We employ microbiological, molecular, biochemical, and cell biological approaches to analyze the detailed architecture and complex genetic regulation of specific virulence determinants of Pseudomonas aeruginosa and Mycobacteria tuberculosis and to understand how these virulence determinants interface with the mammalian host. Both organisms cause serious pulmonary and systemic infections with high mortality rates. The major foci of my research include, the molecular mechanisms of iron and oxygen regulated virulence gene expression in these organisms and the structure-function relationships of distinct phospholipase C (PLC) enzymes expressed by these pathogens and by mammalian cells in response to a variety of stimuli.
Phospholipase C is a powerful signaling enzymatic activity in eukaryotic cell and in P. aeruginosa and M. tuberculosis. PLCs are considered to be potent virulence factors for these pathogens. In eucaryotic cells, enzymes with specific activity against phosphatidylcholine (PC-PLCs) and sphingomyelin (Smase’s) have been associated with critical signal transduction mechanisms in eukaryotic cells. They play a key role in eukaryotic cell transformation, induction of mitogen activated protein (MAP) kinases, IL-4 receptor signaling, induction of nitric oxide synthesis and programmed cell death (apoptosis). We are actively engaged in further characterizing the molecular and cellular biology of PC-PLCs and SMase’s produced by P. aeruginosa and M. tuberculosis. It is remarkable that these microbes express proteins (i.e. PLCs and SMase’s) that outright mimic eukaryotic proteins known to be critical to the production of powerful second messenger signaling molecules in eukaryotic cells. Furthermore, we have recently determined that these bacterial PLCs are also biosynthetic enzymes and are capable of synthesizing sphingomyelin (SM Synthase) that may protect these organisms from host defenses and may disrupt lipid rafts of host cells. This is an extremely clever strategy for an invading microbe to usurp the normal cellular processes of the host by expressing a protein with such a powerful signaling potential, at an inappropriate time with respect to the host. Further studies are directed at understanding how the expression of the microbial PC-PLCs, SMase’s and SM Synthase impact on the eucaryotic cell and how the expression the PC-PLCs and Smase’s by the mammalian host impacts on the pathogenesis of an infection by these microbes. More recently, we have identified that the novel Sec-independent Twin Arginine Translocase system is required for the secretion of these PLCs and other extracellular virulence determinants. Moreover, we determined that this novel secretion system is required for the induction of inflammatory processes in chronic pulmonary infection models. Accordingly, we believe that the Twin Arginine Translocase is an attractive target for the development of novel antimicrobial agents or that TAT mutants may be developed into live vaccines against a variety of diseases including tuberculosis, anthrax and plague.
We are also currently analyzing the molecular architecture of an iron dependent repressor, which is central to iron and oxygen regulated gene expression in a variety of prokaryotic pathogens. The current model relating to this repressor, designated ferric uptake regulator (Fur), is that it is only active when bound to reduced iron (Fe(II), and then it binds to a specific operator in iron regulated genes. Recent data from our laboratory indicate that this regulatory protein has an extremely profound and complex impact on the physiology and pathogenic potential of P. aeruginosa. We have recently determined the crystalline structure of Fur and we are further analyzing its novel architecture and function in detail. Another major effort is directed at further characterizing the multitude of genes we discovered that are targets of Fur repression. Finally, we have identified two small RNA molecules (sRNA) which are regulated by Fur and potentially plays a vital role in iron homeostasis and defenses against oxidative stress. Our goal is to examine the mechanism by which this sRNA specifically controls the gene expression at the post transcriptional level.