Antibody-mediated immunity to Vibrio cholerae at epithelial surfaces.

摘要

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has an estimated worldwide disease burden in the millions and remains a significant public health threat. Immunity to V. cholerae is primarily antibody-mediated and though V. cholerae colonization evokes a mucosal immune response, it is the secretory IgA (SIgA) antibodies produced against bacterial surface antigens, specifically lipopolysaccharide (LPS) that confer protective immunity. SIgA antibodies are thought to function by inhibiting colonization by cross-linking and agglutination of pathogens, thereby limiting access to the epithelium, a process known as immune exclusion. Recent studies in other enteric pathogens have demonstrated that SIgA may function through a range of activities, including inhibiting motility, invasion, and secretion systems required for bacterial pathogenesis. Despite evidence that intestinal immunity to V. cholerae is predominantly mediated by anti-LPS IgA antibodies, the mechanism by which these SIgA antibodies confer protection remains unclear. Thus, the objective of my dissertation was to elucidate the interaction between anti-LPS antibodies and V. cholerae and determine to the impact that antibody binding has on V. cholerae pathogenesis. To characterize the mechanisms of antibody-mediated immunity, I have used monoclonal antibodies (mAbs) that bind epitopes within different regions of the V. cholerae LPS structure and, by comparing the IgA, IgG and Fab fragment derivatives of the mAbs, I was able to assess how antibody-mediated cross-linking and direct antibody binding contribute independently to immunity.;MAb 2D6 IgA binds the O-polysaccharide (O-PS) region of V. cholerae LPS and was previously shown to be sufficient to protect neonatal mice when challenged with V. cholerae. I first sought to identify the mechanism(s) by which 2D6 IgA is protective in vivo. Treatment with 2D6 IgA limited V. cholerae access to the epithelium in vivo and rapidly agglutinated and inhibited motility in vitro. To determine if antibody-mediated motility arrest preceded antibody-mediated agglutination, I produced chimeric IgG derivatives of mAb 2D6 and digested the IgG into monovalent Fab fragments. The 2D6 Fab fragments rapidly inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest were separate phenomena. Exposure of V. cholerae to 2D6 IgA or Fab fragments resulted in an increase in membrane surface-associated blebs and a wrinkled surface morphotype, suggesting antibody binding induced outer membrane stress. Taken together, I linked readouts of protection to a single epitope on V. cholerae O-PS, provided evidence that antibody-mediated motility arrest occurs independently of agglutination, and proposed that protection of the intestinal epithelium from V. cholerae infection by SIgA is multifactorial, including antibody-induced agglutination, motility arrest, and possibly outer membrane stress.;I next sought to examine the potential of antibodies directed against the core/lipid A regions of LPS as it is unknown if they contribute to antibody-mediated protection and they are largely conserved between V. cholerae serotypes. Using mAb ZAC-3, which binds the core/lipid A region of V. cholerae LPS, I showed that treatment with ZAC-3 IgG or Fab fragments led to a reduction in V. cholerae colonization in both classical and El Tor biotypes, providing the first evidence that a core/lipid A antibody was sufficient to limit V. cholerae colonization, resulting from at least, in part, agglutination-independent effects of direct antibody binding. Additionally, ZAC-3 IgG and Fab fragments rapidly reduced V. cholerae motility, which could be correlated with the reduction in colonization in vivo..;Finally, RNA-seq was used to determine if the functional readouts I observed in response to antibody treatment were due to changes in V. cholerae gene transcription and to determine which of those transcriptional changes were agglutination-dependent or independent. Comparing differential gene expression upon ZAC-3 treatment, I observed fewer differentially expressed genes in response to Fab fragment treatment compared to the IgG, suggesting that the ability of an antibody to cross-link may serve as a stronger signal of membrane stress than direct binding of the antibody alone, and this difference is reflected in the degree to which V. cholerae responds at the transcriptional level. Genes involved in motility and chemotaxis were identified in multiple V. cholerae biotypes in response to both 2D6 and ZAC-3 treatment, suggesting a conserved response to anti-LPS antibody treatment. Based on these results, I propose that SIgA antibodies confer protection by binding to the LPS that spans the entire surface of V. cholerae, inducing an outer membrane stress that directly impacts bacterial motility and transcription through agglutination-dependent and independent mechanisms. The results from this dissertation suggest that SIgA antibodies directed against V. cholerae LPS confer immunity in a multifactorial manner, expanding on the model of IgA-mediated protective immunity, into one that goes beyond that of bacterial agglutination, and demonstrates the complex and dynamic interplay between the host and pathogen. Understanding how LPS-specific antibodies interfere with the capacity of V. cholerae to colonize the intestinal epithelium will assist in the design of better-targeted vaccine strategies, and allow for more accurate prediction of the protective efficacy of the mucosal antibody responses elicited by candidate cholera vaccines.