PhD Food Science defence "IMPACT OF PROTEOBIOTICS SECRETED BY LACTOBACILLUS ACIDOPHILUS LA-5 ON CLOSTRIDIUM DIFFICILE VIRULENCE FACTORS"
Date and Time
Food Science lecture room 128
Final Examination for the Degree of PhD - AFSANEH NAJARIAN
Dr. Loong-Tak Lim, Chair
Dr. Mansel Griffiths, Co-Advisor
Dr. Milena Corredig, Advisory Committee Member
Dr. Gisele LaPointe, Department Member
Dr. Jeremy Burton, External Examiner
TITLE: IMPACT OF PROTEOBIOTICS SECRETED BY LACTOBACILLUS ACIDOPHILUS LA-5 ON CLOSTRIDIUM DIFFICILE VIRULENCE FACTORS
ABSTRACT: Clostridium difficile is a leading pathogen of hospital-associated diseases including infectious diarrhea, toxic megacolon, and pseudomembranous colitis. Increasing frequency and severity of diseases besides inconsistent results of antibiotic therapy, demand alternative therapeutic candidates using a different strategy to control pathogenicity of C. difficile. Unlike antibiotics, antivirulence compounds disarm pathogens and selectively impose weaker pressure on them while do not develop antibiotic-resistant. Reducing essential virulence factors in C. difficile including toxin production, adherence, and biofilm formation could substantially minimize its pathogenicity and lead to a faster recovery from the disease.
This study aimed to investigate antivirulence effects of bioactive peptides namely La-5 proteobiotics produced by Lactobacillus acidophilus La-5 on the pathogenicity of C. difficile. Three clinically important strains of C. difficile were incubated with or without reconstituted La-5 cell-free supernatant (CFS). The C. difficile culture filtrates were collected for the assessment of quorum sensing, and expression of several virulence genes performing reverse transcription q-PCR. C. difficile attachment and cytotoxicity in human epithelial cells were monitored in vitro using human epithelial cells HT-29 and Caco-2 monolayers. Additionally, L. acidophilus La-5 was incubated in a medium containing whey protein, and inhibitory effects of its CFS were tested performing quantitative biofilm assay then visualized in scanning electron microscopy (SEM). Proteobiotics caused a significant down-regulation in expression of some of the virulence genes mainly two essential toxin genes tcdA and tcdB and adhesion gene cwp84 in all C. difficile strains. A substantial down-regulation in luxS expression was observed in ribotype 027 and 078 (P<0.05). La-5 proteobiotic also reduced cytotoxicity and cytopathic effect of C. difficile culture filtrate on HT-29 and Caco-2 monolayers significantly (P < 0.0.5). Furthermore, pre-incubation of cell monolayers and La-5 CFS reduced attachment of the C. difficile bacterial cells on both cell monolayers. The biofilm formation and population of C. difficile cells encased in biofilms were significantly decreased within the treated cultures (P < 0.0001). SEM analysis also confirmed quantitative results showing a sufficient reduction in biofilm formation. Our study suggests that La-5 proteobiotics could potentially be used to cure C. difficile infection, prevent relapses, and improve disease outcome.