Bacterial Pathogenesis, Host-Pathogen Interactions at the Molecular and Cellular Level
Our laboratory is interested in the pathogenesis of bacterial lung infections; such as, tuberculosis and Legionnaires' disease. We are examining the virulence mechanisms of bacteria using cellular, molecular and genetic techniques. Our primary research goal is to obtain a better understanding of the roles of the pathogen and host in disease. These studies should contribute to our understanding of host-pathogen interactions at the molecular and cellular level. We hope that through a better understanding of the mechanisms by which these organisms cause disease we can prevent some, if not all, of these infections in the future.
Mycobacterial research in our laboratory also focuses on the mechanisms of entry into eukaryotic cells. The majority of our studies have been carried out on the rapid-growing mycobacteria Mycobacterium marinum due to its ease of use, rapid growth, high frequency of homologous recombination and genetic relatedness to M. bovis and M. tuberculosis. In addition, we are currently conducting virulence studies in M. bovis, M. tuberculosis and M. avium. Use of a rapid-growing pathogenic mycobacteria has allowed more timely progress in our virulence studies than would be possible using other mycobacterial species. Our current studies using these organisms have resulted in the determination of novel growth conditions that dramatically effect the entry mechanisms of mycobacteria. In addition, we have found that pathogenic and nonpathogenic mycobacteria differ in the frequency and mechanisms of entry into monocytes. We used this information to determine the specific genes involved in monocyte entry. Through the use of RICE and other molecular systems we have identified approximately six loci containing more that 15 different genes that affect entry. In addition, we have begun to screen a saturated transposon library for defects in entry and identified two additional loci after screening only about 4% of the M. marinum genome. These observations suggest that, similar to Legionella, a large number of mycobacterial genes are involved in entry into host cells. We plan to characterize the role of each of these genes in virulence using both in vitro and in vivo virulence models. As we develop a better understanding of the biological function of these genes using our rapidly growing mycobacterial species, M. marinum, we plan to evaluate the relevance of those genes that we consider most important to the disease with the highest impact in humans, M. tuberculosis. We feel that this is the most rapid and cost-effective method for investigating the pathogenesis of the slow-growing, yet extremely important, pathogen M. tuberculosis. Through examination of the mechanisms of entry and the factors that regulate them, we hope to further our understanding of how mycobacteria cause disease as well as provide insight into novel methods for their prevention.
Our laboratory has focused on investigation of the mechanisms of Legionella pneumophila entry into host cells. The ability of these bacteria to gain access to the intracellular compartment is critical to Legionella pathogenesis. It remains unclear whether the mechanisms of entry utilized by Legionella, as well as mycobacteria, to enter macrophages, their primary host cell, are critical to subsequent intracellular events. In order to better understand the entry mechanisms used and their effects, we have begun to identify both the bacterial and host genes involved in this process. By examining both sides of this interaction we hope to dissect it at the molecular and cellular levels. Clearly entry into macrophages is a complex process involving multiple bacterial and host cell components. This is due to the fact that multiple receptors are present on macrophages that naturally bind bacteria and other foreign particles in a relatively non-specific manner, i.e. mannose-, LPS-, complement-, Fc- and surfactant-receptors. In addition, bacteria commonly have multiple adherence factors. Thus, we plan to make mutations in both the bacterial and host genes involved to allow evaluation of each mechanism in subsequent intracellular events as well as the disease process. At present, we have identified more than 25 Legionella genes that play a role in entry. The majority of these genes were identified through the use of a novel molecular approach developed in our laboratory, designated Replicating and Integrating Controlled Expression (RICE) systems. Many of the determinants isolated are involved in processing, secretion and regulation of proteins involved in entry. However, our laboratory is primarily interested in those bacterial proteins that may interact directly with host cell proteins. This approach allows us to focus on those genes that are more likely to have evolved specifically for pathogenic interactions. We have constructed in-frame deletions in five of the genes identified, rtxA, enhA, enhB, enhC and enhD. All five of these genes affect the ability of Legionella to enter macrophages and an environmental host for Legionella, Acanthamoeba castellanii. Further characterization of all five of these genes at the molecular, biochemical and cellular level is ongoing. In addition, we have recently found that the Legionella strains currently used for investigation of pathogenesis differ in a number of genes that effect virulence. There are three strains used by nearly every laboratory throughout the world. We have found that none of them are genetically identical and that the differences affect adherence, entry, intracellular replication and virulence in mice. These differences must be taken into account when evaluating data obtained from different laboratories, point toward the need for a standard Legionella strain to be used in all laboratories and provide clues to the role of these genes in pathogenesis.
The Host (Phagocytes)
In order to better understand the host cell factors that are involved in bacterial uptake mechanisms and immunity to Legionella infection we have been taking advantage of the environmental host, A. castellanii. This host cell has several advantages to working with mammalian cells, though we do work with six human and three murine monocytic cell lines in our laboratory. Since amoebae are single-celled and relatively easy to grow and maintain in the laboratory, they represent a useful model system for understanding the virulence mechanisms of L. pneumophila. Because of their ease of manipulation, Acanthamoeba have also been utilized extensively as a model system for basic cell and molecular biological studies. In order to characterize the host-side of the L. pneumophila entry process, we have developed a selection for amoebae mutants that affect this host-pathogen interaction. We have already identified six L. pneumophila-resistant A. castellanii (Lra) clones. Based on their morphology and defects in L. pneumophila adherence, entry, intracellular replication and lysosomal fusion, these clones were classified into four Lra phenotypic groups. Proteomic analysis of these mutants has allowed identification of at least three proteins that differ between these mutants and wild-type amoebae. Characterization of the molecular defects involved is quite important, since these amoebae are the first A. castellanii mutants isolated that affect infection by L. pneumophila and possibly the first A. castellanii mutants of any kind. In addition, we have developed a selection that allows us to isolate populations of mammalian monocytic cells that lack receptor(s) of interest. This approach combined with microarray analysis of gene expression in different macrophage cell lines and transfection of dominant positive and negative receptor mutant genes should allow us to evaluate the role of particular receptors in entry into mammalian cells. Thus, we are taking a multi-faceted approach to the understanding of the interactions of bacteria with phagocytes from the perspective of both the bacterium and its host.