intracellular bacterial infections such as brucellosis.

Salmonella enterica serotype Typhi, the etiological agent of typhoid fever, produces the Vi capsular antigen, a virulence factor absent in Salmonella enterica serotype Typhimurium. Previous studies suggest that the capsule-encoding viaB locus reduces inflammatory responses in intestinal tissue; however, there are currently no data regarding the in vivo expression of this locus. Here we implemented direct and indirect methods to localize and detect Vi antigen expression within polarized intestinal epithelial cells and in the bovine ileal mucosa. We report that tviB , a gene necessary for Vi production in S. Typhi, was significantly upregulated during invasion of intestinal epithelial cells in vitro. During infection of bovine ligated loops, tviB was expressed at levels significantly higher in calf tissue than those in the inoculum. The presence of the Vi capsular antigen was detected in calf ileal tissue via fluorescence microscopy. Together, these results support the concept that expression of the Vi capsular antigen is induced when S. Typhi transits from the intestinal lumen into the ileal mucosa.

Salmonella entericaserotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgKmutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljBmutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3α and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.

Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella -induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus . Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus -induced placentitis.

Salmonella enterica serotype Typhi is a strictly human adapted pathogen that does not cause disease in nonprimate vertebrate hosts, while Salmonella enterica serotype Typhimurium is a broad-host-range pathogen. Serotype Typhi lacks some of the proteins (effectors) exported by the invasion-associated type III secretion system that are required by serotype Typhimurium for eliciting fluid secretion and inflammation in bovine ligated ileal loops. We investigated whether the remaining serotype Typhi effectors implicated in enteropathogenicity (SipA, SopB, and SopD) are functionally exchangeable with their serotype Typhimurium homologues. Serotype Typhi elicited fluid accumulation in bovine ligated ileal loops at levels similar to those elicited by a noninvasive serotype Typhimurium strain (the sipA sopABDE2 mutant) or by sterile culture medium. However, introduction of the cloned serotype Typhi sipA , sopB , and sopD genes complemented the ability of a serotype Typhimurium sipA sopABDE2 mutant to elicit fluid secretion in bovine ligated ileal loops. Introduction of the cloned serotype Typhi sipA , sopB , and sopD genes increased the invasiveness of a serotype Typhimurium sipA sopABDE2 mutant for human colon carcinoma epithelial (HT-29 and T84) cells and bovine kidney (MDBK) cells. Translational fusions between the mature TEM-1 β-lactamase reporter and SipA or SopD demonstrated that serotype Typhi translocates these effectors into host cells. We conclude that the inability of serotype Typhi to cause fluid accumulation in bovine ligated ileal loops is not caused by a functional alteration of its SipA, SopB, and SopD effector proteins with respect to their serotype Typhimurium homologues.

The centisome 63 type III secretion system (T3SS-1) encoded by Salmonella pathogenicity island 1 (SPI1) mediates invasion of epithelial cells by Salmonella enterica serotype Typhimurium. Characterization of mutants lacking individual genes has revealed that T3SS-1 secreted proteins (effectors) SopE2 and SopB are required for invasion while the SipA protein accelerates entry into cells. Here we have revisited the question of which T3SS-1 effectors contribute to the invasion of epithelial cells by complementing a strain lacking all of the effector genes that are required to cause diarrhea in a calf (a sipA sopABDE2 mutant). Introduction of either the cloned sipA , the cloned sopB , or the cloned sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for nonpolarized HT-29 cells. However, a contribution of sopA or sopD to invasion was not apparent when invasion assays were performed with the nonpolarized colon carcinoma cell lines T84 and HT-29. In contrast, introduction of either the sopA , the sopB , the sopD , or the sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells. Furthermore, introduction of a plasmid carrying sipA and sopB increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells significantly compared to the introduction of plasmids carrying only sipA or sopB . We conclude that SipA, SopA, SopB, SopD, and SopE2 contribute to S. enterica serotype Typhimurium invasion of epithelial cells in vitro.

Salmonella enterica serotype Typhimurium causes enterocolitis in humans, a localized infection characterized by diarrhea and by pathological changes that are most severe in the intestine and mesenteric lymph nodes (24, 25). In contrast, S. enterica serotype Typhi causes typhoid fever, a systemic infection characterized by fever, while diarrhea is considered to be an insignificant symptom developing in only one-third of patients (28). A striking difference between the host responses elicited during infections with serotype Typhimurium and serotype Typhi in humans is the type of inflammation observed in the intestine. Analysis of biopsy samples reveals that inflammation in the intestines of patients infected with serotype Typhimurium is characterized by an infiltrate that is composed primarily of polymorphonuclear (PMN) cells (6, 27), while inflammation in typhoid fever patients is caused predominantly by infiltration with monocytes (21, 39). Similarly, the predominant cell type (representing 95% of fecal leucocytes) in stools from typhoid fever patients is mononuclear, whereas PMN cells predominate (representing 75% of fecal leucocytes) in stool samples from enterocolitis patients (13).

Recent studies on the molecular pathogenesis of Salmonella enterica serotype Typhimurium-induced enterocolitis using tissue culture models and the neonatal calf model have led to an improved understanding of key events occurring during the complex series of host-pathogen interactions leading to

TheBrucella abortus virBlocus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whethervirBis involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-typeB. abortusbut not recovery of avirBmutant. In mice lacking either NADPH oxidase or iNOS, however,B. abortusinfected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. AvirBmutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.

Bovine macrophages underwent apoptosis as a result of infection with aMycobacterium bovisfield strain. Macrophages infected with a multiplicity of infection (MOI) of 25:1 developed chromatin condensation and DNA fragmentation at 4 h and 8 h, respectively, whereas changes in chromatin condensation induced by MOIs of 10:1 and 1:1 required more time and had a reduced number of apoptotic cells. Not only infected macrophages underwent apoptosis, but also uninfected bystander macrophages became apoptotic. Increased differential expression of thymosin β-10 was identified inM. bovis-infected bovine macrophages by differential display reverse transcriptase PCR. Phagocytosis of latex beads had no effect on the expression of thymosin β-10, whereas bacterial suspensions upregulated thymosin β-10 expression, suggesting thatM. bovisor mycobacterial products are essential in the process. Heat-inactivatedM. bovisinduced a slight increase in thymosin β-10 mRNA, whereas live virulent and attenuatedM. bovisorganisms increased the gene expression almost twofold. A mouse macrophage cell line (RAW 264.7) overexpressing the bovine thymosin β-10 transgene had spontaneous apoptosis at a higher rate (66.5%) than parental cells (4.7%) or RAW cells harboring the empty vector (22.8%). The apoptotic rates of the overexpressing cells were significantly higher when compared with both the empty vector transfected (P< 0.01) and parental cells (P< 0.001). Our evidence suggests that upregulation of thymosin β-10 inM. bovis-infected macrophages is linked with increased cell death due to apoptosis.

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium ( S. typhimurium ) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10 10 CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP , with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis . These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

It was previously demonstrated thatSalmonella entericaserovar Typhimurium induces cell death with features of apoptosis in murine macrophages. Mice infected withSalmonellaserovar Typhimurium develop systemic disease without diarrhea, whereas the infection in cattle and in humans is localized and characterized by diarrhea. Considering these clinical disease expression differences between mice and cattle, we investigated whether serovar Typhimurium is cytotoxic for bovine macrophages. Macrophages infected with serovar Typhimurium grown in the logarithmic phase quickly underwent cell death. Macrophages infected with stationary-phase cultures or with a mutant lackingsipBunderwent no immediate cell death but did develop delayed cytotoxicity, undergoing cell death between 12 and 18 h postinfection. Both pathways were temporarily blocked by the general caspase inhibitor Z-VAD-Fmk and by the caspase 1 inhibitor Z-YVAD-Fmk. Comparisons of macrophages from cattle naturally resistant or susceptible to intracellular pathogens indicated no differences between these two genetic backgrounds in terms of susceptibility to serovar Typhimurium-induced cell death. We conclude thatSalmonellaserovar Typhimurium induces cell death in bovine macrophages by two distinct mechanisms, earlysipB-mediated and delayedsipB-independent mechanisms.