D-113. Mutations Conferring Staphylococcus aureus Sensitivity to Cathelicidin Antimicrobial Peptides Mapping to the Bla Operon

M. A. Pence, R. L. Gallo, V. Nizet, S. A. Kristian;
Univ. of California, La Jolla, CA.

Background: Staphylococcus aureus (SA) is a pre-eminent Gram-positive bacterial pathogen associated with a wide array of superficial and invasive human diseases increasingly resistant to antibiotic therapies. Production of cathelicidin antimicrobial peptides (AMPs) by epithelial cells and phagocytes are a critical first line element of mammalian innate immune defense. Reduced susceptibility of SA to AMPs may contribute to its virulence potential. Methods: A random mutant library of SA strain Newman was generated by Tn917 transposition. Individual mutants were screened for increased susceptibility to murine cathelicidin mCRAMP. Tn917 insertions were mapped by single primer PCR amplification. Significance of candidate AMP resistance genes were confirmed by targeted plasmid integrational mutagenesis and complementation of the WT strain. Proteolytic activity was measured on skim milk agar and immune resistance in human whole blood survival assay. Results: Of 4,800 Tn917 mutants screened, 19 mutants showed > 4-fold increased sensitivity to mCRAMP, and 5 of these mapped to the S. aureus beta-lactamase (bla) locus, with mutation sites suggesting a primary role of the blaI gene encoding a transcriptional repressor. Targeted disruption of blaI in WT SA Newman and SA and WT MRSA strain Sanger 252 reproduced the phenotype of increased sensitivity to mCRAMP and human cathelicidin LL-37; WT resistance could be restored by complementation of the mutants with blaI expressed on a plasmid. Cathelicidin resistance correlated to secreted proteolytic activity of the respective strains, suggesting blaI regulation of an AMP degrading SA protease as an underlying mechanism. Inactivation of blaI reduced SA survival in human whole blood. Induction of SA by minute, subinhibitory concentrations of penicillin increased MRSA sensitivity to mCRAMP killing (P < 0.005). Conclusions: BlaI is known to repress the expression of the staphylococcal beta-lactamase gene blaZ. Here we describe that BlaI also acts as a immune evasion factor of S. aureus that contributes to cathelicidin resistance and delayed immune clearance. Pharmacological induction of BlaI expression may serve as an adjunctive therapy for MRSA.