Author:

Abstract:

The human body is colonized by a vast number of micro-organisms collectively referred to as the human microbiota. The main microbial niches of the human body include the skin, the oronasopharyngeal cavity, the gastrointestinal tract (GIT) and the genital tract. The microbes in these niches provide various health benefits to the host. Whilst the role of GIT microbiota is well studied in the past few decades, studies on the beneficial effects of vaginal microbiota are limited. Lactobacilli are the most dominant members of the vaginal microbiome playing a crucial role in female health. However, the molecular mechanisms behind the adaptation of these microbes in the vaginal niche and their health promoting effectsare in general not well known. Residing at the site of entry of variouspathogens causing urogenital and sexually transmitted infections, like HIV and Candida, these bacteria play an indispensable role in supportinghost defense.In this PhD research work, we focused on the presence of cell surface-associated mannose binding lectins (MBLs) in vaginal Lactobacillus strains as putative important adaptive and probiotic factors,especially for the design and selection of probiotics to inhibit HIV infection. MBLs constitute a class of carbohydrate binding agents (CBAs) that can bind to mannose rich envelope glycoprotein, gp120, of HIV-1 and related macromolecules exposed on sugar-rich pathogens, and prevent their entry in the host cell. Our first goal was to screen various lactobacilli for the presence of MBLs that could interact with HIV-1 gp120. The screening study revealed only one strain L. plantarum CMPG5300 that boundexclusively to gp120. This strain also exhibited a high degree of auto-aggregation. This vaginal strain was subjected to a genomic and proteomic analysis to search for the putative HIV-1 gp120 binding lectin(s).During genomic analysis, we focused on the annotation of the surface proteome of L. plantarum CMPG5300, in particular candidate genes encoding putative MBL(s) and niche adaptation factors. Sortase dependent proteins (SDPs) form a major class of bacterial surface proteins in these Gram positive bacteria. To investigate if SDP(s) are involved in the gp120 binding capacity of the strain, the sortase gene of CMPG5300 was knocked outusing an optimized electroporation protocol. We observed that the mutant lost its capacity to bind to mannan and HIV-1 gp120. Interestingly, the mutant also became defective in its auto-aggregating property, adhesion to vaginal epithelial cell line and biofilm formation. To investigate the role of the first detected sortase-targeted lectin 1 in gp120 binding of the strain, we constructed a knock-out mutant and also expressed lectin 1 and some of its separate domains in E. coli and we could find some interesting gp120 binding properties.On the other hand, a proteomic analysis was conducted by isolating various protein fractions of CMPG5300. The fractions were separated via affinity chromatography on a mannose-rich sepharose column to elute the MBL(s) of the strain. The chromatographic assays yielded the extracellular form of a moonlighting protein of CMPG5300 as one of the mannose binding adhesins of CMPG5300. This moonlighting protein could however not be purified from supernatant in sufficient amounts. Heterologous expression inside E. coli also did not result in a protein that could bind HIV-1 gp120. However, the recombinant form showed a slight antiviral activity against HIV-2.To conclude, we have identified the auto-aggregating vaginal L. plantarum strain CMPG5300, which shows binding to mannan and the HIV-1 gp120. We could molecularly identify the cell-bound protein lectin 1 and an extracellular released moonlighting protein as putative novel carbohydrate binding agents, but future studies need to further investigate the probiotic potential of CMPG5300, including the ability of the bacterium to lower levels of HIV transmission and infection in vivo.