Genomic and proteomic characterization of two strains of Shigella flexneri 2 isolated from infants' stool samples in Argentina

Abstract

Background: Shigella specie is a globally important intestinal pathogen disseminated all over the world. In this study we analyzed the genome and the proteomic component of two Shigella flexneri 2a clinical isolates, collected from pediatric patients with gastroenteritis of the Northwest region of Argentina (NWA) in two periods of time, with four years of difference. Our goal was to determine putative changes at molecular levels occurred during these four years, that could explain the presence of this Shigella`s serovar as the prevalent pathogen in the population under study.

Results: As previously reported, our findings support the idea of Shigella has a conserved "core" genome, since comparative studies of CI133 and CI172 genomes performed against 80 genomes obtained from the NCBI database, showed that there is a large number of genes shared among all of them. However, we observed that CI133 and CI172 harbors a small number of strain-specific genes, several of them present in mobile genetic elements, supporting the hypothesis that these isolates were established in the population by horizontal acquisition of genes. These differences were also observed at proteomic level, where it was possible to detect the presence of certain secreted proteins in a culture medium that simulates the host environment.

Conclusion: Great similarities were observed between the CI133 and CI172 strains, confirming the high percentage of genes constituting the "core" genome of S. flexneri 2. However, numerous strain specific genes were also determined. The presence of the here identified molecular elements into other strain of our culture collation, is currently used to develop characteristic markers of local pathogens. In addition, the most outstanding result of this study was the first description of a S. flexneri 2 producing Colicin E, as one of the characteristics that allows S. flexneri 2 to persist in the microbial community. These findings could also contribute to clarify the mechanism and the evolution strategy used by this pathogen to specifically colonize, survive, and cause infection within the NWA population.

Keywords: Clinical isolates; Epidemiology; Genomic; Mobilome; Proteomic; Shigella; Virulence.

Fig. 1

Comparison of the newly sequenced S. flexneri 2 strains. A General features of the S. flexneri 2 CI133 and CI172 genomes of sequences. B Comparison between CI133 and CI172 strains genome sequences. The strains are represented by the circumference, while the red ribbons represent the alignments of 100% identity performed by BLAST, the width indicate the alignment length. When the alignment is in opposite orientation of the DNA sequence is indicated by twisted lines in grey colors. C Venn diagram of S. flexneri 2 CI133 and CI172 strains, showing 4,194 shared CDSs at the intersection, the 111 strain specific genes of CI133 represented with an orange circle and the 74 strain specific genes of C172 represented with a green circle

Fig. 2

Differential analysis of the CI133 and CI172 genomes sequence. Pangenomic diversity of the newly sequenced strains of S, flexneri 2 and 80 genomes of NCBI database. Boxplots represented with light blue represent the increasing pangenomic diversity with the addition of the strains whiles the “core” genome is represented by orange boxplots that decreases with the addition of the strains

Fig. 3

Plasmidic content analysis from genome sequence of the CI133 and CI172 strains. A Scale reconstruction of the virulence plasmid identified in the CI133 and CI172 strains, by homology of the sequences obtained with pCP301 and using the DNAMAN program. Red arrows indicate virulence genes identified in each strain; Green arrows indicate elements required for plasmid stability, black arrows indicate insertion sequences and the blue box indicates the origin of replication. The direction of the arrows indicates the orientation of each element with respect to the pCP301 sequence. The node or contigs where each element is found in each strain is indicated on each element. B Strain specific cluster of S. flexneri 2 CI172 strain showing the Colicin related genes and their probable plasmidic origin. C DNA plasmidic profile obtained from virulent CI133 and CI172 strains: 1-λDNA-HindIII molecular marker used to estimate the size of each plasmidic band, the size of each band is in the left indicated; 2- CI133 strain and 3- CI172 strain. The analysis was carried out by agarose gel electrophoresis and stained with ethidium bromide. DNA_ch: chromosomal DNA; p-I, pII and p-III putative plasmids identified by PlasmidFinder software

Fig. 4

CDSs corresponding to the COG functional categories represented in the CI133 and CI172 strains genomes. The letters represent each one of the functional categories: [D] Cell cycle control, cell division, chromosome partitioning; [M] Cell wall/membrane/envelope biogenesis; [N] Cell motility; [O] Post-translational modification, protein turnover, and chaperones; [T] Signal transduction mechanisms; [U] Intracellular trafficking, secretion, and vesicular transport; [V] Defense mechanisms; [W] Extracellular structures; [A] RNA processing and modification; [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replication, recombination and repair; [C] Energy production and conversion; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [G] Carbohydrate transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport, and catabolism; [R] General function prediction only; [S] Function unknown

Fig. 5

Protein–Protein interactive networks. A Proteins present and upregulated in the CI133 strain. B Proteins showing interactions present in the CI172 strain. The proteins are represented with the nodes, the edges represent the interactions between the proteins, and the width of edges the degree of interactions. The circles are showing different COG categories: [O] Post-translational modification, protein turnover, and chaperones; [U] Intracellular trafficking, secretion, and vesicular transport; [C] Energy production and conversion; [E] Amino acid transport and metabolism; [M] Cell wall/membrane/envelope biogenesis; [I] Lipid transport and metabolism; [J] Translation, ribosomal structure and biogenesis; [H] Coenzyme transport and metabolism and [G] Carbohydrate transport and metabolism. The networks were constructed using the STRING v11.5 bioinformatics tool