Contribution of Fimbria and Filamentous hemagglutinin in Bordetella Adherence and Immune Suppression

Pertussis, or whooping cough, is a highly contagious respiratory disease caused by Bordetella pertussis. Recent changes to the vaccine against B. pertussis have led to the re-emergence of this disease. Given the re-emergence of pertussis, it is critical to understand Bordetella pathogenesis. Fimbria and filamentous hemagglutinin (FHA), two proposed bacterial adhesins and components of the current vaccine, have been shown to be important virulence factors required for bacterial colonization of the lower respiratory tract of rats. Bordetella fimbria (Type I pili) are generally considered to function as adhesins despite a lack of supporting experimental evidence. FHA is a large exoprotein that has been shown critical for Bordetella adherence. We developed a novel in vivo adherence assay that confirmed FHA functions as an adhesin and we demonstrated a role for fimbria in adherence to ciliated respiratory epithelium. We further established that fimbria are required for bacterial persistence in the lower respiratory tract of mice and that fimbria contribute to suppression of the immune response. We also observed that wild-type and FHA-deficient bacteria induced cellular infiltrate around the major airways of the lungs and that FIM-deficient bacteria induced cellular infiltrate in the alveoli. A bacterial localization assay established that WT and FHA-deficient bacteria localized to the major airways and that FIM-deficient bacteria localized to the alveoli. Together, these findings suggest that fimbria mediate the first critical step of localizing the bacteria to the ciliated epithelium of the major airways, and that FHA may mediate tight adherence to these cells. We also demonstrated that SCID mice, which contain functional neutrophils and macrophages but lack B and T-cells, are unable to clear FIM-deficient bacteria from the lungs. B and T-cells adoptively transferred in to SCID mice prior to inoculation partially restored clearance of FIM-deficient bacteria from the lungs. Collectively, our data suggest that fimbria and FHA mediate critical adherence steps necessary for Bordetella to colonize and to modulate the immune system and that B and/or T-cells are required to mediate the initial host response to control Bordetella infection.Doctor of Philosoph

Evidence for phenotypic bistability resulting from transcriptional interference of bvgAS in Bordetella bronchiseptica: Phenotypic bistability inBordetella

Bordetella species cause respiratory infections in mammals. Their master regulatory system BvgAS controls expression of at least three distinct phenotypic phases in response to environmental cues. The Bvg+ phase is necessary and sufficient for respiratory infection while the Bvg− phase is required for survival ex vivo. We obtained large colony variants (LCVs) from the lungs of mice infected with B. bronchiseptica strain RBX9, which contains an in-frame deletion mutation in fhaB, encoding filamentous hemagglutinin. RBX9 also yielded LCVs when switched from Bvg− phase conditions to Bvg+ phase conditions in vitro. We determined that LCVs are composed of both Bvg+ and Bvg− phase bacteria and that they result from defective bvgAS positive autoregulation. The LCV phenotype was linked to the presence of a divergent promoter 5′ to bvgAS, suggesting a previously undescribed mechanism of transcriptional interference that, in this case, leads to feedback-based bistability (FBM). Our results also indicate that a small proportion of RBX9 bacteria modulates to the Bvg− phase in vivo. In addition to providing insight into transcriptional interference and FBM, our data provide an example of an in-frame deletion mutation exerting a ‘polar’ effect on nearby genes

Bordetella filamentous hemagglutinin and fimbriae: critical adhesins with unrealized vaccine potential

Pertussis, or whooping cough, is a highly contagious respiratory disease that is caused by the Gram-negative bacterium Bordetella pertussis, which is transmitted exclusively from human to human. While vaccination against B. pertussis has been successful, replacement of the whole cell vaccine with an acellular component vaccine has correlated with reemergence of the disease, especially in adolescents and infants. Based on their presumed importance in mediating adherence to host tissues, filamentous hemagglutinin (FHA) and fimbria (FIM) were selected as components of most acellular pertussis vaccines. In this review, we describe the biogenesis of FHA and FIM, recent data that show that these factors do, in fact, play critical roles in adherence to respiratory epithelium, and evidence that they also contribute to persistence in the lower respiratory tract by modulating the host immune response. We also discuss shortcomings of whole cell and acellular pertussis vaccines and the possibility that FHA and FIM could serve as effective protective antigens in next-generation vaccines

The Role of IL-27 in Susceptibility to Post-Influenza Staphylococcus Aureus Pneumonia

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection

Cooperative Roles for Fimbria and Filamentous Hemagglutinin in Bordetella Adherence and Immune Modulation

ABSTRACTBordetella fimbriae (FIM) are generally considered to function as adhesins despite a lack of experimental evidence supporting this conclusion for Bordetella pertussis and evidence against a requirement for FIM in adherence of Bordetella bronchiseptica to mammalian cell lines. Using B.bronchiseptica and mice, we developed an in vivo adherence assay that revealed that FIM do function as critically important adhesins in the lower respiratory tract. In the first few days postinoculation, FIM-deficient B.bronchiseptica induced a more robust inflammatory response than wild-type bacteria did, suggesting that FIM, like filamentous hemagglutinin (FHA), allow B.bronchiseptica to suppress the innate immune response to infection. Localization analyses indicated that FIM are required for efficient attachment to airway epithelium, as bacteria lacking FIM localized to alveoli. FHA-deficient bacteria, in contrast, localized to airways. Bacteria unable to produce both FIM and FHA localized to alveoli and caused increased inflammation and histopathology identical to that caused by FIM-deficient bacteria, demonstrating that lack of FIM is epistatic to lack of FHA. Coinoculation experiments provided evidence that wild-type B.bronchiseptica suppresses inflammation locally within the respiratory tract and that both FHA and FIM are required for defense against clearance by the innate immune system. Altogether, our data suggest that FIM-mediated adherence to airway epithelium is a critical first step in Bordetella infection that allows FHA-dependent interactions to mediate tight adherence, suppression of inflammation, and resistance to inflammatory cell-mediated clearance. Our results suggest that mucosal antibodies capable of blocking FIM-mediated interactions could prevent bacterial colonization of the lower respiratory tract.IMPORTANCEAlthough fimbriae (FIM) have been shown to be important mediators of adherence for many bacterial pathogens, there is surprisingly little experimental evidence supporting this role for Bordetella fimbria. Our results provide the first demonstration that Bordetella FIM function as adhesins in vivo, specifically to airway epithelium. Furthermore, our results suggest that FIM mediate initial interactions with airway epithelial cells that are followed by tight filamentous hemagglutinin (FHA)-mediated binding and that together, FIM and FHA allow Bordetella to suppress inflammation, leading to prolonged colonization. Given the shortcoming of the current acellular component pertussis (aP) vaccine in preventing colonization, these findings suggest that generation of antibodies capable of blocking FIM-mediated adherence could potentially prevent Bordetella colonization

New Insight into Filamentous Hemagglutinin Secretion Reveals a Role for Full-Length FhaB in Bordetella Virulence

ABSTRACTBordetella filamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, contributes to virulence, but how it functions mechanistically is unclear. FHA is first synthesized as an ~370-kDa preproprotein called FhaB. Removal of an N-terminal signal peptide and a large C-terminal prodomain (PD) during secretion results in “mature” ~250-kDa FHA, which has been assumed to be the biologically active form of the protein. Deletion of two C-terminal subdomains of FhaB did not affect production of functional FHA, and the mutant strains were indistinguishable from wild-type bacteria for their ability to adhere to the lower respiratory tract and to suppress inflammation in the lungs of mice. However, the mutant strains, which produced altered FhaB molecules, were eliminated from the lower respiratory tract much faster than wild-type B.bronchiseptica, suggesting a defect in resistance to early immune-mediated clearance. Our results revealed, unexpectedly, that full-length FhaB plays a critical role in B.bronchiseptica persistence in the lower respiratory tract.IMPORTANCEThe Bordetella filamentous hemagglutinin (FHA) is a primary component of the acellular pertussis vaccine and an important virulence factor. FHA is initially produced as a large protein that is processed during secretion to the bacterial surface. As with most processed proteins, the mature form of FHA has been assumed to be the functional form of the protein. However, our results indicate that the full-length form plays an essential role in virulence in vivo. Furthermore, we have found that FHA contains intramolecular regulators of processing and that this control of processing is integral to its virulence activities. This report highlights the advantage of studying protein maturation and function simultaneously, as a role for the full-length form of FHA was evident only from in vivo infection studies and not from in vitro studies on the production or maturation of FHA or even from in vitro virulence-associated activity assays

Bordetella pertussis pathogenesis: current and future challenges

Pertussis, or whooping cough, has recently reemerged as a major public health threat despite high levels of vaccination against the etiological agent, Bordetella pertussis. In this Review, we describe the pathogenesis of this disease, with a focus on recent mechanistic insights into virulence factor function. We also discuss the changing epidemiology of pertussis and the challenges of vaccine development. Despite decades of research, many aspects of B. pertussis physiology and pathogenesis remain poorly understood. We highlight knowledge gaps that must be addressed to develop improved vaccines and therapeutic strategies

An Improved Recombination-Based In Vivo Expression Technology-Like Reporter System Reveals Differential cyaA Gene Activation in Bordetella Species

ABSTRACT Bordetella pertussis and Bordetella bronchiseptica rely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg − phase, expression of vrg genes, including those required for motility in B. bronchiseptica , is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg + phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg + phase during respiratory infection, Bvg-regulated gene activation in B. bronchiseptica has not been investigated in vivo . To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation both in vitro and in vivo . Using pGFLIP, we demonstrated that cyaA , considered to be a “late” Bvg + phase gene, is activated substantially earlier in B. bronchiseptica than B. pertussis following a switch from Bvg − to Bvg + phase conditions. We show that the altered activation of cyaA is not due to differences in the cyaA promoter or in the bvgAS alleles of B. bronchiseptica compared to B. pertussis , but appears to be species specific. Finally, we used pGFLIP to show that flaA remains repressed during infection, confirming that B. bronchiseptica does not modulate to the Bvg − phase in vivo

Bordetella PlrSR regulatory system controls BvgAS activity and virulence in the lower respiratory tract

Bordetella spp. includes Bordetella pertussis, the causal agent of whooping cough. The Bordetella virulence gene (BvgAS) two-component regulatory system (TCS) is considered the “master virulence regulator” in Bordetella, as it controls expression of all known virulence factor-encoding genes. We show here that another TCS, PlrSR, is required for BvgAS activity in the lower respiratory tract (LRT) and for virulence even when BvgAS is rendered constitutively active, suggesting that it controls critical functions for bacterial survival in the LRT independently of BvgAS. Our data introduce a new layer of complexity to a paradigm of Bordetella virulence control that has held for more than 30 y, and they indicate the existence of previously unknown bacterial factors that may serve as vaccine components and therapeutic targets

Differential Requirement for c-Jun N-terminal Kinase 1 in Lung Inflammation and Host Defense

The c-Jun N-terminal kinase (JNK) - 1 pathway has been implicated in the cellular response to stress in many tissues and models. JNK1 is known to play a role in a variety of signaling cascades, including those involved in lung disease pathogenesis. Recently, a role for JNK1 signaling in immune cell function has emerged. The goal of the present study was to determine the role of JNK1 in host defense against both bacterial and viral pneumonia, as well as the impact of JNK1 signaling on IL-17 mediated immunity. Wild type (WT) and JNK1 −/− mice were challenged with Escherichia coli, Staphylococcus aureus, or Influenza A. In addition, WT and JNK1 −/− mice and epithelial cells were stimulated with IL-17A. The impact of JNK1 deletion on pathogen clearance, inflammation, and histopathology was assessed. JNK1 was required for clearance of E. coli, inflammatory cell recruitment, and cytokine production. Interestingly, JNK1 deletion had only a small impact on the host response to S. aureus. JNK1 −/− mice had decreased Influenza A burden in viral pneumonia, yet displayed worsened morbidity. Finally, JNK1 was required for IL-17A mediated induction of inflammatory cytokines and antimicrobial peptides both in epithelial cells and the lung. These data identify JNK1 as an important signaling molecule in host defense and demonstrate a pathogen specific role in disease. Manipulation of the JNK1 pathway may represent a novel therapeutic target in pneumonia