From 2013 – 2016, I worked as a research assistant in Dr. Vincent Starai’s laboratory where I focused on understanding eukaryotic membrane fusion and analyzing binding interactions in the infection pathway of the bacteria Legionella pneumophila (pictured above) which causes Legionnaires’ Disease, an atypical pneumonia with a 10% rate of mortality.
As part of a $1.5 million grant from the NIH, I submitted and presented two posters to UGA’s Center for Undergraduate Research Opportunities (CURO) detailing my research and findings.
A targeted and unbiased screen for genetic suppressors of Legionella pneumophila protein effector LegC7
Legionella pneumophila is a Gram-negative bacterium that causes a severe form of pneumonia known as Legionnaires’ disease. During infection, L. pneumophila secretes nearly 300 effector proteins into host cells in order to evade lysosomal degradation by modulating vesicle trafficking pathways. One of these effector proteins, LegC7, has been shown to be toxic upon expression in the budding yeast, Saccharomyces cerevisiae. Upon LegC7 expression, S. cerevisiae accumulates membranous structures reminiscent of so called “class E” compartments, which result from defects in multivesicular body function.
The proteins which comprise the Class E VPS family are members of the endosomal sorting complex required for transport proteins (ESCRT) which are responsible for recognizing, sequestering and packaging membrane proteins into vesicles for vacuolar degradation. Because the Class E phenotype was produced in yeast during LegC7 expression, we hypothesize that LegC7 interacts with one or more of the yeast Class E gene products.
We therefore continued a targeted screen of the yeast Class E genes by transforming a plasmid encoding LEGC7 into yeast strains with deletions of vps23, vps28, snf7, or bro1 and found that deletion of these genes did not suppress LegC7 toxicity. We then performed an unbiased screen in an attempt to find genetic suppressors of LegC7 toxicity using ethyl methanesulfonate (EMS) mutagenesis to isolate a strain that exhibits a toxicity reversal due to a genomic mutation. We will sequence the genome from this strain to identify the gene products that LegC7 might require for toxicity.
An overexpression screen to determine how Legionella pneumophila effector protein LegC7 disrupts Class C tethering Complex Formation
I examine the relationship between LegC7 and the Class C Tethering Complexes – CORVET (class C Core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting). The CORVET and HOPS complexes have been identified as a set of universally conserved proteins which are essential for early-to-late endosome transition and endo-lysosomal trafficking pathways by tethering membranes, activating and proof-reading SNARE assembly to drive membrane fusion and interact with Rab GTPases.
Using homologous recombination, 4 different genes will be overexpressed in yeast: vps8, vps16, vps21 and vps41. By assaying whether overexpression of CORVET/HOPS subunits can suppress LegC7 toxicity we can determine which specific subunit(s) LegC7 may have binding interactions with.
Chetan Hebbale 