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1.
Infect Immun ; 77(4): 1664-78, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19179419

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.


Subject(s)
Anti-Bacterial Agents , Bacillus anthracis/drug effects , Chemokines, CXC , Interferons/immunology , Spores, Bacterial/drug effects , Animals , Anthrax/immunology , Anthrax/microbiology , Anti-Bacterial Agents/immunology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacillus anthracis/pathogenicity , Bacillus anthracis/physiology , Chemokine CXCL10/immunology , Chemokine CXCL10/pharmacology , Chemokine CXCL10/therapeutic use , Chemokine CXCL11/immunology , Chemokine CXCL11/pharmacology , Chemokine CXCL11/therapeutic use , Chemokine CXCL9/immunology , Chemokine CXCL9/pharmacology , Chemokine CXCL9/therapeutic use , Chemokines, CXC/immunology , Chemokines, CXC/pharmacology , Chemokines, CXC/therapeutic use , Colony Count, Microbial , Female , Humans , Lung/immunology , Lung/microbiology , Mice , Mice, Inbred C57BL , Spores, Bacterial/pathogenicity
2.
J Bacteriol ; 187(22): 7579-88, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16267282

ABSTRACT

Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.


Subject(s)
Adenylate Cyclase Toxin/genetics , Adenylate Cyclase Toxin/isolation & purification , Bordetella/enzymology , Virulence Factors, Bordetella/genetics , Virulence Factors, Bordetella/isolation & purification , Adenylate Cyclase Toxin/analysis , Adenylate Cyclase Toxin/toxicity , Animals , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/toxicity , Blotting, Western , Bordetella/genetics , Calmodulin/metabolism , Cell Line , Cyclic AMP/analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Expression , Hemolysis , Macrophages/microbiology , Mice , Molecular Sequence Data , Protein Binding , RNA, Bacterial/analysis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Virulence Factors, Bordetella/analysis , Virulence Factors, Bordetella/toxicity
3.
Infect Immun ; 73(11): 7535-40, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16239556

ABSTRACT

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antigens, Differentiation/metabolism , Bacillus anthracis/immunology , Receptors, Immunologic/metabolism , Signal Transduction , Spores, Bacterial/immunology , Toll-Like Receptor 2/physiology , Toll-Like Receptor 4/physiology , Aerosols , Animals , Cell Line , Humans , Mice , Mice, Knockout , Myeloid Differentiation Factor 88 , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Tumor Necrosis Factor-alpha/metabolism
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