Karlyshev, Andrey (2020) Development of novel intervention strategies for fighting bacterial infections using whole genome sequencing of probiotic bacteria. In: 14th International Scientific Conference on Probiotics, Prebiotics, Gut Microbiota and Health - IPC2020; 11 Nov 2020, Prague, Czech Republic (Held online).

Abstract

Introduction Beneficial effects of probiotics on human health are well documented. Investigation of a role of these microorganisms in prevention and treatment of infectious diseases is particularly important due the global problem of multidrug resistance developed by many bacterial pathogens. Since these effects are variable even between the strains of the same species of probiotic bacteria, the investigation of the factors involved is essential. Some insights into the reasons for such variation can be gained from comparative genome analysis. This report presents some of our recent findings based on the genome analysis of specific strains of Lactobacillus spp. In particular, the molecular mechanisms of bacterial attachment to host cells and potential applications of our results are discussed. Inhibition of bacterial attachment by targeting specific adhesins and host cell receptors may prevent colonisation of host organisms by pathogenic bacteria. Investigation of the molecular mechanisms of probiotic activity is important for the development of novel strategies for fighting and prevention of infectious diseases. Methods Potential genes involved in bacterial aggregation and/or attachment to host cells were identified via whole genome sequencing and comparative genomics. The whole genome sequences of probiotic bacteria were generated by using IonTorrent PGM, PacBio technology and Optical genome mapping. The gene products were purified as His-tagged fusion proteins after cloning and expression in E. coli. The binding activities of the purified proteins as well as whole cells were tested using an ELISA-like method. Results The complete genome sequencing of Lactobacillus fermentum strain 3872 revealed the presence of a unique plasmid, containing a gene encoding a collagen-binding protein (CBP). The plasmid was also found capable of producing toxin-antitoxin proteins, which prevents its elimination from the cells. Expression and purification of the CBP from E. coli confirmed its affinity to human collagen 1. In order to investigate a possible biological role of this protein, an in vitro model of adhesion was developed. The results allowed the identification of a collagen-specific adhesin of Campylobacter jejuni (the major causative agent of gastrointestinal infection in humans) and demonstrated competition between these bacteria and L. fermentum for binding to collagen. In addition to CBP encoding genes, the genome analysis of this strain also revealed the presence of genes encoding two collagen-like proteins (CLPs). The presence of CLP encoding genes was detected only in few strains of Lactobacillus spp. Strain L. fermentum 3872 is unique in its ability to produce both CLPs and CBP. Discussion The data suggest that CBP of L. fermentum 3872 plays a role in interaction with host cells, which may lead to competitive exclusion of pathogenic bacteria. In addition, simultaneous production of both CBP and CLPs by this probiotic strain may lead to aggregation and co-aggregation with bacterial pathogens leading to elimination of the latter. A possible application of these finding for the development of novel intervention strategies for fighting bacterial infections is discussed. Keywords: Adhesins, Aggregation, Attachment, Collagen, Genome Sequencing

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