Mechanisms of CXCL10 Antimicrobial Activity against Bacillus anthracis 661 views

Bacillus anthracis is a bacterial pathogen that is the causative agent of anthrax with a high risk for bioweapon development. The chemokine CXCL10 has been described as an important component of the innate and adaptive immune system for its chemoattractant properties influencing various leukocytes. B. anthracis spores and vegetative cells have been shown to be directly killed by the chemokine CXCL10 through previously unknown mechanisms. We first studied the role of the identified bacterial target FtsX, as well as the role of various portions of CXCL10 in killing B. anthracis vegetative cells. We show that CXCL10 acts as a bifunctional molecule that can utilize at least two separate mechanisms for eliciting antimicrobial activity: 1) An FtsE/X-dependent mechanism through interaction of a portion of CXCL10 (presumably, the CXCL10 N-terminal region) other than the C-terminal α-helix with FtsE/X and 2) An FtsE/X-independent mechanism observed at high concentrations of intact CXCL10 and in the absence of FtsX- this latter mechanism requires the presence of the CXCL10 C-terminal α-helix and results in membrane depolarization. Further work was performed to investigate the FtsE/X-dependent antimicrobial mechanism, and we hypothesized that CXCL10 kills through disruption of peptidoglycan processing through the bacterial FtsE/X complex. We characterized the peptidoglycan phenotype of B. anthracis parent strain and the B. anthracis bacterial mutants ΔftsX and ftsE(K123A/D481N) to use as a baseline comparator to CXCL10 treated cells. Studies conducted with CXCL10 and a C-terminal truncated CXCL10 showed that exposure of the B. anthracis parent strain to both molecules resulted in disruption of peptidoglycan processing. The B. anthracis bacterial mutants ΔftsX and ftsE(K123A/D481N) strains did not exhibit disruption of peptidoglycan processing in the presence of CXCL10 or the C-terminal truncated CXCL10. These data indicate that CXCL10 exerts an antimicrobial effect against B. anthracis vegetative cells through the FtsE/X complex by disrupting peptidoglycan remodeling, resulting in bacterial lysis. Finally, studies were conducted to identify key bacterial components involved in CXCL10 activity against B. anthracis spores. A transposon mutant library of B. anthracis Sterne strain spores identified the gene encoding BAS0651, a putative DL-endopeptidase involved in peptidoglycan hydrolysis and cell wall remodeling during cellular elongation. Further analyses supported that CXCL10 causes a delay in B. anthracis spore germination and also leads to an increase in spore permeability, a possible mechanism for the resulting loss of spore viability. In vivo mouse studies were conducted that showed the absence of BAS0651 in B. anthracis parent strain resulted in an increase in spore germination and vegetative cell proliferation during initial pulmonary infection in C57BL/6 mice. It appears CXCL10 targets B. anthracis spores through BAS0651, which may result in the dysregulation of peptidoglycan remodeling during spore germination. The findings in this dissertation reveal novel mechanisms of CXCL10 antimicrobial activity against B. anthracis spores and vegetative cells. Understanding how CXCL10 kills B. anthracis will potentially result in the development of a new class of antimicrobials based on CXCL10, as well as the identification of important new antimicrobial bacterial targets.

PHD (Doctor of Philosophy)

Microbiology; Bacillus anthracis; CXCL10

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2016-06-24