In a complex environment, higher organisms face the constant threat of microbial infection.  Effective first lines of defense against infectious pathogens comprise the innate immune system.  A key component of innate immunity is the production of small, cationic antimicrobial peptides (AMPs), a protection strategy conserved from insects through man.  In mammals, gene families encoding AMPs include defensins and cathelicidins.  Our AMP research efforts are the product of a fruitful collaboration with Richard Gallo, a UCSD dermatologist and investigator whose laboratory studies the cellular biology and immunology of the skin.  The Gallo laboratory has made several important advances in our understanding of the cathelicidin family of antimicrobial peptides, including the first purification of the porcine cathelicidin PR-39 and the discovery of the murine cathelicidin CRAMP.  Our collaboration adopts a combined mammalian and bacterial genetic approach to eludating the contributions of cathelicidins to host immunity.

 

Humans and mice each express a single cathelicidin, which are encoded by similar genes and have similar alpha-helical structures, spectra of antimicrobial activity and tissue distribution.  Cathelicidins effectively kill group A Streptococcus (GAS) in vitro, and cathelicidin production greatly is increased in the skin after infectious challenge with GAS.  To assess directly the function of cathelicidins in vivo, the Gallo laboratory generated mice that are null for CRAMP by targeted recombination.  When challenged with GAS sucutaneously, CRAMP-deficient mice developed much larger areas of necrotic infection the wild-type mice and failed to clear the replicating bacteria from the wound.  To corroborate this finding, we identified a GAS transposon mutant with increased resistance to cathelicidin killing in vitro.  The transposon insertion mapped to a protein with features of a transcriptional regulator and was confirmed by targeted plasmid integrational mutagenesis.  Mice infected with this mutant developed lesions of larger size and longer duration than those infected with the cathelicidin-sensitive GAS parent strain.  In effect, induction of cathelicidin resistance in the bacterial pathogen reproduced the phenotype of the CRAMP-knockout mouse.  The pattern of findings in the skin were mirrored in the results of whole blood killing assays.  These studies represented the first in vivo demonstration that endogenous expression of a mammalian antimicrobial peptide provides defense against an invasive bacterial infection.

Ongoing collaborative projects with the Gallo laboratory explore additional biological functions of the cathelicidin molecule and its regulation in response to infectious challenge, the molecular genetic and phenotypic basis of bacterial sensitivity or resistance to antimicrobial peptide action, and the impact of bacterial antimicrobial peptide resistance on bacterial virulence and the epidemiology of infectious diseases.

 

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