Antimicrobial Effects of Interferon-Inducible CXC Chemokines againstBacillus anthracisSpores and Bacilli

Abstract

Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.Bacillus anthracis is a gram-positive, spore-forming bacterium that causes the disease anthrax. The infectious B. anthracis spore is a dormant, metabolically inactive form of the organism made up of distinct, concentric layers that collectively provide a highly structured casing capable of protecting the spore core from high temperature, UV irradiation, lytic digestion, and numerous reactive agents (31, 59). Spore germination is initiated through receptor-mediated interactions between soluble germinant molecules (typically nutrients such as single amino acids, sugars, or purine nucleosides) and germinant receptors located at the inner membrane of the dormant spore (20,36). Although the molecular mechanism(s) linking germinant binding to the loss of dormancy is undefined, germinant receptor engagement initiates a cascade of processes, including dipicolinic acid (DPA) release, that promote core rehydration and result in the controlled degradation of the protective spore structures; as germination concludes, metabolic activity resumes, and vegetative outgrowth is initiated (58). Fully virulent B. anthracis bacilli generate several virulence factors including an antiphagocytic, poly-D-glutamic acid capsule encode...

Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis

“…In this system, vegetative bacilli were prepared from a B. anthracis Sterne strain 7702 transposon mutant library generated by random insertion of the mariner transposable element Himar1 into genetic loci of B. anthracis. This pooled library of transposon mutants (≈100,000 mutants per screen) was treated with CXCL10 at 48 μg/mL (5.6 μM), a chemokine concentration shown to consistently and completely kill Sterne inocula (11). In this way, CXCL10 would eliminate organisms lacking the targeted phenotype, CXCL10 resistance, and allow the isolation of surviving bacilli harboring Himar1-disrupted genes whose products are important in the ability of CXCL10 to effectively kill vegetative B. anthracis.…”

“…1A), resulting in the recovery of 18 individual isolates, designated Tnx1-Tnx18. To confirm resistance to CXCL10, each recovered Tnx mutant was tested individually in an Alamar blue-based assay of bacterial growth and viability, a measure shown to correlate well with viable cfu levels (11). Of the 18 primary screen isolates, 15 were confirmed to be significantly resistant to CXCL10 compared with B. anthracis Sterne strain, and 10 of these isolates demonstrated near complete resistance to CXCL10-mediated bactericidal activity at the chemokine concentration examined (Fig.…”

“…We also examined a potential role for FtsX in B. anthracis spore susceptibility to chemokine-mediated disruptions in spore germination and the maintenance of viability, as reported (11). Spores were prepared from Sterne and ΔftsX bacteria and treated with several concentrations of CXCL10.…”

“…Recent studies by our laboratory have also shown each of these CXC chemokines to exert antimicrobial effects against Bacillus anthracis, the Gram-positive, sporeforming bacterium that causes the disease anthrax. More particularly, CXCL9, CXCL10, and CXCL11 were found to target both the spore and bacillus forms of B. anthracis Sterne strain, causing disruptions in spore germination and marked reductions in spore and bacilli viability (11); additionally, human CXCL10 has also been found to directly kill encapsulated Ames strain bacilli (12). In vivo, these chemokines likely reach antimicrobial concentrations at local sites of infection as previously discussed (11).…”

“…More particularly, CXCL9, CXCL10, and CXCL11 were found to target both the spore and bacillus forms of B. anthracis Sterne strain, causing disruptions in spore germination and marked reductions in spore and bacilli viability (11); additionally, human CXCL10 has also been found to directly kill encapsulated Ames strain bacilli (12). In vivo, these chemokines likely reach antimicrobial concentrations at local sites of infection as previously discussed (11). In support of this notion, neutralization of CXCL9, CXCL10, and CXCL11, but not their shared cellular receptor CXCR3, significantly increased host susceptibility to inhalational anthrax in a murine model of infection (12), indicating that these host chemokines mediate biologically relevant contributions to host defense against pulmonary B. anthracis infection.…”