The long-term objective of this research is to determine the mechanisms by which the environmental bacterium Listeria monocytogenes (Lm) maximizes life within mammalian cells through the host-induced expression and secretion of bacterial virulence factors. Lm is a facultative intracellular bacterium that survives as a saprophyte in soil but transitions into a pathogen upon entry into a mammalian host. Lm remains an increasingly important agent of serious food-borne invasive infections and has been responsible for some of the largest food safety recalls in U.S. history. Key to the success of Lm in establishing infection is the elaboration of numerous surface proteins and secreted virulence factors. While the process of protein secretion across the inner and outer membrane has been well studied in Gram-negative bacteria, much less remains known about the process in Gram-positives where proteins must fold in the environment-exposed space between the membrane and the thick Gram-positive cell wall. PrsA2 is a secreted post-translocation chaperone that is dispensable for bacterial growth in broth culture but essential for Lm virulence. Lm strains lacking PrsA2 function are severely attenuated in mouse models of infection, exhibit reduced secretion and activity of a number of virulence factors, and are significantly more sensitive to drugs that target the bacterial cell wall. PrsA2 and its related family members in other Gram-positives are attractive drug targets given that PrsA is surface exposed and inhibition of PrsA function not only impairs virulence factor secretion but also increases sensitivity to a number of antibiotics. This proposal will mechanistically define the role of PrsA2 in regulating the activity of Lm secreted proteins and in maintaining cell wall integrity. The working hypothesis is that PrsA2 regulates the proper folding, stabilization, and activity of proteins that are induced and secreted during host infection, thus contributing to bacterial virulence as well as viability within the host cytosol, whereas the related chaperone PrsA1 contributes to survival under stress conditions that lead to protein aggregation.
Aim 1 will decipher multiple facets of PrsA1/PrsA2 activity based on structural analyses and targeted mutations. The PrsA1 crystal structure has been used to model PrsA2 and to construct targeted mutations within the PrsA1/2 foldase, PPIase, membrane binding and dimerization regions. Characterization of Lm PrsA1/PrsA2 variants will define how different attributes of PrsA1/2 function contribute to Lm physiology and virulence.
Aim 2 will elucidate fundamental aspects of PrsA2 function based on substrate interactions. Experiments outlined in this aim seek to define the molecular basis of PrsA2-substrate recognition and folding.
Aim 3 will examine how loss of Lm cytosolic viability resulting from chaperone deletion impacts immune signaling, and will provide a proof of principle experiment of chaperone mutants as vaccine vectors.
The aims set forth in this proposal will provide insight into essential yet poorly defined aspects of Gram-positive protein secretion and cell wall homeostasis and will have broad relevance not only for Lm but for other important Gram-positive bacterial pathogens.

Public Health Relevance

Listeria monocytogenes continues to be an increasingly significant health threat as it has been associated with numerous multi-state food-borne outbreaks that have resulted in thousands of illnesses and several hundred deaths within the past few years. The largest and most expensive recalls of food products in history have occurred as a result of L. monocytogenes contamination, and the bacterium generally ranks as the third or fourth most common cause of bacterial meningitis in North America. This proposal is focused on deciphering how L. monocytogenes secreted chaperones contribute to the elaboration of active virulence factors critical for intracellular bacterial survival as well as bacterial stress resistance; this Information will have direct relevance for similar systems in other Gram-positive bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Mills, Melody
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University of Illinois at Chicago
Schools of Medicine
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