Performing Department
Molecular Biology
Non Technical Summary
Listeriosis has the highest mortality rate (20-25%) among foodborne diseases. The majorsource of food contamination by listeria is food processing facilities. At present, we poorlyunderstand factors allowing listeria to persist in such facilities despite intensive sanitationmeasures. It is generally known that exopolysaccharides (EPS) secreted by bacteria significantlyincrease their resistance to various chemical stresses and dehydration. However, until recently,listeria were believed to be unable to synthesize EPS. The Gomelsky and Miller laboratories in theMolecular Biology Department showed that listeria can synthesize a unique EPS that promotes cellaggregation (biofilms), and that bacteria "coated" by EPS are multi-fold more resistant todehydration and multi-million fold more resistant to commonly used disinfectants. We hypothesizethat this EPS plays an important role in listerial persistence in food processing facilities. In thisproject, we intend to test this hypothesis. We have identified and patented an enzyme thatdegrades listerial EPS.The goal of this project is to develop and characterize performance of a probe that can be used to detectEPSprevalence in produce storage and processing facilities.Thisprojectwill be carried out by a Ph.D. student supervised by a molecular microbiologist andbiochemist involved in listerial EPS discovery, and a food microbiologist.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The Gomelsky and Miller laboratories have recently discovered that Listeria monocytogenes, the causative agent of listeriosis, synthesizes a novel EPS. This EPS coats bacterial aggregates and significantly enhances listerial resistance to desiccation (dehydration) and disinfectants used in the food industry (Chen et al., 2014). At present, we don't know how prevalent the listerial EPS is in food-processing facilities and what role it plays in listerial survival. To address these questions, we intend to develop a method for listerial EPS detection and apply it to samples from listeria-contaminated facilities and from contaminated fresh produce (where EPS presence is expected). Because the likelihood that EPS enhances listerial survival in food processing facilities is high, we also plan to develop the means to degrade this EPS and prevent its formation de novo. We have identified, characterized and patented an enzyme, glycosylhydrolase PssZ from L. monocytogenes, which hydrolyzes listerial EPS. When purified PssZ protein is added to listerial cultures, it disperses pre-formed EPS-based aggregates (biofilms) and prevents new EPS formation, which makes bacteria vulnerable to disinfectants and desiccation (Köseoglu et al., 2015). For potential industrial applications, in the future we intend to identify PssZ analogs (homologs) with properties that are more compatible with sanitation solutions and/or produce washing solutions, than the properties of listerial PssZ. The Objective of this project is to develop a listerial EPS detection probe and begin determining the presence of EPS in food processing facilities with listerial contamination.
Project Methods
MethodsTwo approaches are commonly applied for detecting EPS. One approach relies on EPS-specific antibodies. A PNAG (poly-N-acetyl-glucosamine)-specific antibody for detecting EPS synthesized by Staphylococcus aureus and Staphylococcus epidermidis is a good example (reviewed in Skurnik et al., 2016). Another approach relies on non-antibody-based EPS recognition. Because sensitivities of antibodies are difficult to predict and we already have a candidate protein for the non-antibody approach, here we will focus on the latter approach. By using site-directed mutagenesis, we have constructed a PssZ mutant (PssZ E72Q) that retains EPS binding capacity but lacks hydrolytic ability (Köseo?lu et al., 2015). Here we intend to develop a listerial EPS-specific detection probe based on this mutant protein. As the main analytical tool for detecting listerial EPS via a non-antibody approach we will use surface Plasmon resonance (SPR), a sensitive and reliable method for detecting and monitoring interactions between biomolecules (Hoa et al., 2007; Mayer & Hafner, 2011). SPR measures the binding of molecules immobilized to the surface of a biosensor chip to their soluble binding partners via optically monitoring the change in refractive index near the sensor surface as the binding complex forms (Linman & Cheng, 2010). This technique has been applied successfully to examine interactions between carbohydrate and carbohydrate-modifying enzymes (Shen et al., 2003; Jelinek & Kolusheva 2004), and to detect various pathogenic bacteria (Fratamico et al., 1998; Bokken et al., 2003), including listeriae (Leonard et al., 2004) in contaminated food products and other sources. We will attach PssZ E72Q to a SPR chip via a commonly used biotin-streptavidin tethering method (Mayer & Hafner, 2011). The SPR device (Nicoya Lifesciences) will be available in the laboratory of a colleague in the Department of Molecular Biology. Assays will be optimized by varying the concentration of added EPS, buffer components, and temperature. We have described a method for listerial EPS purification from the L. monocytogenes EPS overproducing strain, ΔpdeBCD (Köseo?lu et al., 2015). In brief, bacterial aggregates cells will be collected from culture medium by centrifugation, resuspended and washed to remove media and sloughed cell components, suspended in water and boiled to remove cell-bound listerial EPS from the cell surface. EPS then will be quantified and concentrated by ultrafiltration prior to use in SPR experiments. To control for non-specific binding in the assays we will perform negative control experiments using related but non-cognate polysaccharides, such as carboxymethyl cellulose and poly-N-acetyl-glucosamine (available from Sigma-Aldrich), which are not synthesized by L. monocytogenes. A boiled preparation obtained from a non-EPS-producing laboratory strain, ΔpdeBCD ΔpssC, in which the EPS synthase enzyme gene, pssC, has been deleted (Chen et al., 2014), will also be used as a negative control.Expectations/ potential pitfalls/ alternative solutionsEPS-based biofilms significantly increase L. monocytogenes tolerance to desiccation and disinfectants, and EPS synthesis is expected to occur on plant-derived matter, yet the prevalence of EPS-containing biofilms in food processing facilities is completely unknown. Upon completion of Objective 1 we expect to generate a probe specific for listerial EPS and optimize EPS detection. If we face significant problems with the PssZ E72Q approach, we will turn to developing listerial EPS-specific antibodies. The purified EPS will be used to inoculate rabbits for production of polyclonal antibodies. In addition to using aspurified EPS, we will de-acetylate listerial EPS (using the protocol described by us earlier [Köseo?lu et al., 2015]) because de-acetylation has proved to be effective for enhancing immunogenicity of anti-PNAG antibodies (Skurnik et al., 2016). Polyclonal antibodies will be produced commercially. Upon the completion of Objective 1, if time permits, we will begin applying our detection system to determine the presence of EPS in listerial biofilms obtained from contaminated food processing facilities. Because EPS is poorly synthesized by listeria grown in rich culture medium (Chen et al., 2014), we will work directly with contaminated samples (and not with cultured cells). Samples from contaminated facilities will be obtained by using swabs soaked in sterile saline solution. We have made arrangements for obtaining listeriae-contaminated samples from the FDA. For sample collection we will work with the FDA and when permitted, with the management from the facilities experiencing L. monocytogenes contamination. For those samples that contain listerial EPS, aliquots will be set aside for culturing bacteria for strain archival and phenotypic analyses. Genotypic analyses of L. monocytogenes strains are now commonly done by the FDA. Super-EPS producing strains will be collected for future analysis that may involve measurements of the expression of the EPS synthesis pss gene cluster (by RT-PCR) and intracellular concentrations of c-di-GMP (by mass-spectrometry), the second messenger that activates EPS synthesis. The methodology for such analyses has been described by us earlier (Chen et al., 2014; Köseo?lu et al., 2015).