Mechanisms and role of actin-based motility in Burkholderia virulence

University Of California Berkeley, Berkeley CA

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Abstract

? DESCRIPTION (provided by applicant): Burkholderia in the `pseudomallei group' are Gram-negative bacteria that cause melioidosis in humans (B. pseudomallei), glanders in equines and humans (B. mallei), or are rarely pathogenic (B. thailandensis). Central features of Burkholderia pathogenesis include the ability to invade host cells, escape from the phagosome, and grow in the cytosol, where bacteria mobilize the actin cytoskeleton to power intracellular motility. Movement facilitates host cell fusion and the formation of multinucleate giant cells (MNGCs), which enable continued bacterial growth and are essential for virulence. Although motility is a central feature of Burkholderia infection, the mechanism of actin polymerization and its correlation with movement, host cell fusion and virulence is poorly understood. The Burkholderia intracellular motility A (BimA) protein is required for actin-based motility. Interestingly, BimA differs considerably in sequence between B. pseudomallei, B. mallei, and B. thailandensis. BimA sequence differences mirror those found in host actin polymerizing proteins, suggesting that different Burkholderia species hijack distinct host actin polymerization pathways. Despite this information, we have yet to answer the following fundamental questions about BimA. How does each BimA ortholog nucleate actin? How do species differences in BimA contribute to differences in motility and host cell fusion? Does motility driven by each BimA ortholog require a unique set of host proteins? How does BimA contribute to virulence and how do the different orthologs impact BimA function during infection? In the exploratory experiments proposed here, we will test the hypothesis that BimA orthologs polymerize actin by different mechanisms, leading to differences in intracellular motility, host cell fusion, and virulence in animals. In Ai 1, we will determine how each BimA ortholog nucleates actin, test for differences in the efficiency with which each ortholog drives B. pseudomallei motility and MNGC formation, and define the host protein requirements for motility driven by each ortholog. These experiments will reveal the molecular mechanism(s) of actin assembly by BimA from different species, and will uncover how differences in actin polymerization mechanisms contribute to Burkholderia motility and cellular infection. In Aim 2, we will determine whether BimA-driven motility is important for B. pseudomallei infection in mice by examining the ability of a bimA mutant to colonize the lung, liver and spleen, as well as promote virulence. Moreover, we will assess the efficiency with which each BimA ortholog contributes to B. pseudomallei virulence. These experiments will reveal whether BimA is a key virulence factor, and whether BimA orthologs differ in their ability to promote infection and virulence in an animal model. Taken together, the proposed experiments will reveal the role of actin-based motility in Burkholderia pathogenesis, and may uncover new mechanisms of host-pathogen interactions.