Infection and Immunity

Bacterial and parasitic urinary tract infections(UTI) affect many, but our understanding of associated immunity remains poor. Prior work indicates interleukin-22(IL-22) is a key cytokine during infection of other epithelia. We hypothesized IL-22 is crucial during UTI. IL-22-null(KO) and wild-type(wt) mice underwent bladder wall injection with S. haematobium eggs or transurethral infection with uropathogenic E. coli UTI89. IL-22 and its soluble binding protein, IL-22BP, were increased in the bladder after S. haematobium egg injection. Genes typically induced by IL-22(CXCL2, REG3G[antimicrobial defense protein]], S100A8, S100A9, and Areg) were expressed at higher levels following S. haematobium egg injection. IL-22 stimulation of bladder tissue induced REG3B expression. IL-22 receptor alpha-1 expression was detectable in the urothelium by immunofluorescence and qPCR. Injection of S. haematobium eggs into IL-22-KO vs wt mice triggered differential expression of genes related to transferase activity(transferring alkyl or aryl groups) and epithelial cell development. Numerous uroplakin genes were downregulated in egg-injected, IL-22-KO mice versus their wt counterparts. These decreases in uroplakin expression suggest that, as in the gut, IL-22 is important for replenishing the epithelium during infection-related injury. Following transurethral infection with UTI89, IL-22-KO mice had lower bacterial counts in their urine, bladder, and kidneys. Giving stabilized IL-22 cytokine (IL-22-Fc) to UTI89-infected mice led to higher kidney bacterial counts and increased morbidity. Our data suggest that IL-22 is indeed important in urinary tract immunity, and may interfere with clearance of bacteria from the urinary tract, potentially through its role in maintenance of mature urothelium.

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA. Previous bioinformatic studies have indicated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. We confirm that hTLR9 colocalizes with chlamydial inclusions in the pro-monocytic cell line, U937. Utilizing HEK293 reporter cell lines, we demonstrate that purified genomic DNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in non-phagocytic HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion and exacerbated by the inhibition of lipooligosaccharide biosynthesis. The induction of aberrance / persistence also significantly alters Chlamydia-specific TLR9 signaling. Our observations support the hypothesis that chlamydial gDNA is released at appreciable levels by the bacterium during the conversion between its replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly.

The obligate intracellular bacterial pathogen Chlamydia trachomatis (Ctr) is reliant on an unusual developmental cycle consisting of two cell forms termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but does not replicate. The RB replicates in the host cell but is non-infectious. This developmental cycle is central to chlamydial pathogenesis. In this study we developed mathematical models of the chlamydial developmental cycle that account for potential factors influencing the timing of RB to EB cell type switching during infection. Our models predicted that two broad categories of regulatory signals for RB to EB development could be differentiated experimentally; an "intrinsic" cell autonomous program inherent to each RB or an "extrinsic" environmental signal to which RBs respond. To experimentally differentiate between these hypotheses, we tracked the expression of Ctr developmental specific promoters using fluorescent reporters and live cell imaging. These experiments indicated that EB production was not influenced by increased MOI or by superinfection, suggesting the cycle follows an intrinsic program that is not influenced by environmental factors. Additionally, live cell imaging of these promoter constructs revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a cell type gene expression progression with the promoters for euo and ihtA active in RBs, while the promoter for hctA was active in early EBs/intermediate cells and finally the promoters for the true late genes, hctB, scc2, and tarp active in the maturing EB. ImportanceChlamydia trachomatis is an obligate intracellular bacteria that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells Chlamydia must complete a multi cell type developmental cycle. The developmental cycle consists of two specialized cells; the EB which mediates infection of new cells and the RB which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB to EB cell type switching. We demonstrate that RB to EB development follows a cell autonomous program that does not respond to environmental cues. Additionally, we show that RB to EB development is a function of cell growth and cell division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacteriums pathogenesis.

Chlamydia muridarum has been used to study chlamydial pathogenesis since it induces mice to develop hydrosalpinx, a pathology observed in C. trachomatis-infected women. We identified a C. muridarum mutant that is no longer able to induce hydrosalpinx. In the current study, we evaluated the mutant as an attenuated vaccine. Following an intravaginal immunization with the mutant, mice were protected from hydrosalpinx induced by wild type C. muridarum. However, the mutant itself productively colonized the mouse genital tract and produced infectious organisms in vaginal swabs. Nevertheless, the mutant failed to produce infectious shedding in the rectal swabs following an oral inoculation. Importantly, mice orally inoculated with the mutant mounted transmucosal immunity against challenge infection of wild type C. muridarum in the genital tract. The protection was detected as early as day 3 following the challenge infection and the immunized mice were protected from any significant pathology in the upper genital tract. However, the same orally immunized mice failed to prevent the colonization of wild type C. muridarum in the gastrointestinal tract. The transmucosal immunity induced by the oral mutant was further validated in the airway. The orally vaccinated mice were protected from both lung infection and systemic toxicity caused by intranasally inoculated wild type C. muridarum although the same mice still permitted the gastrointestinal colonization by the wild type C. muridarum. These observations suggest that the mutant C. muridarum may be developed into an intracellular oral vaccine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Staphyloccocus aureus (S. aureus) is a major bacterial pathogen in orthopedic periprosthetic joint infection (PJI). S. aureus forms biofilms that promote persistent infection by shielding bacteria from immune cells and inducing an antibiotic-resistant metabolic state. We developed an in vitro system to study S. aureus biofilm interactions with primary human monocytes in the absence of planktonic bacteria. In line with previous in vivo data, S. aureus biofilm induced expression of inflammatory genes such as TNF and IL1B, and their anti-inflammatory counter-regulator IL-10. S. aureus biofilm also activated expression of PD-1 ligands that suppress T cell function, and of IL-1RA that suppresses differentiation of protective Th17 cells. Gene induction did not require monocyte:biofilm contact and was mediated by a soluble factor(s) produced by biofilm-encased bacteria that was heat resistant and > 3 kD in size. Activation of suppressive genes by biofilm was sensitive to suppression by Jak inhibition. These results support an evolving paradigm that biofilm plays an active role in modulating immune responses, and suggest this occurs via production of a soluble vita-PAMP. Induction of T cell suppressive genes by S. aureus biofilm provides insights into mechanisms that suppress T cell immunity in PJI, and suggest that anti-PD-1 therapy that is modeled on immune checkpoint blockade for tumors may be beneficial in PJI.

By far most urinary tract infections are caused by genetically diverse uropathogenic Escherichia coli (UPEC). Knowledge of the virulence mechanisms of UPEC is critical for drug development, but most studies focus on only a single strain of UPEC. In this study, we compared the virulence mechanisms of four antibiotic-resistant and highly pathogenic UPEC isolates in human blood monocyte-derived macrophages and a bladder epithelial cell (BEC) line: ST999, ST131, ST1981 and ST95. We found that while non-pathogenic E. coli strains are efficiently killed by macrophages in bactericidal single membrane vacuoles, the UPEC strains survive within double-membrane vacuoles. On side-by-side comparison, we found that whereas ST999 only carries Fe3+ importers, ST95 carries both Fe2+ and Fe3+ importers and the toxins haemolysin and colibactin. Moreover, we found that ST999 grows in the Fe3+ rich vacuoles of BECs and macrophages with concomitant increased expression of haem receptor chuA and the hydrogen peroxide sensor oxyR. In contrast, ST95 produces toxins in iron-depleted conditions similar to that of the urinary tract. Whereas ST95 also persist in the iron rich vacuoles of BECs, it produces colibactin in response to low Fe3+ contributing to macrophage death. Thus, iron regulates the contrasting toxicities of UPEC strains in macrophages and bladder epithelial cells due to low and high labile iron concentrations, respectively. Key findingsO_LIAntibiotics resistant uropathogenic E. coli strains ST999, ST131, ST1981, and ST95 survive within spacious double membrane vacuoles. Non-pathogenic E. coli strains XL1 blue and MG1655 are cleared in single membrane vacuoles in macrophages. C_LIO_LIST999 lacks Fe2+ importer and toxins, and grows in iron rich vacuoles of macrophages and bladder epithelial cells. C_LIO_LIST95 carries both Fe2+ and Fe3+ importers and grows in iron low conditions. C_LIO_LIST95 expresses toxins and induces cell death of infected macrophages, but not of bladder epithelial cells. C_LIO_LIBladder epithelial cells have a higher pool of labile iron than macrophages. Differential expression of virulence factors by ST999 and ST95 in bladder epithelial cells and macrophages is dependent on iron concentration. C_LI

Previous research demonstrated that Campylobacter jejuni encodes a heme utilization system that facilitates heme-dependent growth under iron-limiting conditions and that transcription of this system is induced during human infection. Despite these observations, it remained unknown whether the heme transport system is required for colonization and disease in a susceptible host. To address this, we created individual non-polar deletion mutants of each component of the heme transport system, as well as a total deletion of the inner membrane transporter, ChuBCD, and examined their ability to promote heme-dependent growth and iron uptake. From this work, we found that only the heme receptor, ChuA, was required for heme-dependent growth and iron acquisition, which supports earlier work of another group. Further, we examined whether intestinal colonization, immune activation, and pathology were altered during infection with these mutants. After establishing that elevated heme and chuABCD expression occurs during C. jejuni infection of IL-10-/- mice, we found that heme transport mutants exhibited significantly reduced fecal shedding and colonization of the cecum and colon. In addition, we found that neutrophil and macrophage recruitment and intestinal pathology often remained intermediately elevated despite decreased bacterial loads. These results suggest that heme utilization promotes efficient colonization and full pathogenicity in C. jejuni, but that neither is completely abrogated in its absence.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species N. lactamica and N. mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the pro-inflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed significant release of PG monomers. The mouse colonizer N. musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of pro-inflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Despite advances made in our understanding of Neisseria gonorrhoeae pathogenesis, factors dictating the divergent presentation of gonococcal disease observed between men and women, in vivo, remain unclear. Clinical data indicate that gonococcal pathogenesis of the female genital tract is influenced by steroid hormones. Notwithstanding, there are limited data addressing how steroid hormones modulate gonococcal pathogenesis. Hence, we investigated the effect(s) of physiological concentrations of estrogens and progestogens on N. gonorrhoeae viability and on complement-mediated infection of primary cervical cells. In contrast to previous studies that showed a bacteriostatic effect of non-physiological concentrations of steroid hormones on gonococci, our data indicate that physiological concentrations of estrogens and progestogens do not inhibit gonococcal growth in vitro or during infection of primary cervical cells. Estradiol promoted complement receptor 3 recruitment to the cervical cell surface and, thus, the ability of gonococci to associate with these cells. Progesterone-predominant assay conditions resulted in decreased expression of Opa proteins by gonococci, increased complement production by cervical cells, and increased iC3b opsonization of gonococci during cervical cell challenge. Collectively, our data support clinical observations and demonstrate that estrogens and progestogens distinctly modulate gonococcal cervical infection.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram- positive encapsulated bacterium that colonizes the gastrointestinal tract of 30-50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates expression of the gene, npx, which encodes a NADH peroxidase. GBS mutants with a npx deletion ({Delta}npx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invasion of reproductive tissues and is critical for inducing disease progression including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in {Delta}npx infected animals compared to those infected with wild type (WT)-GBS. Complementation in trans reversed this phenotype, indicating npx is critical for GBS survival and initiation of proinflammatory signaling in the gravid host.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is also a leading cause of preventable blindness. Limited mouse model of chronic C.tr infection are available to study the host immune response. We have developed in vivo mice models of acute and chronic infections for C. trachomatis to explore the significance of macrophage-directed response in mediating immune activation/suppression. During chronic and repeated infections, IFN{gamma} secretion from T cells is abated while TGF{beta} and IL-10 secretion is enhanced. An increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is also observed during chronic infection. It was observed that alternatively-activated macrophages with low CD40 expression promote Th2 and Treg differentiation and lead to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to alternately-activated phenotype. C. trachomatis infection prevents increase in CD40 expression. Low IFN{gamma}, as observed in chronic infection leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFN{gamma} responsiveness in macrophages via hampering IFN{gamma}RI and IFN{gamma}RII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFN{gamma} supplementation. Alternatively-activated macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even after IFN{gamma} supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFN{gamma}, thus promoting immunosupression. Thus, C. trachomatis modulate the innate immune cells attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF{kappa}B luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NF{kappa}B signalling. Although two isolates were associated with swarm sizes of >7 cm and NF{kappa}B activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NF{kappa}B signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4{+/-}1.8 to 46.1{+/-}2.5 fold, P = NS) NF{kappa}B signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

Chlamydia trachomatis has adapted to subvert signaling in epithelial cells to ensure successful intracellular development. Interferon-{gamma} (IFN{gamma}) produced by recruited lymphocytes signals through the JAK/STAT pathway to restrict chlamydial growth in the genital tract. However, during Chlamydia infection in vitro, addition of IFN{gamma} does not fully induce nuclear localization of its transcription factor STAT1 and target gene, IDO1. We hypothesize that this altered interferon response is a result of Chlamydia targeting components of the IFN{gamma}-JAK/STAT pathway. To assess the ability of replicating Chlamydia to dampen interferon signaling, HEp2 human epithelial cells were infected with C. trachomatis serovar L2 for 24 hours prior to exposure to physiologically relevant levels of IFN{gamma} (500 pg/mL). This novel approach enabled us to observe reduced phospho-activation of both STAT1 and its kinase Janus Kinase 2 (JAK2) in infected cells compared to mock-infected cells. Importantly, basal JAK2 and STAT1 transcript and protein levels were dampened by infection even in the absence of interferon, which could have implications for cytokine signaling beyond IFN{gamma}. Additionally, target genes IRF1, GBP1, APOL3, IDO1, and SOCS1 were not fully induced in response to IFN{gamma} exposure. Infection-dependent decreases in transcript, protein, and phosphoprotein were rescued when de novo bacterial protein synthesis was inhibited with chloramphenicol, restoring expression of IFN{gamma}-target genes. Similar Chlamydia-dependent dampening of STAT1 and JAK2 transcript levels were observed in infected END1 endocervical cells and in HEp2s infected with C. trachomatis serovar D, suggesting a conserved mechanism of dampening the interferon response by reducing the availability of key signaling components. ImportanceAs an obligate intracellular pathogen that has evolved to infect the genital epithelium, Chlamydia has developed strategies to prevent detection and antimicrobial signaling in its host to ensure its survival and spread. A major player in clearing Chlamydia infections is the inflammatory cytokine interferon-{gamma} (IFN{gamma}), which is produced by immune cells that are recruited to the site of infection. Reports of IFN{gamma} levels in vaginal and cervical swabs from Chlamydia-infected patients range from 1-350 pg/mL, while most in vitro studies of the effects of IFN{gamma} on chlamydial growth have used 15-85 fold higher concentrations. By using physiologically-relevant concentrations of IFN{gamma} we were able assess Chlamydias ability to modulate its signaling. We found that Chlamydia decreases the expression of multiple components that are required for inducing gene expression by IFN{gamma}, providing a possible mechanism by which C. trachomatis can attenuate the immune response in the female genital tract to cause long-term infections.

The inflammation induced by Chlamydia trachomatis infection in the female genital tract (FGT) can have severe consequences. Recent observations in women and mice infected with Chlamydia suggest that type I interferon (IFN-I) may have deleterious effects. This study aimed at elucidating the consequences of IFN-I production on C. trachomatis-induced inflammation in epithelial cells and the molecular pathway(s) involved. We showed that combination of IFN-I and Chlamydia resulted in a stronger induction of inflammation than Chlamydia alone, while IFN-I alone had no effect. Inhibiting AKT and mTOR, but not silencing STAT1, significantly attenuated the synergetic effect between IFN-I and bacteria on inflammation. Inhibition of ERK also blocked this synergistic effect, although ERK were not activated by IFN-I. We hypothesized that IFN-I enhanced the expression of a pathogen recognition receptor of the host, thereby potentiating detection of the bacteria and the subsequent inflammatory response. IFN-I, but not C. trachomatis infection, increased the expression of Toll-like receptor 3 (TLR3), and silencing or knocking-out TLR3 prevented the synergetic effect between infection and IFN-I. Furthermore, our data support the presence of dsRNA in infected cells and the activation of the MAPK/ERK and AP-1/ATF2 signaling cascades downstream of TLR3. Taken together, our data demonstrate that IFN-I exacerbates the host inflammatory response triggered by Chlamydia by increasing TLR3 expression and activation, leading to hyperinflammation. The identified signaling cascades represent potential targets for therapeutical intervention to limit tissue damage upon Chlamydia infection.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cause most bacterial sexually transmitted infections (STIs) worldwide. CT/NG co-infection is more common than expected due to chance, suggesting CT/NG interaction. However, CT/NG co-infection remains largely unstudied. Obligate intracellular CT has a characteristic biphasic developmental cycle consisting of two bacterial forms, infectious elementary bodies (EBs) and non-infectious, replicating reticulate bodies (RBs), which reside within host-derived, membrane-bound intracellular inclusions. Diverse stressors cause divergence from the normal chlamydial developmental cycle to an aberrant state called chlamydial persistence. Persistence can be induced by host-specific factors such as intracellular nutrient deprivation or cytokine exposure, and exogenous factors such as beta-lactam exposure, which disrupts RB to EB conversion. Persistent chlamydiae are atypical in appearance and, as such, are called aberrant bodies (ABs), but remain viable. The primary hallmark of persistence is reversibility of this temporary non-infectious state; upon removal of the stressor, persistent chlamydiae re-enter normal development, and production of infectious EBs resumes. The beta-lactam amoxicillin (AMX) has been shown to induce chlamydial persistence in a murine vaginal infection model, using the mouse pathogen C. muridarum (CM) to model human CT infection. This remains, to date, the sole experimentally tractable in vivo model of chlamydial persistence. Recently, we found that penicillinase-producing NG (PPNG) can alleviate AMX-induced CT and CM persistence in vitro. We hypothesized that PPNG vaginal co-infection would also alleviate AMX-induced CM persistence in mice. To evaluate this hypothesis, we modified the CM/AMX persistence mouse model, incorporating CM/PPNG co-infection. Contradicting our hypothesis, and recent in vitro findings, PPNG vaginal co-infection failed to alleviate AMX-induced CM persistence.

The impact of endemic parasitic infection on vaccine efficacy is an important consideration for vaccine development and deployment. We have examined whether intestinal infection with the natural murine helminth Heligmosomoides polygyrus bakeri alters antigen-specific antibody and cellular immune responses to oral and parenteral vaccination in mice. We found that oral vaccination of mice with a clinically relevant, live, attenuated, recombinant Salmonella vaccine that expresses chicken egg ovalbumin (Salmonella-OVA) disrupts ovalbumin-specific regulatory T cell networks in the gut associated lymphoid tissue and promotes T-effector responses to OVA. Chronic intestinal helminth infection significantly reduced Th1-skewed antibody responses to oral vaccination with Salmonella-OVA. Activated, adoptively-transferred, OVA-specific CD4+ T cells accumulated in draining mesenteric lymph nodes (MLN) of vaccinated mice, irrespective of their helminth-infection status. However, helminth infection increased the frequencies of adoptively-transferred OVA-specific CD4+ T cells producing IL-4 and IL-10 in the MLN. Chronic intestinal helminth infection also significantly reduced Th2-skewed antibody responses to parenteral vaccination with OVA adsorbed to alum. These findings suggest helminth-induced impairment of vaccine antibody responses may be driven by the development of IL-10-secreting CD4+ T regulatory cells. They also underscore the potential need to treat parasitic infection before mass vaccination campaigns in helminth-endemic areas.

Clostridioides difficile infection (CDI) is responsible for the majority of identifiable antibiotic-associated diarrhea. Women are more susceptible to CDI than men. In this study, we show that female mice developed more severe CDI than males. Furthermore, females in estrus developed only mild CDI 1-2 days later, while females in proestrus developed deadly disease. Mirroring the delayed effect of the estrous cycle, pre-infection prolactin levels formed a complex network with immunoglobulins and cytokines that affected early CDI severity one day after challenge. Similarly, pre-infection progesterone and luteinizing hormone formed a network that affected CDI two days after challenge. As expected, immune effectors early in the infection formed a hormone-independent network that concurrently correlated with CDI severity. Interestingly, early infection follicular stimulating hormone levels created a network that affected the CDI recovery phase. In summary, murine sexual hormones affect CDI progression by affecting the immune system both before and during disease progression.

A C. muridarum mutant designated as intrOv was evaluated as an intracellular Oral vaccine vector because it can induce protection in the genital tract following oral inoculation but does not elicit genital pathology following intravaginal infection. However, the mechanism of intrOvs attenuation is unclear. Here we report that few live organisms were recovered from vaginal swabs during the early stage of intrOv intravaginal infection in mice. At a low inoculating dose, an isogenic wild-type control strain established a productive infection, while intrOv failed to do so. Although a higher inoculating dose allowed intrOv and its control to productively infect mice, fewer live intrOv than the control organisms were recovered from the lower genital tract tissues on day 3 post-infection. By day 7, animals infected with intrOv or the control shed similar numbers of live organisms, suggesting the intrOvs deficiency on day 3 was transient. Consistently, intrOv reduced invasion of epithelial cells but maintained as robust intracellular replication as its control. Our results correlate intrOvs delay in infecting the lower genital tissues and reduction in invading epithelial cells with its attenuation in genital pathogenicity, laying the foundation for further revealing the mechanisms of the intrOvs attenuation in pathogenicity during genital tract infection.

Listeria monocytogenes is a Gram-positive pathogen that causes the severe foodborne disease listeriosis. Following oral infection of the host, L. monocytogenes disseminates from the gastrointestinal tract to peripheral organs, including the gallbladder, where it replicates to high densities. The gallbladder then becomes the primary bacterial reservoir and source of fecally excreted bacteria. Despite its importance in pathogenesis, little is known about how L. monocytogenes survives and replicates in the gallbladder. In this study, we assessed the L. monocytogenes genes required for growth and survival in ex vivo non-human primate gallbladders using a transposon sequencing approach. The screen identified 43 genes required for replication in the gallbladder, some of which were known to be important for virulence, and others had not been previously studied in the context of infection. We evaluated the roles of 19 genes identified in our screen both in vitro and in vivo, and demonstrate that most were required for replication in bile in vitro, for intracellular infection of murine cells in tissue culture, and for virulence in an oral murine model of listeriosis. Interestingly, strains lacking the mannose phosphoenolpyruvate-dependent phosphotransferase system (PTS) permeases Mpt and Mpo exhibited no defects in intracellular growth or intercellular spread but were significantly attenuated during murine infection. While the roles of PTS systems in vivo were not previously appreciated, these results suggest that PTS permeases are necessary for extracellular replication during infection. Overall, this study demonstrates that L. monocytogenes genes required for replication in the gallbladder also play broader roles in disease.

Urinary tract infections (UTIs) are a significant public health burden that impact millions of people every year and are highly prevalent among in hospital-acquired infections. Klebsiella pneumoniae is the second most common cause of UTIs after uropathogenic Escherichia coli (UPEC). Thus far, the molecular mechanisms underlying pathogenesis is better understood in UPEC than K. pneumoniae. UPEC is known to have fitness factors such as fimbrial adhesion and evasion of complement-mediated killing. In other infection types, K. pneumoniae fitness has been associated with mucoidy and diverse capsular serotypes. To establish K. pneumoniae virulence factors contributing to UTI, we examined how environmental cues regulate urovirulence-associated phenotypes in clinical K. pneumoniae UTI strains. These factors included capsular polysaccharide properties, hemagglutination, serum resistance, adherence to bladder epithelial cells, and in vivo fitness. We found that clinical K. pneumoniae UTI isolates phenotypes are highly heterogeneous and can change in response to human urine. Despite K. pneumoniae clinical isolates presenting heterogeneous fitness properties, all similarly colonize the urinary tract. These results suggest that additional fitness factors contribute to K. pneumoniae uropathogenesis. Identifying these shared fitness factors will provide mechanistic insights into Klebsiella uropathogenesis and reveal candidate therapeutic targets.