Development of Chimeric Type Iv Secretion Systems For Transfer of Heterologous Substrates Across The Gram-Negative Cell Envelope

<p>Many bacteria use Type IV Secretion Systems (T4SSs) to aid in pathogenesis by translocating virulence factors across the cell envelope and into eukaryotic cells. These systems are structurally and functionally diverse, but are often compared to the archetypal VirB/VirD4 T4SS of <em>Agrobacterium tumefaciens</em>. This system is composed of the VirD4 type IV coupling protein (T4CP) and 11 VirB subunits (VirB1-11) that assemble as the secretion channel and an extracellular pilus. The T4CP is an inner membrane ATPase that interacts with T4SS substrates and the secretion channel, and is thought to link substrates with the secretion channel and possibly energize transfer through the channel lumen. In this thesis, I sought to adapt T4SSs in the surrogate hosts <em>A.</em> <em>tumefaciens </em>and <em>Escherichia coli</em> for use in identification of novel T4SS effector proteins from genetically-intractable Rickettsial species. I first constructed chimeric T4SSs in <em>A. tumefaciens</em> by substituting native VirD4 with Rickettsial VirD4 homologs. However, I was unable to demonstrate transfer of the promiscuous IncQ plasmid pML122 or known <em>A. tumefaciens</em> effector proteins. I next tested the <em>E. coli</em> pKM101-encoded T4SS, which is known to transfer DNA substrates, for the capacity to deliver heterologous protein substrates to <em>E. coli</em> recipients. Using the Cre-recombinase reporter assay for translocation (CRAfT), I showed that pKM101 translocates effector proteins from <em>A. tumefaciens</em> and two Rickettsial species, <em>Anaplasma phagocytophilum</em> and <em>Wolbachia pipientis</em>. I next created chimeric T4CPs by joining the transmembrane domain (TMD) of pKM101-encoded TraJ with the soluble domains (SDs) of VirD4 homologs from <em>A. tumefaciens</em> and the Rickettsial species. I showed that all of these chimeric systems translocate protein substrates, although less efficiently than the native pKM101 T4SS. Finally, I demonstrated that a variable C-terminal extension (CTE) that is present on the <em>A. tumefaciens</em> and Rickettsial T4CPs plays a modulatory role for secretion of different protein substrates. My findings showed for the first time that a T4SS encoded by an <em>E. coli </em>conjugative plasmid is capable of translocating a variety of protein substrates from phylogenetically diverse alphaproteobacterial species, including <em>A. tumefaciens</em>, <em>A. phagocytophilum</em>, and <em>W. pipientis</em>.</p>

Name thesis:degree_name

Masters of Science (MS)

Level thesis:degree_level

Thesis (MS)

Year dc:date.available

2014

Author dc:creator

• Berry, Trista M

Contributors dc:contributor

• Peter J Christie
• Theresa M Koehler
• Jeffrey Actor

• VirD4
• type IV secretion
• T4SS
• effector
• chimera
• virulence factors
• Bacteriology
• Biology
• Laboratory and Basic Science Research
• Molecular Genetics
• Pathogenic Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/490

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1529