Regulation of Bacterial Virulence and Catabolism
Research in my laboratory is aimed at understanding the role of bacterial metabolism in regulation of both virulent and non-virulence interactions of bacteria with eukaryotes. The focus of current research is to understand the intertwined regulation of bacterial virulence with bacterial detection and metabolism of host-derived compounds during lung infection by Pseudomonas aeruginosa.
<strong>The organism and clinical importance:</strong>
Pseudomonas aeruginosa is a Gram-negative bacterium nearly ubiquitous in the environment. It is found in freshwater, soils, and man-made water fixtures (particularly faucets and showers), and is the most prevalent antibiotic resistant, Gram-negative, opportunistic pathogen of humans. However, as denoted by the term ‘opportunistic pathogen’, while we are continuously exposed to P. aeruginosa at a low level, healthy individuals do not develop disease. Infection by P. aeruginosa requires some breakdown of the innate immune system: immune deficiency, burns, chemotherapy, or inhibition of physical clearance mechanisms. Lung infection caused by P. aeruginosa is a source of significant morbidity and mortality in ventilated patients and is the primary cause of chronic progressive respiratory failure and death in patients with cystic fibrosis. It is the lung infections caused by P. aeruginosa that my lab studies.
<strong>Hypotheses guiding our research:</strong>
• Chemical communication between bacteria and eukaryotes alter the interaction.
• Bacteria can use host-derived metabolites to guide behaviors towards colonization, pathogenesis, symbiosis, or commensalism.
• The metabolic flexibility of P. aeruginosa enables it to thrive in the lung.
• Specific virulence genes of P. aeruginosa are induced by the lung environment, in particular, by metabolism of host-derived compounds.
• Development of novel agents to inhibit P. aeruginosa infections within the mammalian lung will require a thorough understanding of the regulation of bacterial virulence within the lung environment.
<strong>Below are specific projects currently underway in the laboratory:
The interaction between choline catabolism and P. aeruginosa virulence in the lung </strong>
Upon inhalation into the lung, P. aeruginosa comes in contact with the airway surface liquid, which is composed primarily of the phospholipid phosphatidylcholine (PC, ~75% of surfactant by weight). P. aeruginosa secretes phospholipases that can cleave PC, releasing the polar head-group (phosphorylcholine) and the lipid tail (diacylglycerol), both of which can be taken up by the bacterium and utilized as a nutrient source. The phosphorylcholine molecule (ChoP) can be further catabolized to glycine betaine (GB). GB, via the transcription factor GbdR, leads directly to transcriptional induction of one of the major PC-specific phospholipases, PlcH.
We have identified the GbdR transcription factor, the enzymes involved in choline catabolism in P. aeruginosa, and demonstrated the direct transcriptional regulation of plcH by GbdR. However, there remain many related questions, including:
• What other virulence factors are directly regulated by GbdR?
• Are there additional transcription factors that bind GB?
• Since GB can be catabolized, how does the bacterium regulate the balance between catabolism and induction of virulence?
These questions are being addressed using standard molecular genetics techniques, as well as cDNA microarrays, genetic screens, chromatin-affinity purification, and biochemical purifications.
<strong>Identification and characterization of novel genes induced by pulmonary surfactant.</strong>
Like many bacteria, the majority of open-reading frames in P. aeruginosa are predicted to encode proteins with no known or predicted function. Therefore, in any given genetic screen or microarray experiment, you will identify proteins that appear to be important for your system, but must be fully characterized in order to better understand them. Using a series of microarray experiments examining P. aeruginosa exposure to pulmonary surfactant, we have identified a number of candidate genes that are highly induced by surfactant, but have either have vague predictions of function or no predicted function. The goal of these projects is to exploit the tractability of the P. aeruginosa system (easy genetics, multiple sequenced genomes, mutant libraries) in conjunction with biochemical methods and bioinformatics to determine the function of these proteins of interest, many of which have homologues in other bacteria. Once a function is described, we are interested in understanding the transcriptional or post-transcriptional regulation of any genes that appear to be directly involved in interactions with eukaryotes.
<strong>Identification and characterization of unknown steps in regulation and metabolism of host-derived compounds.</strong>
While we are awash in genomic data and structural predictions for proteins, it is still staggering that >40% of bacterial proteins remain without a function. To advance our understanding of basic bacterial metabolism and begin to reduce that percentage, we have been using genetic screens and genome context-directed experiments to identify the genes that encode proposed metabolic or regulatory steps for which no gene is annotated.
Hampel KJ, Labauve AE, Meadows JA, Fitzsimmons LF, Nock AM, Wargo MJ 2014. Characterization of the GbdR Regulon in Pseudomonas aeruginosa. Journal of Bacteriology 196(1):7-15
Wargo, MJ 2013. Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection. PLoS One. 2013;8(2):e56850
Meadows JA and Wargo MJ 2013. Characterization of Pseudomonas aeruginosa growth on O-acylcarnitines and identification of a short-chain acylcarnitine hydrolase. Applied and Environmental Microbiology 79(11):3355-63
Fitzsimmons LF, Hampel KJ, Wargo MJ 2012. Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa. Journal of Bacteriology 194(17):4718-26
Fitzsimmons LF, Flemer S, Wurthmann AS, Deker PB, Sarkar IN and Wargo, MJ 2011. Small molecule inhibition of choline catabolism in Pseudomonas aeruginosa and other aerobic choline catabolizing bacteria. Applied and Environmental Microbiology 77(13):4383-9
All Wargo publications