Regulation of virulence gene expression in B. anthracis
Anthrax progresses rapidly and asymptomatically to fatal systemic infection characterized by massive bacteremia. The fact that B. anthracis readily grows in human blood (serum) suggests that this pathogen is very efficient in acquiring nutrients from nutrient-limited serum and is intrinsically resistant to the bactericidal activity of serum. However, the physiology of B. anthracis during its growth in human blood remains essentially unknown.
I have analyzed the gene expression profile of B. anthracis cells grown in human serum as compared to that grown in rich medium using microarrays. About ~380 B. anthracis genes are identified as being differentially expressed (> 2 fold) in human serum, revealing that certain metabolic pathways (e.g., nucleotide and amino acid biosynthesis) become critical for the growth of B. anthracis in human serum. Of particular interest are six genes, each of which encodes a putative transcriptional regulator with unknown function. DNA and amino acid sequence analyses of one putative transcriptional regulator suggests that this regulator, together with its downstream unannotated small open reading frame, constitutes a novel peptide-based quorum sensing system in B. anthracis.
In most peptide-based quorum sensing systems known to date, a signal peptide is produced as pre-pro-peptide, which is processed into pro-peptide during secretion and further into mature signal peptide outside of the cell. The signal peptide is typically reimported into the cell, in which it binds and activates its cognate transcriptional regulator, controlling expression of target genes.
The peptide-based quorum sensing system has only been identified in Gram-positive bacteria, and it regulates expression of genes involved in various cellular processes such as conjugation, biofilm formation, and virulence. However no such system has been found in B. anthracis. Currently I am characterizing the putative quorum sensing system consisting of the regulator named aqsR (anthrax quorum sensing Regulator) and its cognate signal peptide aqsP (anthrax quorum sensing Peptide). A mutant strain deleted for aqsR displays three notable phenotypes: its growth in human serum is impaired, it is hypersusceptible to cationic antimicrobial peptides, and its virulence is attenuated in the mouse model of pulmonary anthrax. These findings are the first evidence suggesting that the peptide-based quorum sensing system controls virulence gene expression in B. anthracis.
I plan to characterize the AqsR-AqsP system in detail. Specifically, I will identify genes regulated by AqsR, determine the role of AqsR-regulated genes, and establish the molecular mechanism of AqsR-AqsP action. A first submitted R01 grant proposal entitled “peptide-based quorum sensing controlling virulence in B. anthracis” is currently funded via R56.
Antimicrobial peptide resistance in enteric Gram-negative bacteria
This part of my research is focused on mechanisms of Gram-negative bacterial resistance to human antimicrobial peptides such as -helical LL-37 (also called cathelicidin) and -sheet defensin (e.g., human neutrophil peptide; HNP-1) produced by human neutrophils. It is well known that bacterial resistance to these endogenous peptides is critical for bacterial survival in the host; however, resistance mechanisms for defensin, in particular, remain largely unknown.
We have previously developed a microarray-based genomic technique, named MGK for monitoring of gene knockouts1. MGK can simultaneously track the abundance of thousands of mutants competitively grown in a control and a selective condition, and identify mutants with growth (dis)advantage in a selective condition.
Applying MGK to an E. coli deletion mutant library (known as Keio library)2, we have identified E. coli genes previously unknown for their roles in defensin resistance. Two genes identified encode a protein (YibP) that activates the amidase activity of both AmiA and AmiB, and a transcriptional regulator (CysB) only known to activate expression of cysteine biosynthetic genes. Inactivation of yibP leads to hyper-susceptibility, whereas cysB knockout confers hyper-resistance to antimicrobial peptides in E. coli and S. enterica. The immediate goal of this project is to investigate how YibP and CysB contribute to antimicrobial peptide resistance at biochemical and genetic level, and determine their contribution to Salmonella pathogenesis.
In addition to the major projects outlined above, I am also pursuing collaborative research. A current collaborative R21 project, with Dr. Peter Zuber at Oregon Health & Science Univ., investigates the role of Spx paralogs in B. anthracis stress response, and I as co-investigator provide my expertise in microarray experiments and data analyses. Dr. Taeok Bae in Indiana Univ. Medical School has created a defined library of Staphylococcus aureus transposon mutants (called Phoenix library). Using MGK and the Phoenix library, a collaborative R21 proposal (submitted in June, 2011) aims to identify Staphylococcal genes essential for survival in human neutrophils and mouse models of infection.