Infection and Immunity最新文献

Relapsing fever Borrelia (RFB) employs antigenic variation to alter its surface protein structure in response to host immune pressure. This process occurs by the single translocation of archived variable major protein (Vmp) pseudogenes into a vmp expression locus. Borrelia miyamotoi, phylogenetically grouped with RFB, has the genetic makeup for antigenic variation, but it has not been determined whether B. miyamotoi can create new variant serotypes in vivo. We inoculated mice with a non-clonal parental B. miyamotoi CT13-2396 strain with a known Vmp majority serotype with spirochete isolation at various days post-infection. The vmp that determined the reisolated variant serotype was identified by PCR of the expression locus followed by DNA sequencing of the amplified product. For each mouse reisolate, new variants replaced the parent majority serotype. Moreover, some mice produced additional variant reisolates days apart, indicative of the presentation seen in relapsing fever infections. Infection of mice with a clonal population resulted in the elimination of the inoculated serotype and isolation of new variants. Mouse serum obtained following infection revealed IgM antibodies reactive to the parent Vmp serotype, suggesting that the immune response eliminated or greatly reduced the majority population. These results demonstrated that B. miyamotoi reisolated from infected mice exhibited serotype populations differing from the inoculated strain, indicating the spirochetes underwent antigenic variation to evade the host's immune response. However, whether the observed variation occurred by way of outgrowth of minority populations or by translocation of archived pseudogenes to the expression locus creating new variants awaits further study.

Enteroaggregative Escherichia coli (EAEC) is a common cause of diarrhea worldwide and is associated with growth faltering in developing countries. EAEC are defined by a characteristic adherence pattern mediated by the aggregative adherence fimbriae (AAFs). Despite the critical role of AAF in the definition of the EAEC pathotype, it is not known what host molecules mediate adherence and EAEC pathogenesis during infection of the human gastrointestinal tract. Multiple receptor candidates have been proposed based on in vitro experimentation. We propose that AAFs interact with multiple receptors during colonization of the human gastrointestinal mucosa, and we hypothesize that structural features of the AafA protein (the major subunit of AAF variant II produced by EAEC strain 042) promote these diverse interactions. In this study, we utilize a panel of AafA variants encoding single amino acid substitutions to understand the role of individual residues in biofilm formation as well as adherence to mucin, fibronectin, and human intestinal cells. We identify both charged and uncharged residues that participate in these interactions, and these residues cluster in two regions of the protein that may define a binding pocket at the junction of polymerized subunits. Although both bovine submaxillary mucin and human fibronectin are sialylated molecules, adherence to mucin is diminished by the removal of sialic acid residues while adherence to fibronectin is not, suggesting that the mechanisms of adherence to these molecules are distinct. Overall, our data provide insight into the structural features that determine AAF/II binding to mucin, sialic acid, and human intestinal cells.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningitis in immunocompromised individuals. Both host- and pathogen-specific factors are known to affect patient outcome, and recent studies showed that strain-specific differences in C. neoformans clinical isolates can influence virulence in A/J mice. However, it is unclear how the immunologic and genetic background of inbred mouse strains affects disease outcome during C. neoformans infection. In this study, we show that a hypervirulent phenotype is dependent on the host immune response and mouse genetic background. A/J mice intranasally infected with the hypervirulent isolates, UgCl247, UgCl422, and UgCl236, have increased neutrophil and T-cell recruitment when compared with infection with the reference strain KN99α. In addition, the cytokine profile of the hypervirulent isolates in A/J mice had a profound IFNγ and IL-17 response, and lung resident CD4 T-cells isolated from A/J mice expressed significantly increased Th1 (CXCR3, Tbet) and Th17 (RORγT) markers compared with KN99α infection. Intriguingly, when C57BL/6J mice were infected with these isolates, the hypervirulent phenotype was not evident, and all isolates had virulence comparable to the KN99α control. The immune response in C57BL/6J mice was also nearly identical in response to infections with the hypervirulent isolates and the KN99α control strain. Finally, we determined that the hypervirulent phenotype in A/J mice is not caused by known genetic mutations in the A/J inbred mouse background. Overall, this study demonstrates that an inbred mouse inhalation model can be used to identify host- and pathogen-specific factors that affect C. neoformans disease progression.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that commonly colonizes the gastrointestinal and lower female genital tracts but can cause sepsis and pneumonia in newborns and is a leading cause of neonatal meningitis. Despite the resulting disease severity, the pathogenesis of GBS is not completely understood, especially during the early phases of infection. To investigate GBS factors necessary for bloodstream survival, we performed a transposon (Tn) mutant screen in our bacteremia infection model using a GBS mariner transposon mutant library previously developed by our group. We identified significantly underrepresented mutations in 623 genes that contribute to survival in the blood, including those encoding known virulence factors such as capsule, the β-hemolysin, and inorganic metal ion transport systems. Most of the underrepresented genes have not been previously characterized or studied in GBS, including gloA and gloB, which are homologs for genes involved in methylglyoxal (MG) detoxification. MG is a byproduct of glycolysis and a highly reactive toxic aldehyde that is elevated in immune cells during infection. Here, we observed MG sensitivity across multiple GBS isolates and confirmed that gloA contributes to MG tolerance and invasive GBS infection. We show specifically that gloA contributes to GBS survival in the presence of neutrophils and depleting neutrophils in mice abrogates the decreased survival and infection of the gloA mutant. The requirement of the glyoxalase pathway during GBS infection suggests that MG detoxification is important for bacterial survival during host-pathogen interactions.IMPORTANCEA transposon-mutant screen of group B Streptococcus (GBS) in a bacteremia mouse model of infection revealed virulence factors known to be important for GBS survival such as the capsule, β-hemolysin/cytolysin, and genes involved in metal homeostasis. Many uncharacterized factors were also identified including genes that are part of the metabolic pathway that breaks down methylglyoxal (MG). The glyoxalase pathway is the most ubiquitous metabolic pathway for MG breakdown and is only a two-step process using glyoxalase A (gloA) and B (gloB) enzymes. MG is a highly reactive byproduct of glycolysis and is made by most cells. Here, we show that in GBS, the first enzyme in the glyoxalase pathway, encoded by gloA, contributes to MG resistance and blood survival. We further demonstrate that GloA contributes to GBS survival against neutrophils in vitro and in vivo and, therefore, is an important virulence factor required for invasive infection.

Neisserial surface protein A (NspA) is a small, conserved outer membrane protein that has been investigated as a vaccine antigen against meningococcal disease. After NspA had been tested in humans, this antigen was discovered to recruit the human complement regulator Factor H (FH). Previous studies in transgenic mice showed that human FH decreased the protective antibody responses to NspA. The purpose of the present study was to map the binding sites for human FH and anti-NspA antibodies. We found that an anti-NspA monoclonal antibody (mAb), AL-12, inhibits binding of FH to NspA by enzyme-linked immunosorbent assay (ELISA). Based on this result, we tested the roles of the 10 charged residues on the external loops of NspA in binding these two molecules by site-specific mutagenesis and binding experiments. Through ELISA and surface plasmon resonance experiments, we show that three aspartate (D) residues, D77 on loop 2 and D113 and D118 on loop 3, are important for binding human FH. Further, residues D113 and D118, as well as lysine 79 and arginine 109, are involved in binding mAb AL-12, which binds to a conformational epitope. The results have implications for strategies to increase NspA immunogenicity by decreasing binding to human FH, as has been done with other antigens that recruit this complement regulator.

Cullin-1 (Cul1), a cullin-RING ubiquitin ligase component, represses c-MYC activity in the nucleus. Orientia tsutsugamushi causes the potentially fatal rickettsiosis, scrub typhus. The obligate intracellular bacterium encodes an arsenal of ankyrin repeat-containing effectors (Anks), many of which carry a eukaryotic-like F-box motif that binds Cul1. O. tsutsugamushi reduces Cul1 levels in the nucleus. This phenomenon is not due to an alteration in Cul1 neddylation but is bacterial burden- and protein synthesis-dependent. Five of the 11 Anks capable of binding Cul1 (Ank1, Ank5, Ank6, Ank9, Ank17) sequester it in the cytoplasm when each is ectopically expressed. Ank1 and Ank6 proteins with alanine substitutions in their F-boxes that render them unable to bind Cul1 cannot exclude Cul1 from the nucleus. Coincident with the reduction of Cul1 in the nuclei of Orientia-infected cells, c-MYC nuclear levels are elevated, and Cul1 target genes are differentially expressed. Several of these genes regulate apoptosis. The resistance of O. tsutsugamushi-infected cells to staurosporine-induced apoptosis is recapitulated in uninfected cells expressing Ank1 or Ank6 but not alanine-substituted versions thereof that cannot bind Cul1. Other F-box-containing Anks that cannot bind or exclude Cul1 from the nucleus also fail to confer resistance to apoptosis. Overall, O. tsutsugamushi modulates the Cul1:c-MYC intranuclear balance as an anti-apoptotic strategy that is functionally linked to a subset of its F-box-containing Anks.

The postmenopausal population usually suffers from more severe periodontal disease than non-menopausal women due to the decrease and low levels of estrogen, especially β-estradiol (E2). While additional estrogen therapy can effectively relieve alveolar bone resorption, this suggests that estrogen has played an important role in the development of periodontitis. The integrity of the gingival epithelial barrier plays a key role in protecting gingival tissue from inflammatory injury caused by pathogens. However, it remains unclear whether estrogen can maintain the integrity of the gingival epithelial barrier to reduce inflammatory injury. Here, using an infection model established with Porphyromonas gingivalis lipopolysaccharide (LPS) in human gingival epithelial cells (hGECs) and ovariectomized or Sham mice, we assessed the protective effect of estrogen on the gingival barrier using qPCR, western blotting, immunohistochemistry, and transcriptome analysis. The results showed that estrogen restored epithelial barrier function to inhibit P. gingivalis-LPS invasion and further downregulate the inflammatory reaction (P < 0.05) by upregulating expressions of tight junction proteins (such as JAM1 and OCLN) at mRNA and protein levels in both hGECs and ovariectomized or Sham mice (P < 0.05). Estrogen also protected against alveolar bone resorption and preserved barrier integrity in both ovariectomized and Sham mice (P < 0.05). In conclusion, E2 prevented the progression of gingival epithelial barrier damage and inflammation induced by P. gingivalis-LPS by increasing the expression of tight junction proteins. The protective effect of estrogen on gingival epithelial barrier injury highlighted its potential application in treating periodontitis and inflammatory diseases involving epithelial barrier dysfunction.