Lysozyme resistance of Listeria monocytogenes

Bacterial pathogens must survive exposure to antibacterial molecules of the innate immune system in order to successfully establish infection. Such is the case with Listeria monocytogenes, a Gram-positive bacterial pathogen that is the causative agent of listeriosis, a deadly foodborne disease dangerous to pregnant women and immunocompromised individuals. L. monocytogenes is highly resistant to antibacterial molecules of the innate immune system such as lysozyme, a potent cell wall-degrading enzyme found throughout the body of all animals. Nearly 100 years ago, Alexander Fleming discovered lysozyme and observed that pathogenic bacteria were lysozyme-resistant, while non-pathogens were lysozyme-sensitive. Curiously, non-pathogenic lysozyme-sensitive bacteria encode the same cell wall enzymes as pathogenic lysozyme-resistant bacteria. It therefore remained unclear what distinguished pathogens from non-pathogens in regards to lysozyme resistance. In this work, a forward genetic screen was performed to identify additional factors required for lysozyme resistance in L. monocytogenes and subsequent investigations were made to uncover how these molecules regulated one another./nThe screen for lysozyme-sensitive mutants identified a number of cell wall-related enzymes, such as the peptidoglycan deacetylase pgdA; however no enzymes were identified that were unique to L. monocytogenes. Rather, the screen identified two regulators of gene expression, the non-coding RNA Rli31 and the transcription factor DegU, which upregulated expression of cell wall-modifying enzymes. Mutants of rli31 and degU were similarly sensitive to lysozyme as mutants of pgdA, suggesting that the regulators were absolutely required for lysozyme resistance. These data suggested that high lysozyme resistance of L. monocytogenes was not due to the acquisition of novel cell wall-modifying enzymes, but was due to upregulation of cell wall enzymes that were distributed among both pathogenic and non-pathogenic bacteria. This logic provides an alternative example of how virulence phenotypes are acquired by bacteria, many of which originate via horizontal gene transfer. We propose that pathogen-specific regulation of broadly distributed genes represents another mechanism by which pathogens acquire virulence./nNon-coding RNAs are an increasingly appreciated class of gene regulators in bacteria, with over 200 non-coding RNAs having been identified to date in L. monocytogenes. The mechanism of Rli31 function was investigated and multiple lines of evidence suggested that the uncharacterized protein SpoVG was as an intimately related, opposing regulator to Rli31. Neither Rli31 nor SpoVG was epistatic with any of the fourteen lysozyme-sensitive mutants, and the data suggested that Rli31 and SpoVG both regulated an unknown lysozyme-resistance gene. The cell wall morphology of the spoVG mutant suggested that this unknown gene may be required for producing extracellular polysaccharide. Separately from the lysozyme-resistance phenotypes of these mutants, Rli31 inhibited expression of the carbohydrate import protein Lmo0901, while SpoVG was independently required for Lmo0901 expression. The 5’ UTR of SpoVG contained 14/14 nucleotides of perfect complementarity to Rli31, yet Rli31 did not regulate SpoVG mRNA nor protein abundance. This suggested that the SpoVG mRNA may function as an Rli31 decoy target that regulates its activity. In summary, these data described a complex model whereby Rli31 and SpoVG independently and oppositely regulate multiple target genes, but may also regulate one another./nAttempts were made to better characterize SpoVG, whose function remained unclear. SpoVG was required for swarming motility of L. monocytogenes, and suppressor mutations of this phenotype mapped to RNAse J1, Rho, and NusG. SpoVG was then described as an RNA-binding protein that interacted with multiple sRNAs in vitro but did not bind RNAs with known protein-binding partners such as SRP RNA. Together, these results suggested that SpoVG is a pleiotropic RNA-binding protein that interacts closely with sRNAs, and we propose that SpoVG is similar in function to the well-described RNA chaperone Hfq in Gram-negative bacteria./nIn conclusion, this study described the genes required for lysozyme resistance of L. monocytogenes, and identified a complex regulatory mechanism required for their expression. From these data, we determined that high lysozyme resistance in pathogens is due to upregulation of common cell wall enzymes. We conclude that the non-coding RNA Rli31 and the RNA binding protein SpoVG are important regulators of lysozyme resistance genes in L. monocytogenes. Together, this study provides a comprehensive examination of lysozyme resistance genes in a bacterial pathogen.

Advisor: Portnoy, Daniel A