Identifying the mycobacterial genes controlling bacteriolysis in the macrophage cytoplasm

PROJECT SUMMARY
Pathogenic mycobacteria damage the phagosome and interact with the macrophage cytoplasm. The mycobac-
terial factors that control bacteriolysis in the cytoplasm are unknown. The objective of this proposal is to define
the genes controlling mycobacterial bacteriolysis in the macrophage cytoplasm. The central hypothesis for this
application is that mycobacteria actively control bacteriolysis in the cytoplasm. To test this hypothesis, the fol-
lowing specific aims will be tested. The applicant has adapted an established reporter for cytoplasmic bacteriol-
ysis from Listeria monocytogenes for use with Mycobacterium. Under the first aim, the applicant proposes a
targeted approach to define the impact of known virulence factors on mycobacterial bacteriolysis. The objective
of Aim 1 is to define if known virulence pathways protect M. marinum from bacteriolysis. The applicant will test
the working hypothesis that known virulence pathways that modulate the inflammasome protect Mycobacterium
from bacteriolysis in the macrophage cytoplasm. M. marinum strains lacking specific virulence pathways will be
generated and bacteriolysis will be measured during macrophage infection. Under the second aim, the applicant
proposes an unbiased approach to identify genes required for cytoplasmic adaptation of mycobacterial patho-
gens. The objective of Aim 2 is to Identify genes the control mycobacterial bacteriolysis. The applicant will test
working hypothesis that several conserved pathways protect mycobacterial pathogens from bacteriolysis in the
cytoplasm. Two complementary genetic screens to identify molecular pathways controlling mycobacterial bacte-
riolysis in the cytoplasm during macrophage infection. The applicant expects that the successful completion of
Aim 1 will determine if known virulence pathways protect mycobacteria from bacteriolysis. The completion of
Aim 2 is expected to identify new pathways that promote mycobacterial survival in the cytoplasm. Completion of
the proposed aims will contribute an initial understanding of the mechanisms required for preventing mycobac-
terial bacteriolysis in the macrophage cytoplasm, moving the field in a new direction. This contribution will be
significant because it will identify a new molecular mechanism underlying mycobacterial pathogenesis. The ap-
plication is conceptually innovative because it focuses on mycobacterial determinants that protect mycobacteria
from bacteriolysis in the cytoplasm, which represents a shift in focus for the field. The experimental design is
innovative because it applies a novel indirect reporter of cytoplasmic bacteriolysis that has not previously been
used to study mycobacterial species.

Grant Number: 5R21AI181133-02
NIH Institute/Center: NIH
Principal Investigator: Patricia Champion