MCDB Home > Faculty > David Low > Current Research

My current major research goals are to:

• Identify genes within the DNA adenine methylase (DAM) regulon of bacterial pathogens that play important roles in virulence
• Determine the roles of DAM in regulating gene expression and virulence of bacterial pathogens
• Understand at the biochemical level how bacterial pathogens regulate adhesin gene expression in response to host environmental stimuli such as temperature and amino acid levels
• Explore the influence of bacterial "chromatin" structure on activation and silencing of transcription
• Identify and understand the mechanisms of action of virulence factors that facilitate the colonization of the urinary tract

Our laboratory has focused on the regulation of the pyelonephritis-associated pili (pap) operon, which codes for hair like fibers on the bacterial cell surface that play a critical role in attachment of uropathogenic E. coli to mucosal tissues in urinary tract infections. The expression of these pili is regulated by a heritable switch mechanism in which some cells in an E. coli population actively transcribe pap pilin whereas other cells do not. We have shown that the pap phase switch operates by a mechanism involving DNA methylation pattern formation. In contrast to all other known genetic switch mechanisms, Pap phase variation occurs in the absence of DNA sequence rearrangements or changes in the base-pair sequence, and thus is epigenetic.

Our initial studies on Pap phase variation were the first to show that bacteria have DNA methylation patterns, and that these methylation patterns are directly involved in the regulation of gene expression. More recent studies have focused on understanding the biochemical basis for Pap fimbrial phase variation. The Pap switch mechanism is quite complex, involving at least six regulatory factors: leucine-responsive regulatory protein (Lrp), PapI, CAP, PapB, and deoxyadenosine methylase (Dam). At the core of this genetic switch is the translocation of Lrp, a 19 kDa DNA binding protein, between binding sites located over 100 base-pairs apart. This translocation regulates the DNA methylation pattern by virtue of the fact that a GATC sequence is present within each Lrp binding site. Binding of Lrp inhibits methylation of these GATC sites by Dam. Conversely, methylation of the adenosine residue of a single GATC site inhibits the cooperative binding of Lrp. In phase OFF cells, Lrp binds with highest affinity to Lrp binding sites 1-2-3 containing GATC-prox (proximal to the papBA promoter). Thus, GATC-prox is fully nonmethylated and GATC-dist (distal to the papBA promoter, 100 base-pairs upstream of GATC-prox) is methylated (the phase OFF methylation pattern). Under environmental conditions that induce PapI, this small (8 kDa) regulatory protein binds specifically to Lrp-pap DNA complexes (but not to free Lrp), decreasing the affinity of Lrp for sites 1-2-3, and increasing Lrp's affinity for upstream sites 4 and 5, which contain the GATC-dist site. Dam methylase is also required for the phase OFF to ON transition by methylating GATC-prox Current genetic evidence indicates that methylation of this promoter-proximal GATC site may alter interaction of H-NS with pap DNA sites overlapping sites 1-2-3, indirectly decreasing the affinity of Lrp for this pap DNA region. Following Lrp translocation, GATC-prox becomes methylated by Dam and the GATC-dist site is protected from methylation due to binding of Lrp. This forms the phase ON methylation pattern in which GATC-dist is fully unmethylated and GATC-prox is methylated. We have developed a number of genetic and biochemical approaches to identifying the regions of PapI and Lrp that interact with each other. Our overall goal is to understand how PapI induces the translocation of the Lrp-pap nucleoprotein complex from sites 1-2-3 to sites 4-5-6. Many different bacterial fimbrial systems are under phase variation control, most operating epigenetically similar to pap. To determine the roles of phase variation in pathogenesis we studied the pef operon in Salmonella typhimurium, whose Pef fimbrial product plays a role in attachment of these pathogens to the mouse intestine. Our goal is to disrupt normal switch control by altering switch frequencies by mutation, and to determine what effects these mutations have on the disease process.

We have identified nucleoid-associated proteins including H-NS that silence pap transcription in response to low temperature as well as activate transcription at 37°C. It appears that these chromosome-binding factors alter the structure of DNA which, in turn, affects the interactions between Lrp, PapI, pap DNA, and RNA polymerase. In addition, methylation of GATC-prox, which is required for transcription, alters the interaction of these factors with pap DNA. These protein-DNA interactions share certain features with gene regulation in eukaryotes, in which acetylation of histones and methylation of DNA alters protein-DNA interactions to control transcription.

Since arriving at UCSB I have collaborated with Drs. Mike Mahan and Bob Sinsheimer in investigations of the roles of DNA adenine methylase in the virulence of Salmonella, Yersinia, and other enteric pathogens. A major goal of this work is to develop vaccines and antimicrobials against a wide variety of pathogens. Towards this end, Drs. Mahan, Sinsheimer, and I have helped start a company (Remedyne) to develop real world applications of this technology.

Recently, we have collaborated with Diane McClure (Campus Veterinarian) to develop an animal model of urinary tract infections in which E. coli that naturally cause urinary tract infections in rats. We have identified a rat uropathogen that can colonize the intestines and urinary tracts of these animals. We plan on using this model to assess the roles of adhesins and other bacterial products on the pathogenesis of urinary tract infections. Our long-range goal is to develop antimicrobials and vaccines that are effective at treating and preventing urinary tract infections, respectively.