Comparative analysis of Q-stx regions of stx-phages encoding stx1a, stx2a and stx2c subtypes

KRÜGER, ALEJANDRA; ZHOU, KAI; FRIEDRICH, ALEXANDER W; LUCCHESI, PAULA M. A.; ROSSEN , JOHN WA

Zürich

Conferencia; EMBO CONFERENCE: VIRUSES OF MICROBES: STRUCTURE AND FUNCTION, FROM MOLECULES TO COMMUNITIES; 2014

EMBO

Introduction and objectives Shiga toxin producing E. coli (STEC) is a group of E. coli strains carrying virulence factors that confer them the capacity to cause severe human diseases, including hemorrhagic colitis and hemolytic-uremic syndrome (HUS). The common characteristic of all STEC strains, and their main virulence factor, is the production of Shiga toxins, which are generally encoded by prophages (here named stx-phages). The stx-phages share similar gene organization and regulation with bacteriophage λ, though a mosaic structure and diversity have been described among stx-phages. The stx genes are located in the late phage region downstream the Q gene, a transcriptional antiterminator that controls expression of late phage genes. Therefore, stx-phages have not only a role in the transfer of stx genes but also in the regulation of its expression. Our objectives were to examine the Q-stx region from stx-phages present in STEC strains isolated from cattle in Argentina and to perform a comparative analysis including stx-phage sequences available from the GenBank. Materials and Methods The genome of 9 bovine STEC strains (belonging to O157:H7, O145:H- and O26:H11 serotypes) was sequenced through a paired-end read protocol on the Illumina Miseq platform. Sequence reads were de novo assembled with CLC Genomics Workbench. The contigs containing stx genes were detected by BLAST, and Q and stx genes were identified by using ORF Finder and CD-Search bioinformatic tools. Subsequently, comparative analysis was performed including 34 sequences obtained from the GenBank by sequence alignments and phylogenetic trees constructed independently for Q and stx genes and the intermediate region (IRQ-stx) using Mega6 software. Results One or two Q-stx regions (comprising Q, IRQ-stx and stx sequences) were detected in each genome, according to the number of stx-subtypes present in each of the 9 strains. The sequence analysis showed 100% of identity among phages harboring the same stx subtype and integrated in strains of the same serotype, while 98-99% of identity was observed among phages harboring the same stx subtype but integrated in strains of different serotypes. The Q-stx sequences of Argentine phages analysed in this study were similar to that of phages from strains isolated in other countries. Interestingly, the Q-stx region of stx2a- and stx2c-phages integrated in O157:H7 strains from Argentina were identical to those from TW14359 and EC4115, two O157:H7 STEC strains associated with a spinach outbreak in the US. The comparative analysis, including also 34 sequences of stx1a-, stx2a- and stx2c-phages from GenBank, showed three main clusters of Q sequences, one including Q sequences from stx1a and most of stx2a-phages, another from the remaining stx2a-phages and the other with Q sequences from stx2c-phages. Interestingly, the regions between Q and stx genes (IRQ-stx) showed differences in size but some partial identities in sequence. Five IRQ-stx subtypes were identified: IR506 and IR1298 present in stx1a-phages, IR783 and IR2005 in stx2a-phages and IR649 in stx2c-phages. The database information for IR783 sequence includes putative promoters, tRNAs, a putative DNA methylase and other hypothetical proteins. At present, there is little information reported about the other IR sequences. Conclusion A Q-IR-stx structure is shared among stx-phages, including those present in STEC strains isolated from cattle in Argentina. Heterogeneity in Q has been previously described. However, more studies are necessary to evaluate associations between stx subtypes and Q variants, and also to determine the role of different Q proteins in Stx2 expression levels and severity of disease. Our results demonstrate that there are also different IRQ-stx sequences, detecting five subtypes among the stx1a-, stx2a- and stx2c-phages. The present study highlights the variability in Q-stx region and supports the hypothesis of the mosaic nature of stx-phages.