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Bordetellae are gram-negative bacteria that cause respiratory infections. Bordetella pertussis and Bordetella parapertussis_hu, the causative agents of whooping cough, are human adapted variants of Bordetella bronchiseptica, which naturally infects a broad range of mammals causing infections that range from asymptomatic to severe. We are using these three closely related subspecies to investigate mechanisms of signal transduction, the roles of virulence gene regulation and of specific regulated factors in pathogenesis, and mechanisms of protein secretion. In addition, we have recently begun a new project aimed at understanding mechanisms of pathogenesis used by Burkholderia pseudomallei, the causative agent of melioidosis.

Phosphorelay Control of Virulence Gene Expression

Two-component regulatory systems represent the most common mechanism by which bacteria sense and respond to changes in their environment. Phosphorelays are sophisticated variants of two-component regulatory systems that use a four-step phosphotransfer mechanism. The BvgAS phosphorelay controls expression of virulence genes and many other factors in Bordetella and is prototypical of phosphorelays containing polydomain sensor proteins (see Fig. 1). We have shown that rather than functioning like an ON/OFF switch, the BvgAS phosphorelay functions like a rheostat and controls expression of at least four classes of genes and at least three distinct phenotypic phases. We are investigating the molecular mechanisms underlying phosphorelay signaling by constructing and characterizing mutants of B. pertussis and B. bronchiseptica that express BvgA and BvgS proteins containing single amino acid substitutions. We are using in vitro phosphorylation assays with purified proteins to determine how these amino acid changes affect the kinetics of individual steps of the phosphorelay. And we are collaborating with Dr. Linda Petzold, Professor and Chair of Computer Science and Professor of Mechanical and Environmental Engineering, who is developing mathematical tools to model the BvgAS phosphorelay. We are also investigating the role of positive autoregulation in Bvg-mediated signal transduction. Our results indicate that both positive autoregulation and rapid dephosphorylation kinetics play critical roles in determining how BvgAS controls various phenotypic states in response to temporal and spatial cues.

Role of Filamentous hemagglutinin in the development of respiratory infection

Filamentous hemagglutinin (FHA) is a large, surface-associated and secreted protein that is expressed by all bordetellae that infect mammals. It is highly immunogenic and is included as a primary component in all acellular pertussis vaccines. In vitro studies using B. pertussis suggested that FHA functions as an adhesin and several binding domains, including a heparin binding domain (HBD), a carbohydrate recognition domain (CRD), and an Arg-Gly-Asp (RGD) motif, were identified (Fig. 2). Lack of a suitable animal model for studies with this human-adapted Bordetella subspecies, however, precluded analyzing the importance of these domains in vivo. Using B. bronchiseptica and its natural hosts, we have shown that FHA is critical for lower respiratory tract colonization, immunomodulation, and the ability to overcome innate immunity. Recent work in our lab demonstrated that the C-terminus of mature FHA is oriented away from the bacterial surface (see below), suggesting it may interact with host cell(s) during infection. By constructing and characterizing B. bronchiseptica strains expressing FHA proteins with alterations in the HBD, CRD, RGD, or C-terminal domain in vitro and in vivo, we are investigating the importance of these in vitro-identified domains in the development of respiratory infection.

Mechanism of Two-Partner Secretion

Two-Partner Secretion (TPS) is the most widely distributed secretion pathway known. These systems export large exoproteins (the TpsA proteins) through highly conserved channel-forming b-barrel proteins (the TpsB proteins). FHA is a prototypical TPS family member. It is first synthesized as a large preproprotein called FhaB (the TpsA family member) and is exported by FhaC (the TpsB family member). Previous models indicated that FhaB emerged from the FhaC pore N-terminus first and that after ~2/3 of the protein was exposed on the cell surface it was cleaved by a serine protease called SphB1, generating mature FHA which was consequently released from the bacterial cell. We have shown, however, that the C-terminus of mature FHA, not the N-terminus, is exposed on the cell surface and is required for mediating adherence to cultured epithelial cells. We also showed that the C-terminus of FhaB is required for FHA function in vitro and in vivo and that cleavage of FhaB to form FHA is not the mechanism by which FHA is released from the cell. Our data support a new model for TPS (see Fig. 3). This model provides an explanation for the energetics of export of globular protein domains across membranes in the absence of ATP and it suggests a new mechanism for the control of protein folding. Future studies include those aimed at determining how the C-terminus of FhaB controls the activity of mature FHA.

Burkholderia pseudomallei

Burkholderia pseudomallei is a gram-negative, soil saprophyte and the etiological agent of melioidosis, a severe and invasive human disease associated with high morbidity and mortality. B. pseudomallei naturally inhabits moist soils and pooled surface waters in Southeast Asia, Northern Australia and other tropical and subtropical regions. It infects humans through inhalation, oral ingestion or direct contact with skin abrasions, and causes disease symptoms that range from localized skin abscesses to chronic or acute pneumonia to fulminant septicemia. Because of its low dose of infectivity, aerosol transmission, severe course of disease, and easy cultivation in the laboratory, B. pseudomallei has recently been classified as a category B potential agent of bioterrorism by the US Centers for Disease Control and Prevention. Deciphering the molecular basis of the early steps in the infection process will be crucial to the development of rapid diagnostics, effective vaccines and therapeutics for melioidosis. The B. pseudomallei genome contains open reading frames that are predicted to encode products with significant amino acid similarity to members of the Two Partner Secretion (TPS) and Autotransporter (AT) families of bacterial proteins. TPS and AT exoproteins can be both surface-associated as well as secreted into the extracellular milieu, and have been shown to be important virulence factors in a wide number of pathogenic gram-negative bacteria. We are currently characterizing the expression, maturation and cellular localization of the B. pseudomallei TPS- and AT-like proteins as well as their potential roles in adherence, immunomodulation, and other aspects of B. pseudomallei pathogenesis.