Presentation Abstract

Session: 1-Poster Session 1 (A)
Thursday, Nov 08, 2012, 2:30 PM - 3:45 PM
Presentation Title: A096 - Study of Autolysins and Autolysis Patterns of Clostridium botulinum
Location: Microbiology
Scientific Discipline: + 8. Microbiology -> a. Bacteriology
Author(s): Jessica Jackson and Wei-Jen Lin, Ph.D.
California State Polytechnic University, Pomona, Pomona, CA
Abstract: Introduction: Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium that produces the most potent toxin known to man, the botulinum neurotoxin (BoNT). BoNT causes botulism, a neuroparalytic disease found in humans and animals. The toxin poses severe health risks to humans, and is a major concern for public health officials. Our previous studies show that the toxin is produced during cell growth and released as early as mid-log phase of bacterial growth. We believe autolysins, which hydrolyze the cell wall, are responsible for the cell leakage and early release of the toxin. In this study we analyzed the autolysins of C. botulinum and the different autolysis patterns of three type A C. botulinum strains.
Methods: Autolysis was studied in three C. botulinum strains by monitoring optical density throughout the growth curves to compare the autolysis of these strains grown in two different media. Another method of studying autolysis used fluorescent dyes to determine cell membrane permeability. Bioinformatics research was done comparing C. botulinum type A strain ATCC3502 to Bacillus subtilis strain 168, a model strain with characterized autolysin functions. BLAST was performed on the protein sequences of B. subtilis compared to C. botulinum. Transglycosylase CBO 3012 was further analyzed for its phylogenetic comparison to other species using ClustalW.
Results: Our results show that autolysis occurred in all three strains of C. botulinum, and the rate of autolysis varied due to the strains and growth media. The BLAST results of the genomic sequence analysis showed over 100 C. botulinum genes with homologies to B. subtilis str. 168. Further analysis of CBO 3012, a transglycosylase, showed greater than 98% identity and homology to other C. botulinum type A strains. Different C. botulinum serotypes showed greater than 53% identity and 73% homology to CBO 3012.
Conclusions: Phylogenetic analysis of the transglycosylase CBO 3012 showed the evolutionary relationship of this autolysin in a variety of bacteria, which could lead to a better understanding of the function of this gene, as well as this enzyme’s target site. The expression of the autolysin genes will be further analyzed using microarray analysis of the bacterium grown in the two media that exhibited different autolysis patterns. This data could possibly help correlate autolysins being responsible for bacterial cell lysis and early release of the toxin. The protein sequence alignment and microarray analysis will bring us one step closer to understanding the functions of autolysins in C. botulinum. A better understanding of the mechanism of toxin release and the role of autolysins may lead to better treatment options against this bacteria and its neurotoxin.




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