Ribosomal Hibernation as a Novel Biological Mechanism that Promotes Listeria Persistence During Treatment with Antibiotics and Disinfectants

<p>NON-TECHNICAL SUMMARY:<br/> Listeria monocytogenes (Lm) is a significant public health hazard, responsible for 2,500 cases of food-borne illness and 500 deaths annually in the US. A major challenge to combating Lm transmission and infection is this ubiquitous organism's ability to survive in a variety of environments including soil, processed foods, and within persistent biofilms that are highly resistant to killing by disinfectants. Despite the significance for human health, relatively little is known about the molecular determinants that facilitate Lm persistence. Our lab has identified a conserved ribosome factor called Hibernation Promoting Factor (HPF) that is critical for Lm survival during treatment with disinfectants and clinically relevant antibiotics. The objective of this proposal is to better understand how HPF allows Lm to survive harsh environments and
resist killing by antimicrobial agents. The proposed studies are designed to i) identify conditions that trigger HPF activity ii) evaluate the requirement for HPF in biofilm formation and resistance, and iii) define the proteins whose translation is regulated by HPF. Since HPF is conserved in nearly all foodborne pathogens, the overall expected outcome of these studies is the development of control strategies broadly applicable to bacterial pathogens.<p>
APPROACH: <br/>Specific Aim 1 Methods: Survival curve analyses will be performed on wildtype and hpf cultures grown in defined minimal media. Specifically, CFU will be determined from stationary phase cultures treated with the following stress conditions: carbon starvation (1% w/v ?-methylglucoside, a competitive inhibitor of glucose uptake), oxidative stress (50mM hydrogen peroxide), acid stress (pH 2.5), salt (4% NaCl w/v), ethanol (4% v/v) cold shock (4°C), and heat shock (42°C). <br/>Specific Aim 2 Methods: To test the hypothesis that HPF contributes to resistance in biofilms, wildtype and hpf biofilms will be evaluated for resistance to benzalkonium chloride (BKC) and cadmium chloride (CDC) under conditions that simulate the food-processing environment. In addition, the ability of the mutant to form biofilms will be evaluated using crystal violet staining
which provides a quantitative measure of biofilm density. Wildtype and mutant strain biofilms will be formed on stainless steel and Teflon coupons or in microtiter plates in a manner previously described43. Briefly, the coupons and the microplate wells are seeded with stationary phase Lm suspensions (108 CFU/ml) for 3 hours at 37°C, washed to remove unattached cells, and then incubated at 37°C in rich media without agitation for 48 hours. The resulting biofilms will be repeatedly exposed to BKC or CDC in a manner that simulates the food-processing environment. Specifically, every 24 hours, the coupons are exposed to the disinfectant for 60 seconds, then stored in water for 15 hours at 22°C to simulate starvation, and incubated in growth media for 8 hours. Given the enhanced resistance of biofilms to killing by antimicrobial agents BKC and CDC will be added to each sample in
1X, 5X, and 10X concentrations of those used in the studies involving planktonic cells (preliminary results). CFU will be enumerated every 24 hours for 1 week by dislodging the adherent cells with a swab soaked in a peptone solution containing 0.1% tween 80. As an alternative way to assess the role of HPF in biofilm resistance, cells from untreated coupons will be dislodged, washed, and resuspended in saline to assay their susceptibility to the disinfectants. These latter analyses will determine whether cells within the biofilm are phenotypically different from planktonic cells. Finally, the ability of HPF to form biofilms and the effect of each disinfectant condition on biofilm density will be assessed by staining wells from the microtiter plate with a 0.8% crystal violet solution, washed, dried, and destained with ethanol. The biofilm density of each well is then determined by reading
the absorbance at 580nm. <br/>Specific Aim 3 Methods: To identify factors that are regulated post-transcriptionally by HPF, stable isotope labeling of amino acids in cell culture (SILAC) will be performed on stationary phase hpf, IPTG-inducible hpf-overexpressing, and wildtype cultures according to a standard protocol51, 52. HPF overexpression is achieved by adding IPTG to the media 15 minutes prior to analysis. Here, newly synthesized proteins are labeled in vivo by supplementing the growth media with lysine and arginine (the "light" culture), or with lysine and arginine that have been labeled with stable heavy isotopes of carbon and nitrogen (the "heavy" culture). The hpf cultures are then combined with the wildtype cultures and processed for analysis by mass spectrometry. The incorporation of stable-isotopes allows for peptides with the same sequence present in the wildtype and hpf
preparations to be differentiated on the basis of differences in mass. Data will be analyzed in collaboration with Dr. Lori Kohlstaedt at the California Institute For Quantitative Biosciences Proteomics/Mass Spectrometry Laboratory here on the UC Berkeley campus. The relative abundance of a protein is determined from the ratio of peak intensities in the mass spectrum for each peptide. Student's T-test is used to identify which peptides are significantly altered in the absence (hpf) and presence (WT or HPF-overexpression) of hibernating ribosomes. Evaluation Plan. Two key milestones of this project will be collaborating with Dr. Lisa Gorski at the USDA to identify environmental conditions where ribosomal hibernation is required for survival and evaluating mutant hpf biofilms (please consult the Timeline above). Once these are met, the first manuscript from this work will be prepared
for publication. The third milestone is completion of analysis of the ribosomal hibernation proteome by SILAC and mass spectrometry. We anticipate 1-2 further manuscripts from the proteomics work identifying genes whose translation is regulated by the process of ribosomal hibernation. To monitor progress toward these milestones, regular review of my work will be carried with my mentor Dr. Portnoy and my collaborator Dr. Gorski. This will include presentation at lab meetings and department seminars, and meetings to discuss data, experimental procedures, and research goals. Dissemination plan. Results from this study will be published in peer-reviewed journals and presented at professional meetings.</p>