title: Capsule depolymerase overexpression reduces Bacillus anthracis virulence.
abstract: Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
authors.forename: Angelo
authors.initials: A
authors.lastname: Scorpio
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Chabot
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Day
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Hoover
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
authors.forename: Arthur M
authors.initials: AM
authors.lastname: Friedlander
mesh_terms: D000818:Animals; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D016667:Bacterial Capsules / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006168:Guinea Pigs; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D009876:Operon; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014774:Virulence / Q000235:genetics
chemical_list: D018834:Chaperonin 60; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C022562:capsular-polysaccharide galactohydrolase