K-115. Functional Characterization of Two Putative Myosin Cross Reactive Antigen Genes from Lactobacillus acidophilus NCFM

S. O'Flaherty, T. Klaenhammer;
North Carolina State Univ., Raleigh, NC.

Two genes (LBA0555 and LBA0649) annotated as myosin cross reactive antigens (MCRA) have been identified in the genome of Lactobacillus acidophilus NCFM. Although both are annotated as MCRAs, the function of these proteins in L. acidophilius is unknown. The objective of this study was to make a double mutation in LBA0555 and LBA0649 in L. acidophilus NCFM and phenotypically analyze the mutant strains in an attempt to assign a function to these proteins. In previous microarray experiments, LBA0555 was one of the most highly up regulated genes under acidic conditions imposed with lactic acid at pH 4.5. LBA0649 shares 58% identity with LBA0555. Both LBA0555 and LBA0649 are 67 kDa proteins and neither have signal peptides or LPXTG cell wall anchoring motifs. Further bioinformatic analysis of both proteins revealed that they have similar transmembrane profiles and possess a N-terminal anchor which could function to anchor these proteins to the cell membrane. Recent BLASTp analysis with LBA0649 showed that this protein has homology to another protein reported previously in an L. acidophilus. This protein was annotated as a linoleic isomerase and shares 99% identity with LBA0649. However, L. acidophilus NCFM did not exhibit linoleate isomerase activity when analyzed by gas-liquid chromatography analysis. An in-frame deletion was made in LBA0555, LBA0649 and both genes in a single strain. These three strains (ΔLBA0555, ΔLBA0649 and Δ LBA0555:LBA0649) were compared to the control strain in phenotypic assays. Interestingly, a reduction in adherence to Caco2 cells was observed for all three mutants compared to the control. In addition, reduced growth for the mutants was observed in the surfactants sodium dodecyl sulfate and triton X100. These results suggest that these proteins, especially LBA0649 plays a role in membrane functionality and possibly adherence to intestinal epithelial cells, in vitro.