Mechanism of type VI secretion system 5-dependent intercellular spread of Burkholderia pseudomallei

• Applicant: Dr. Sandra Schwarz
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2013 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 235688424

Secretion systems that deliver effector proteins into host cells are essential virulence factors in a number of bacterial pathogens. The recently discovered type VI secretion system (T6SS) is one such system, which was shown to critically affect the bacteria-host interaction of various Gram negative human pathogens. We will use Burkholderia pseudomallei, a facultative intracellular human and animal pathogen that causes the life-threatening disease melioidosis, as a model organism for the study of the role of T6S in bacterial pathogenesis. The virulence strategies of B. pseudomallei to establish an infection are poorly defined; T6S however, has emerged as a key virulence factor in B. pseudomallei. In particular, the inactivation of one of the T6SSs found in the bacterium, T6SS-5, renders it avirulent at a usually lethal infection dose and unable to proliferate in murine lung tissue. Further, it is known that the T6SS-5 is required to promote the fusion of host cells, a process that facilitates intercellular spread of B. pseudomallei and their escape from extracellular immune defenses. However, the mechanism of T6SS-5 and its substrates that give rise to the observed phenotypes are unknown. Our long-term goal is to define the molecular basis of T6SS-5-dependent interactions with the host that contribute to disease. We previously identified VgrG-5 as a T6SS-5-secreted protein in B. thailandensis, which is a homolog of VgrG-5 in B. pseudomallei. Specific truncations of and amino acid deletions in VgrG-5 result in a host cell fusion defect. Our central hypothesis is that VgrG-5 plays a key role in the virulence of B. pseudomallei by mediating the fusion of host cells in a direct or indirect manner. The objective of this proposal is to perform a comprehensive analysis of B. pseudomallei VgrG-5 activity during infection. This will be accomplished by pursuing the following specific aims: (1) Define the spatiotemporal requirements of T6SS-5 activation and localization of VgrG-5 within host cells using time lapse and immunofluorescence microscopy, (2) Performing a genome-wide survey of the transcriptional host response to VgrG-5 utilizing RNA-Seq of infected macrophages and (3) Determine the cellular target of VgrG-5 and define the relevance of this interaction in intercellular spread and virulence of B. pseudomallei. This study will provide a mechanistic insight into the function of T6SS-5 and the pathogenesis of melioidosis. Finally, we expect that a better understanding of how B. pseudomallei counteracts clearance by the host will reveal targets for anti-infective agents.

• DFG Programme: Research Grants