Comparative genomics of Lactobacillus crispatus suggests novel mechanisms for the competitive exclusion of Gardnerella vaginalis

Abstract

Background: Lactobacillus crispatus is a ubiquitous micro-organism encountered in a wide range of host-associated habitats. It can be recovered from the gastrointestinal tract of animals and it is a common constituent of the vaginal microbiota of humans. Moreover, L. crispatus can contribute to the urogenital health of the host through competitive exclusion and the production of antimicrobial agents. In order to investigate the genetic diversity of this important urogenital species, we performed a comparative genomic analysis of L. crispatus.

Results: Utilizing the completed genome sequence of a strain ST1 and the draft genome sequences of nine other L. crispatus isolates, we defined the scale and scope of the pan- and core genomic potential of L. crispatus. Our comparative analysis identified 1,224 and 2,705 ortholog groups present in all or only some of the ten strains, respectively. Based on mathematical modeling, sequencing of additional L. crispatus isolates would result in the identification of new genes and functions, whereas the conserved core of the ten strains was a good representation of the final L. crispatus core genome, estimated to level at about 1,116 ortholog groups. Importantly, the current core was observed to encode bacterial components potentially promoting urogenital health. Using antibody fragments specific for one of the conserved L. crispatus adhesins, we demonstrated that the L. crispatus core proteins have a potential to reduce the ability of Gardnerella vaginalis to adhere to epithelial cells. These findings thereby suggest that L. crispatus core proteins could protect the vagina from G. vaginalis and bacterial vaginosis.

Conclusions: Our pan-genome analysis provides insights into the intraspecific genome variability and the collective molecular mechanisms of the species L. crispatus. Using this approach, we described the differences and similarities between the genomes and identified features likely to be important for urogenital health. Notably, the conserved genetic backbone of L. crispatus accounted for close to 60% of the ortholog groups of an average L. crispatus strain and included factors for the competitive exclusion of G. vaginalis, providing an explanation on how this urogenital species could improve vaginal health.

Figure 1

Whole-genome alignment of the L. crispatus genomes. The contigs of the draft genomes were ordered with MAUVE using the ST1 genome as a reference. Matching genome regions were identified with BLASTN and visualized using the Artemis Comparison Tool (ACT). Vertical bands represent the BLASTN matches (bit score ≥ 1500). Prophinder-predicted prophage-like genomic regions and IslandViewer predicted GIs are represented as blue boxes on the bottom and red boxes on the top strand of each genome, respectively.

Figure 2

Pan- and core genomes of L. crispatus . Development of the pan- (A) and core (B) genomes as a function of the number of sequenced L. crispatus strains. The total number of genes found according to the pan- and core genome analysis is shown for increasing numbers of sequenced genomes. The dashed lines represent least squares fits to the medians and the R ² describes the suitability of the fit. The box plots present median (horizontal line), 25th and 75th percentiles (solid box), with the data extremes shown by whiskers outside the box. C) The distribution of core and accessory L. crispatus CDSs within COG functional categories. For each category, the top and bottom bars show the percentage of the assigned core and accessory CDSs relative to the entire core and the accessory L. crispatus CDSs, respectively. The proportion of the strain-specific CDSs is highlighted (light blue) in the accessory bars. COGs significantly enriched (p-value ≤ 0.01, hypergeometric distribution) in core (1), shared accessory (2), or strain-specific (3) CDSs are marked next to the COG identifiers. Only COG functional categories with more than 20 members are shown. The COG categories are given in the inset at the bottom of the figure. D) Distribution of ortholog groups at different levels of conservation in each strain. The OrthoMCL-defined ortholog groups were classified into different levels of conservation according to the number of strains they were detected in. Ortholog groups found in all the ten genomes represent the current core (red). Conservation levels are represented by different colors.

Figure 3

Variation in CRISPR/Cas locus in L. crispatus. The arrows represent different genes and their orientation within a locus. Orthologous genes are positioned vertically. The cas genes with conserved function are of the same color, whereas grey describes genes not matching a Cas model. Outlines of genes orthologous to some cas gene, but not matching a Cas model are color coded according to the ortholog groups. Dashed lines represent contig breaks. Diamonds represent direct repeats and boxes different spacer sequences. Identical spacers are represented by the same color. The direct repeats of the top nine genomes are identical.

Figure 4

Variation in EPS gene cluster in L. crispatus . The organization and conservation of the exopolysaccharide synthesis regions in L. crispatus. Orthologous genes are represented with the same color and stars indicate genes found in different loci. Dashed lines represent contig breaks in the MV-1A-US and SJ-3C-US clusters.

Figure 5

Inhibition of L. crispatus or G. vaginalis adhesion to HeLa cells by LEA-specific Fab fragments. Cells of L. crispatus EX533959VC06 (A) or G. vaginalis 101 (B) were pretreated with LEA-specific IgG Fab fragments or unrelated anti-flagellum Fab fragments or left untreated in PBS supplemented with 5 mM PMSF before the adhesion assays. The number of adherent bacteria per epithelial cell in 20 randomly chosen microscopic fields was determined. The assay was performed twice with duplicate samples and the results show mean values of adherent bacteria. The asterisk indicates P < 0.05 as calculated by Student’s t test.

Figure 6

Phylogenetic tree. Phylogenetic relations of the selected L. crispatus (green), L. helveticus (blue) and L. acidophilus (purple) strains based on the SNPs of the core genome. The B. subtilis genome was used as the out-group to root the tree, but is not shown in the figure. In the inset, the branching pattern of the L. crispatus strains is highlighted.