With the arrival of the HIV epidemic and the increase in individuals undergoing chemotherapy for various cancers, the understudied zoonotic opportunistic pathogen Rhodococcus equi has emerged as cause of life- threatening pneumonia in persons of compromised immunity. Rhodococcal pneumonia is characterized by pyogranulomatous inflammation with cavitation, which can be misdiagnosed as Mycobacterium tuberculosis infection. R. equi is a common soil-borne facultative actinomycete which, when inhaled, resists innate killing mechanisms and readily multiplies in macrophages of susceptible hosts, likely by perturbation of endosomal trafficking. Little is known about the molecular basis for R. equi pathogenesis, and prior to our recent work, few tools existed to allow a thorough genetic analysis of this bacterium. Recently, we have developed the genetic methods to construct defined mutants of R. equi and have also created an efficient transposon mutagenesis system. Using these new tools, we demonstrated that vapA (virulence associated p/otein A), a gene present on the virulence plasmid of R. equi, is required for intracellular growth and full virulence. Deletion of vapA attenuates the bacterium, rendering it incapable of growth in vivo and unable to replicate in macrophages cultured in vitro. Our long-term goal is to completely dissect the genetic basis for R. equi virulence. We propose to do so by first expanding our understanding of vapA, which is to date the only known R. equi virulence determinant.
The first AIM of this work is to identify the mechanism of action of vapA. To do so, we will define the intracellular events post phagocytosis of both wild type R. equi and the vapA deletion mutant. Specifically we will characterize the process of phagosomal maturation in wild type R. equi infected macrophages and test the influence of VapA on phagosome acidification and fusion with lysosomes. We will identify interacting host protein partners of VapA.
The second AIM i s to characterize the R. equi virulence regulon through the examination of regulators of vapA expression, VirR (yjrulence regulator) and VarA (yap regulator). We will identify the specific signals transduced by these regulators and identify additional genes controlled by them. We will establish the effects of deletion mutants of virR and varA on R. equi virulence. Finally, we will identify the binding sites of the regulators to the vapA promoter. These studies addressing the molecular mechanisms of R. equi pathogenesis will further our understanding of macrophage cell biology and will yield insight into the biology of actinomycete host-pathogen relationships in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI060469-05
Application #
7758845
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Jacobs, Gail G
Project Start
2006-02-01
Project End
2013-01-31
Budget Start
2010-02-01
Budget End
2013-01-31
Support Year
5
Fiscal Year
2010
Total Cost
$312,330
Indirect Cost
Name
University of Georgia
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
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Wang, Xiaoguang; Coulson, Garry B; Miranda-Casoluengo, Aleksandra A et al. (2014) IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth. Infect Immun 82:1793-800
Miranda-Casoluengo, Raúl; Coulson, Garry B; Miranda-Casoluengo, Aleksandra et al. (2012) The hydroxamate siderophore rhequichelin is required for virulence of the pathogenic actinomycete Rhodococcus equi. Infect Immun 80:4106-14
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