| The probiotic Lactobacillus acidophilus NCFM interacts with intestinal and immune cells, inducing beneficial immune profiles in its host. In order to expand the benefits elicited from this strain, it is imperative to understand the factors involved in its association with host cells. Multiple genes have previously been implicated in adherence of this strain to Caco-2 cells and several proteins found in the extracellular matrix of intestinal cells, in vitro..;In this study, genes encoding a fibronectin binding protein ( fbpA) and three mucus binding proteins (muc) have been progressively deleted in order to determine the contribution of these factors to adherence. The resulting single (NCK2046, ΔfbpA), double (NCK2104, ΔfbpAΔmub), triple (NCK2131, ΔfbpAΔΔmub), and quadruple (NCK2133, ΔfbpAΔΔΔmub ) deletions did not consistently demonstrate altered adherence to Caco-2, HT29-5FU, or IPEC-J2 intestinal epithelial cells, in vitro, in comparison to the parental reference strain. Gene expression analysis revealed that multiple transport proteins and membrane-associated factors were upregulated in NCK2133 compared with the reference strain. The upregulation of these surface-associated proteins indicates the complexity involved in adherence of L. acidophilus to host cells, and suggests that other proteins may compensate for the loss of the FbpA and Muc proteins. In vivo studies are necessary to determine the impact of the deletions to gastrointestinal transit and retention under true physiological conditions. The potential to exploit the intimate association between L. acidophilus and the host immune system has been previously demonstrated by a derivative strain that harbored an expression plasmid encoding an antigen for vaccination. However, plasmids are routinely genetically unstable and often require an antibiotic resistance marker for selection and maintenance, restricting their potential use in human clinical settings. Integration of antigen genes into the chromosome is expected to eliminate these issues. In this study, a β-glucuronidase (gusA3 ) reporter gene was integrated into four intergenic locations throughout the L. acidophilus chromosome, where it was expressed from the native upstream promoter. Integration downstream of two highly expressed constitutive genes, LBA0169 (slpA) and LBA0889 ( eno) resulted in GusA3 activity four logs and three logs higher than the gusA3 negative parent, respectively. Expression of gusA3 downstream of slpA was also nearly a log higher than from a plasmid reference. Integration downstream of an inducible β-galactosidase, LBA1462 (lacZ), resulted in GusA3 activity that was one log higher after growth in lactose than in glucose. Integration into a low expression region, downstream of LBA0645, generated only a basal level of expression. The range of expression levels observed demonstrated the importance of rational targeting of potential chromosomal integration sites. Additionally, chromosomal integration provided stability in the absence of antibiotics, when compared to the plasmid reference. The expression levels observed, combined with the increased stability of the genetic cassettes provided by chromosomal integration, expand the potential for use of lactobacilli as an oral vaccine delivery vehicle in clinical applications. |
