Functional Roles of Aggregation-Promoting-Like Factor in Stress Tolerance and Adherence of Lactobacillus acidophilus NCFM

Abstract

Aggregation-promoting factors (Apf) are secreted proteins that have been associated with a diverse number of functional roles in lactobacilli, including self-aggregation, the bridging of conjugal pairs, coaggregation with other commensal or pathogenic bacteria, and maintenance of cell shape. In silico genome analysis of Lactobacillus acidophilus NCFM identified LBA0493 as a 696-bp apf gene that encodes a putative 21-kDa Apf protein. Transcriptional studies of NCFM during growth in milk showed apf to be one of the most highly upregulated genes in the genome. In the present study, reverse transcriptase-quantitative PCR (RT-QPCR) analysis revealed that the apf gene was highly induced during the stationary phase compared to that during the logarithmic phase. To investigate the functional role of Apf in NCFM, an Delta apf deletion mutant was constructed. The resulting Delta apf mutant, NCK2033, did not show a significant difference in cell morphology or growth compared to that of the NCFMDelta upp reference strain, NCK1909. The autoaggregation phenotype of NCK2033 in planktonic culture was unaffected. Additional phenotypic assays revealed that NCK2033 was more susceptible to treatments with oxgall bile and sodium dodecyl sulfate (SDS). Survival rates of NCK2033 decreased when stationary-phase cells were exposed to simulated small-intestinal and gastric juices. Furthermore, NCK2033 in the stationary phase showed a reduction of in vitro adherence to Caco-2 intestinal epithelial cells, mucin glycoproteins, and fibronectin. The data suggest that the Apf-like proteins may contribute to the survival of L. acidophilus during transit through the digestive tract and, potentially, participate in the interactions with the host intestinal mucosa.