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. 2009;1(5):494-506.
doi: 10.1159/000225955. Epub 2009 Jun 18.

ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis

Shauna M McGillivray  1 , Celia M Ebrahimi, Nathan Fisher, Mojgan Sabet, Dawn X Zhang, Yahua Chen, Nina M Haste, Raffi V Aroian, Richard L Gallo, Donald G Guiney, Arthur M Friedlander, Theresa M Koehler, Victor Nizet
Affiliations

ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis

Shauna M McGillivray et al. J Innate Immun. 2009.

Abstract

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.

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Figures

Fig. 1
Fig. 1
B. anthracis β-hemolytic and proteolytic phenotypes are dependent upon ClpX. Hemolytic activity of parent B. anthracis Sterne and the anthrolysin O (ALO)-deficient strain (Δalo) in 1% human blood solution (a) or 5% human blood agar plates incubated under anaerobic conditions (b). c Transposon mutants were screened for loss of hemolytic activity on human blood agar plates under anaerobic conditions. NH = Nonhemolytic. d Schematic representation of the site of transposon insertion within the B. anthracis chromosome. e Semi-quantitative RT-PCR showing expression of the clpX, lon and fusA genes from cDNA prepared from either parental B. anthracis Sterne (Sterne) or an isogenic ΔclpX allelic replacement mutant. f Human blood agar plate showing hemolytic activity of parent B. anthracis Sterne + empty pUTE29, the ΔclpX mutant + empty pUTE29, and the ΔclpX mutant + pUTE29 containing the clpX gene (pClpX). g Proteolytic activity of the same strains on casein agar plates.
Fig. 2
Fig. 2
Contribution of ClpX to B. anthracis Sterne growth, vegetative morphology and sporulation. a Growth curve at 37°C in BHI for parental B. anthracis Sterne + pUTE29, the ΔclpX + pUTE29, and the ΔclpX complemented in trans by pClpX. b Gram stain of vegetative parent B. anthracis Sterne and ΔclpX in early log growth phase. Magnification × 100. c Sporulation efficiency of parental B. anthracis Sterne + pUTE29, the ΔclpX + pUTE29 and the ΔclpX + pClpX as determined by number of viable spores formed after 24 h growth under nutrient limiting conditions. d Using the same series of strains, germination of spores as determined by loss of heat resistance after 30 min growth in BHI. e Semi-quantitative RT-PCR showing expression of the atxA, lef, cya and fusA genes from cDNA prepared from either parent B. anthracis Sterne (Sterne) or the ΔclpX mutant.
Fig. 3
Fig. 3
ClpX function protects B. anthracis Sterne against antimicrobial peptide killing. a Killing kinetics of parent Sterne, the ΔclpX mutant, and ΔclpX + pClpX incubated with 16 μM of murine cathelicidin CRAMP. b Survival of parent Sterne + pUTE29, the isogenic ΔclpX mutant + pUTE29, or ΔclpX complemented by pClpX incubated with 0.5 μM of human cathelicidin LL-37. c Growth of parent Sterne + pUTE29, the isogenic ΔclpX mutant + pUTE29 or ΔclpX complemented by pClpX in 0.8 or 1.6 μM of human α-defensin-2 (HNP-2). d Growth of parent Sterne + pUTE29, the isogenic ΔclpX mutant + pUTE29 or the ΔclpX complemented by pClpX in 5 mg/ml or 10 mg/ml μM of lysozyme. e Western blot analysis of LL-37 (2 μM final concentration) exposed to culture supernatants from B. anthracis Sterne or its isogenic ΔclpX mutant. f B. anthracis Sterne incubated with either 0.25 μM LL-37 or 0.25 μM LL-37 plus 0.5 m M o-phenanthroline (o-phen). Statistically significant changes between parent Sterne and the ΔclpX mutant: ∗p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Statistically significant differences between complemented strain and ΔclpX mutant: # p < 0.05 or ### p < 0.001.
Fig. 4
Fig. 4
ClpX is required for B. anthracis Sterne virulence. a Kaplan-Meyer survival curve of C57Bl/6 mice after s.c. injection with parent Sterne (11 mice), ΔclpX (11 mice) or ΔclpX + pClpX (10 mice). b Enumeration of surviving cfu of parent or ΔclpX mutant at the site of inoculation 3 days after s.c. challenge. c Histopathologic analysis of lesional biopsies by Gram stain (left) and hematoxylin-eosin (HE) stain (right) from mice challenged s.c. with parent B. anthracis Sterne or ΔclpX mutant 3 days after challenge. d Kaplan-Meyer survival curve of A/J mice following inhalation of parent B. anthracis Sterne (9 mice) or ΔclpX mutant (6 mice); B. anthracis spores administered intranasally.
Fig. 5
Fig. 5
Cathelicidin contributes to innate immune defense against B. anthracis. a Kaplan-Meyer survival curve of wild type (WT; 10 mice) and CRAMP−/− (4 mice) challenged subcutaneously s.c. with B. anthracis Sterne. b Magnitude of inflammatory changes on gross examination of site of s.c. inoculation in wild-tpye and CRAMP−/− mice 5 days after s.c. challenge with wild-type B. anthracis. 0 = Absent; + = trace; ++ = mild; +++ = moderate; ++++ = severe. c Representative images of individual mice.
Fig. 6
Fig. 6
ClpX contributes to in vivo survival of fully virulent B. anthracis Ames. a Competitive index of the ΔclpX mutant compared to the parental strain, B. anthracis Ames, grown overnight in BHI or after infection in guinea pigs. Results are the mean ± standard deviation of 3 overnight growth experiments or 3 spleens. b Kaplan-Meier survival curves of guinea pigs after i.p. injection with B. anthracis Ames or the isogenic ΔclpX mutant. For statistical analysis, p values are positioned below the curves corresponding to animals given B. anthracis Ames and indicate the significance by log-rank test between the outcomes of that group and the group given a similar dose of the ΔclpX mutant.
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