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, 10 (8), e0136605
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Beta-Lactamase Repressor BlaI Modulates Staphylococcus Aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence

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Beta-Lactamase Repressor BlaI Modulates Staphylococcus Aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence

Morgan A Pence et al. PLoS One.

Abstract

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mapping of the Tn917 mutant and subsequent inactivation of blaI leading to elevated beta-lactamase production.
(A) (1) In the absence of beta-lactams, the blaZ-blaR1-blaI genes are repressed by BlaI. (2) When beta-lactam molecules are sensed by BlaR1, the cytoplasmatic domain of this transmembrane protein is autoproteolytically cleaved. (3) Following this event, the repressor protein BlaI is proteolytically cleaved and dissociates from its binding site, enabling transcription of the beta-lactamase-encoding gene blaZ. (4) Finally, the active beta-lactamase BlaZ is secreted, leading to hydrolysis of the beta-lactam molecules. (B) Organization of the blaZ-blaR1-blaI locus in S. aureus Newman. blaR1 and blaI are located in a two-gene operon. blaZ is divergently transcribed. In the cathelicidin susceptible S. aureus Newman mutant G2E3, Tn917 integration occurred 12 bp downstream of the blaI start codon. (C) S. aureus Newman WT and blaI mutant or MRSA252 WT and blaI mutant were incubated with 50 μg/ml nitrocefin for 30 min at 37°C, and the absorbance at 490 nm (A490) was read. A higher A490 value reflects higher beta-lactamase activity. Mean A490 values ± SD of duplicates of one representative experiment of at least three performed for each strain are shown. ***, p<0.001. A representative picture for the MRSA252 WT and blaI mutant bacteria after incubation with nitrocefin are shown.
Fig 2
Fig 2. Effect of blaI on the cathelicidin susceptibility of S. aureus.
(A, B) S. aureus Newman and (C, D) MRSA252 WT, blaI mutant or complemented mutant strains were incubated with CRAMP or LL-37 and the numbers of surviving CFUs were determined at the indicated time points. Samples were run in triplicate, and average CFU/mL values ± SD for one representative experiments of at least two performed for each data set is shown on a log scale. *, p<0.05; **, p<0.01, ***, p<0.001.
Fig 3
Fig 3. BlaI contributes to survival in human whole blood and virulence in vivo.
(A) S. aureus Newman WT with empty complementation vector pDC123, the blaI mutant with pDC123, and the complemented blaI mutant strain were incubated for 1 h in human whole blood and CFU numbers enumerated. Samples were run in triplicate and data were plotted as the average percentage ± SD for each strain as compared to the initial inocula. A representative experiment of three performed is shown. **, p<0.01. (B) CD-1 mice (n = 8) were injected subcutaneously on one flank with S. aureus Newman WT and on the opposite flank with blaI mutant bacteria, and lesion sizes were monitored for 7 days. The lesions for each individual mouse at Day 7 are plotted and the average value indicated. Overall, the blaI mutant lesions were significantly smaller compared to the WT (p<0.04; paired t-test). (C-D) Survival of CD-1 mice (n = 10) after intraperitoneal infection with (C) 1 x 106 CFU of S. aureus Newman WT or Newman blaI mutant or (D) 6 x 108 CFU of MRSA252 or MRSA252 blaI mutant. Survival was monitored for 3 days. The survival for Newman or MRSA252 blaI mutant strain infected mice was significantly higher than for the WT infected strains as assessed by log-rank (Mantel Cox) test; the p values are shown in the respective graphs.
Fig 4
Fig 4. Potential mechanisms of cathelicidin resistance.
(A) Surface charge of S. aureus Newman and MRSA252 strains was compared by poly-L-lysine binding. The S. aureus strain Sa113 and an isogenic mprF mutant with increased negative surface charge were used as controls. (B, C) Hydrophobicity was measured using a modified version of the MATH (microbial adhesion to hydrocarbons) assay. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. (D) Proteolytic activity of Newman WT + pDC123, the blaI mutant + pDC123 and the blaI mutant complemented with pBlaI on skim milk agar plates. Clearance zones around colonies indicate secreted protease activity. (E) Degradation of CRAMP or LL-37 by overnight cell-free supernatants analyzed by SDS-PAGE.
Fig 5
Fig 5. Pre-incubation with subinhibitory concentrations of the beta-lactam antibiotic 6-APA increases the beta-lactamase activity and LL-37 susceptibility of S. aureus Newman.
Bacteria were incubated for 60 min at 37°C in the absence or presence of 6-APA. For an aliquot of each sample the beta-lactamase activity was subsequently determined with nitrocefin as test reagent (A). The residual bacteria were incubated with or without LL-37 for another 60 min and the surviving CFUs were quantified (B). Samples were run in triplicate and one representative experiment of three performed is shown. ***, p<0.001.

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