Functional Analysis of Adhesion Factors and Signaling Mechanisms in Lactobacillus acidophilus NCFM

BUCK, BROCK LOGAN. Functional analysis of adhesion factors and signaling mechanisms in Lactobacillus acidophilus NCFM. (Under the direction of Professor Todd R. Klaenhammer). The lactobacilli are traditionally used for the fermentation of food products, but have recently been investigated for their potential health promoting factors. One important property of these probiotic lactobacilli is their ability to adhere to the intestinal mucosa. Lactobacillus acidophilus NCFM is a probiotic organism which has the ability to adhere to intestinal cells in vitro, although the mechanisms for this adhesion are not clear. A functional genomic approach was utilized to identify specific genes related to the adhesion process of L. acidophilus. The Caco-2 cell model was employed for analysis of the adhesive properties of multiple isogenic mutant strains of L. acidophilus. Mutation of genes encoding a fibronectin-binding protein (FbpA, LBA1148), mucusbinding protein (Mub, LBA1392), and a surface layer protein (SlpA, LBA0169) resulted in a significant decrease in adhesion to Caco-2 cells compared to the control strain. It appears that multiple factors work together to contribute to the adhesive ability of L. acidophilus NCFM. When then environmental conditions of L. acidophilus were modified immediately before adhesion, an explosive adhesive state was induced, termed the Adhesion Adaptive Response (AAR). Microarray analysis was used to study the transcriptional response of the L. acidophilus population to AAR conditions. Interestingly, multiple stress-related genes were overexpressed under AAR conditions along with the quorum sensing related gene, luxS. LuxS acts to synthesize autoinducer-2 (AI-2) which acts as an interspecies signaling molecule. A mutant strain of L. acidophilus deficient in LuxS activity was constructed and analyzed for its adhesive ability. The LuxS mutant strain exhibited a decrease in adhesion compared to the control when harvested directly from logarithmic phase, but still presented an explosive adhesive response following exposure to AAR conditions. Further analysis of the effect of LuxS on L. acidophilus was performed using microarray analysis and phenotypic confirmation studies. L. acidophilus was found to produce AI-2 throughout the logarithmic growth phase. As such, three points were selected for transcriptional analysis of both the wild type and LuxSmutant strain. Following a mixed model statistical analysis we discovered that the majority of differential expression related to LuxS occurs in the early-log stages of growth. Multiple genes related to the stress response, host-microbe interactions, and general growth and metabolism were positively differentially expressed in the presence of AI-2. Multiple surface proteins of were identified that contributed to the ability of L. acidophilus NCFM to adhere to intestinal epithelial cells, in vitro. An explosive adhesive response was induced by altering the environmental conditions in a process called the Adhesion Adaptive Response. Finally, the impact of the autoinducer AI-2 was studied using a novel microarray loop design. We reported that AI-2 influenced the transcriptional response of L. acidophilus at the early stages of growth, suggesting that AI-2 acts as a unique quorum sensing molecule. FUNCTIONAL ANALYSIS OF ADHESION FACTORS AND SIGNALING MECHANISMS IN LACTOBACILLUS ACIDOPHILUS NCFM