Probiogenomics Analysis of 97 Lactobacillus crispatus Strains as a Tool for the Identification of Promising Next-Generation Probiotics

Abstract

Members of the genus Lactobacillus represent the most common colonizers of the human vagina and are well-known for preserving vaginal health and contrasting the colonization of opportunistic pathogens. Remarkably, high abundance of Lactobacillus crispatus in the vaginal environment has been linked to vaginal health, leading to the widespread use of many L. crispatus strains as probiotics. Nevertheless, despite the scientific and industrial relevance of this species, a comprehensive investigation of the genomics of L. crispatus taxon is still missing. For this reason, we have performed a comparative genomics analysis of 97 L. crispatus strains, encompassing 16 strains sequenced in the framework of this study alongside 81 additional publicly available genome sequences. Thus, allowing the dissection of the L.crispatus pan-genome and core-genome followed by a comprehensive phylogenetic analysis based on the predicted core genes that revealed clustering based on ecological origin. Subsequently, a genomics-targeted approach, i.e., probiogenomics analysis, was applied for in-depth analysis of the eight L. crispatus strains of human origin sequenced in this study. In detail their genetic repertoire was screened for strain-specific genes responsible for phenotypic features that may guide the identification of optimal candidates for next-generation probiotics. The latter includes bacteriocin production, carbohydrates transport and metabolism, as well as a range of features that may be responsible for improved ecological fitness. In silico results regarding the genetic repertoire involved in carbohydrate metabolism were also validated by growth assays on a range of sugars, leading to the selection of putative novel probiotic strains.

Evaluation of Modulatory Activities of Lactobacillus crispatus Strains in the Context of the Vaginal Microbiota

“…Bacteriocins are antimicrobial peptides produced by bacteria that are released into the surrounding environment in order to enhance competitiveness toward other microorganisms for niche colonization ( 39 ). Screening for the presence of genes associated with bacteriocin production in the genomes of L. crispatus species allowed the identification of eight putative bacteriocin genes/loci (LCBs) distributed across L. crispatus strains of both human and poultry origin, with the only exception of LCBs 4 and 5, which were exclusively observed in isolates from human hosts, and LCBs 7 and 8, which were only identified in a strain of poultry origin, as previously reported ( 24 , 40 ) ( Fig. 2 ; Table S2).…”

“…Furthermore, LCB 3 was negatively correlated with Streptococcus agalactiae inhibition, while LCB 4 was negatively correlated with Escherichia coli . Interestingly, LCB 3 is comprised of six genes that are predicted to encode a small peptide pheromone along with a bacteriocin immunity protein, normally transcribed together with the bacteriocin in order to protect the producer bacteria ( 41 ), a putative ABC transporter, and a two-component regulatory system ( 24 ). Bioinformatic analysis suggests that this locus corresponds to the inhibition activity against Streptococcus species ( 41 ).…”

“…Moreover, members belonging to this bacterial species produce various antimicrobial compounds ( 21 , 22 ). The production of bacteriocins by lactic acid bacteria has been reported for several decades ( 23 ), and only recently the genetic determinants of bacteriocins produced by L. crispatus (LCB) have been characterized by in silico studies ( 24 , 25 ). In the context of the latter studies, a total of six putative bacteriocin-encoding genes/loci, named LCB 1 to 6, were predicted to be harbored by eight genomes of L. crispatus strains isolated from vaginal swabs.…”

“…In the context of the latter studies, a total of six putative bacteriocin-encoding genes/loci, named LCB 1 to 6, were predicted to be harbored by eight genomes of L. crispatus strains isolated from vaginal swabs. In addition, two bacteriocin-encoding gene clusters, termed LCB 7 to 8, were found to be located on the genomes of seven L. crispatus strains, which had been isolated from chicken fecal samples ( 24 ).…”

Resolution of glycogen and glycogen-degrading activities reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women

“…With the commonly-employed fluorometric starch degradation assay, which we describe as detecting amylopullulanase activity (encompassing both pullulanase and amylase activities), the unique relationship between pullulanase activity and lactic acid production was masked. Recent in vitro cultivation studies have shown that full length type I pullulanase ( pulA ) alleles with an intact signal peptide sequence are necessary for L. crispatus to exhibit robust growth when glycogen is supplied as a sole carbon source [46]; however, pulA was not needed for L. crispatus growth on glucose and the species has been documented growing on maltose and maltodextrin substrates in vitro [98]. Thus, although L. crispatus pullulanases may be found at low concentration or exhibit lower activity than amylases in vivo , they appear to contribute to the production of D-lactic acid in unique, but as yet poorly understood ways.…”