Description: Factors and mechanisms determining the differences in virulence and host specificity between the zoonotic agents Chlamydia psittaci and Chlamydia abortus are still largely unknown. In the present study, two strains were compared for their invasiveness, virulence, and capability of eliciting an immune response in chicken embryos. On breeding day 10, embryonated chicken eggs were inoculated with 5 x 10 4 inclusion-forming units. As shown by immunohistochemistry and quantitative real-time PCR, C. psittaci displayed a significantly better capability of disseminating in the chorioallantoic membrane (CAM) and internal organs than C. abortus . The higher infectious potential of C. psittaci in birds was underlined by significantly higher mRNA expression rates of essential chlamydial genes, such as incA , groEL (in CAM, liver, and spleen), cpaf , and ftsW (in CAM). Although the immune responses to both pathogens were similar, C. psittaci elicited higher macrophage numbers and a stronger expression of a subset of immune-related proteins. The data imply that invasiveness of Chlamydia spp. and propagation in the host are not solely dependent on the level of host immune response but, even to a greater extent, on the expression of bacterial factors related to virulence. The fact that C. psittaci has coped far better than C. abortus with the avian embryo's response by upregulating essential genes may be a key to understanding the mechanisms underlying host adaptation and etiopathology.

Description: Clostridium difficile can infect the large intestine and cause colitis when the normal intestinal microbiota is altered by antibiotic administration. Little is known about the innate immune signaling pathways that marshal inflammatory responses to C. difficile infection and whether protective and pathogenic inflammatory responses can be dissociated. Toll-like receptors predominantly signal via the MyD88 adaptor protein and are important mediators of innate immune signaling in the intestinal mucosa. Here, we demonstrate that MyD88-mediated signals trigger neutrophil and CCR2-dependent Ly6C hi monocyte recruitment to the colonic lamina propria (cLP) during infection, which prevent dissemination of bystander bacteria to deeper tissues. Mortality is markedly increased in MyD88-deficient mice following C. difficile infection, as are parameters of mucosal tissue damage and inflammation. Antibody-mediated depletion of neutrophils markedly increases mortality, while attenuated recruitment of Ly6C hi monocytes in CCR2-deficient mice does not alter the course of C. difficile infection. Expression of CXCL1, a neutrophil-recruiting chemokine, is impaired in the cLP of MyD88 –/– mice. Our studies suggest that MyD88-mediated signals promote neutrophil recruitment by inducing expression of CXCL1, thereby providing critical early defense against C. difficile -mediated colitis.

Description: Lack of an adequate animal model of Plasmodium falciparum severe malarial anemia (SMA) has hampered the understanding of this highly lethal condition. We developed a model of SMA by infecting C57BL/6 mice with P. chabaudi followed after recovery by P. berghei infection. P. chabaudi / P. berghei -infected mice had an initial 9- to 10-day phase of relatively low parasitemia and severe anemia, followed by a second phase of hyperparasitemia, more profound anemia, reticulocytosis, and death 14 to 21 days after infection. P. chabaudi / P. berghei -infected animals had more intense splenic hematopoiesis, higher interleukin-10 (IL-10)/tumor necrosis factor alpha and IL-12/gamma interferon (IFN-) ratios, and higher antibody levels against P. berghei and P. chabaudi antigens than P. berghei -infected or P. chabaudi -recovered animals. Early treatment with chloroquine or artesunate did not prevent the anemia, suggesting that the bulk of red cell destruction was not due to the parasite. Red cells from P. chabaudi / P. berghei -infected animals had increased surface IgG and C3 by flow cytometry. However, C3 –/– mice still developed anemia. Tracking of red cells labeled ex vivo and in vivo and analysis of frozen tissue sections by immunofluorescence microscopy showed that red cells from P. chabaudi / P. berghei -infected animals were removed at an accelerated rate in the liver by erythrophagocytosis. This model is practical and reproducible, and its similarities with P. falciparum SMA in humans makes it an appealing system with which to study the pathogenesis of this condition and explore potential immunomodulatory interventions.

Description: Clostridium perfringens type B causes enteritis and enterotoxemia in domestic animals. By definition, these bacteria must produce alpha toxin (CPA), beta toxin (CPB) and epsilon toxin (ETX) although most type B strains also produce perfringolysin O (PFO) and beta2 toxin (CPB2). A recently identified Agr-like quorum-sensing (QS) system in C. perfringens controls all toxin production by surveyed type A, C, and D strains, but whether this QS is involved in regulating toxin production by type B strains has not been explored. Therefore, the current study introduced agrB null mutations into type B strains CN1795 and CN1793. Both type B agrB null mutants exhibited reduced levels of CPB, PFO, and CPA in their culture supernatants, and this effect was reversible by complementation. The reduced presence of CPB in culture supernatant involved decreased cpb transcription. In contrast, the agrB null mutants of both type B strains retained wild-type production levels of ETX and CPB2. In a Caco-2 cell model of enteritis, culture supernatants of the type B agrB null mutants were less cytotoxic than supernatants of their wild-type parents. However, in an MDCK cell in vitro model for enterotoxemic effects, supernatants from the agrB null mutants or wild-type parents were equally cytotoxic after trypsin activation. Coupling these and previous results, it is now evident that strain-dependent variations exist in Agr-like QS system regulation of C. perfringens toxin production. The cell culture results further support a role for trypsin in determining which toxins contribute to disease involving type B strains.

Description: Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN- stimulation of uninfected and M. avium subsp. paratuberculosis -infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN- signaling, in uninfected monocytes. In contrast, IFN- stimulation of M. avium subsp. paratuberculosis -infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN- to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN- receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN- receptors SOCS1 and SOCS3 as well as decreased expression of IFN- receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis -infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

Description: Placental infection with Plasmodium falciparum is associated with increased levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-), and previous studies have associated increased levels of these cytokines with low birth weight (LBW), especially for malaria-infected primigravidae. To define the contribution of TNF-α and IFN- networks to placental-malaria-associated LBW, we measured chemokines induced by TNF-α and IFN- and related them to birth weight in a birth cohort of 782 mother-infant pairs residing in an area of P. falciparum holoendemicity in Tanzania. Among primigravidae, levels of CCL2, CXC ligand 9 (CXCL9), and CXCL13 were significantly higher during malaria infection in both the placenta and peripheral blood. Placental CXCL9 and CXCL13 levels were also higher in placental blood from secundigravidae and multigravidae. In multivariate analyses adjusted for known predictors of birth weight, malaria-infected primigravidae with placental CXCL9 levels in the lowest tertile gave birth to babies who weighed 610 g more than babies born to mothers with high CXCL9 levels. CXCL9 expression is induced by IFN-, and the strong association between birth weight and placental CXCL9 is consistent with previous observations relating IFN- to poor pregnancy outcomes.

Description: In this paper, we show that the larvae of the greater wax moth, Galleria mellonella , can be used as a model to study enteropathogenic Escherichia coli (EPEC) virulence. G. mellonella larvae are killed after infection with EPEC type strain E2348/69 but not by an attenuated derivative that expresses diminished levels of the major virulence determinants or by a mutant specifically defective in type III secretion (T3S). Infecting EPEC inhabit the larval hemocoel only briefly and then become localized to melanized capsules, where they remain extracellular. Previously, it was shown that mutations affecting the Cpx envelope stress response lead to diminished expression of the bundle-forming pilus (BFP) and the type III secretion system (T3SS). We demonstrate that mutations that activate the Cpx pathway have a dramatic effect on the ability of the bacterium to establish a lethal infection, and this is correlated with an inability to grow in vivo . Infection with all E. coli strains led to increased expression of the antimicrobial peptides (AMPs) gloverin and cecropin, although strain- and AMP-specific differences were observed, suggesting that the G. mellonella host perceives attenuated strains and Cpx mutants in unique manners. Overall, this study shows that G. mellonella is an economical, alternative infection model for the preliminary study of EPEC host-pathogen interactions, and that induction of the Cpx envelope stress response leads to defects in virulence.

Description: Mycobacterium tuberculosis remains a significant global pathogen, causing extensive morbidity and mortality worldwide. This bacterium persists within granulomatous lesions in a poorly characterized, nonreplicating state. The two-component signal transduction systems MprAB and DosRS-DosT (DevRS-Rv2027c) are responsive to conditions likely to be present within granulomatous lesions and mediate aspects of M. tuberculosis persistence in vitro and in vivo . Here, we describe a previously uncharacterized locus, Rv1813c-Rv1812c , that is coregulated by both MprA and DosR. We demonstrate that MprA and DosR bind to adjacent and overlapping sequences within the promoter region of Rv1813c and direct transcription from an initiation site located several hundred base pairs upstream of the Rv1813 translation start site. We further show that Rv1813c and Rv1812c are cotranscribed, and that the genomic organization of this operon is specific to M. tuberculosis and Mycobacterium bovis . Although Rv1813c is not required for survival of M. tuberculosis in vitro , including under conditions in which MprAB and DosRST signaling are activated, an M. tuberculosis Rv1813c mutant is attenuated in the low-dose aerosol model of murine tuberculosis, where it exhibits a lower bacterial burden, delayed time to death, and decreased ability to stimulate proinflammatory cytokines interleukin-1β (IL-1β) and IL-12. Interestingly, overcomplementation of these phenotypes is observed in the M. tuberculosis Rv1813c mutant expressing both Rv1813c and Rv1812c , but not Rv1813c alone, in trans . Therefore, Rv1813c and Rv1812c may represent general stress-responsive elements that are necessary for aspects of M. tuberculosis virulence and the host immune response to infection.

Description: The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa , (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.