Investigation on the Evolution of Shiga Toxin-Converting Phages Based on Whole Genome Sequencing

Abstract

Bacteriophages are pivotal elements in the dissemination of virulence genes. The main virulence determinants of Shiga Toxin producing E. coli, Shiga Toxins (Stx), are encoded by genes localized in the genome of lambdoid bacteriophages. Stx comprise two antigenically different types, Stx1 and Stx2, further divided into subtypes. Among these, certain Stx2 subtypes appear to be more commonly occurring in the most severe forms of the STEC disease, haemorrhagic colitis and haemolytic uremic syndrome (HUS). This study aimed at obtaining insights on the evolution of Stx2 bacteriophages, due to their relevance in public health, and we report here on the analysis of the genomic structure of Stx2 converting phages in relation with the known reservoir of the E. coli strains harboring them. Stx2-converting phages conveying the genes encoding different stx2 subtypes have been isolated from STEC strains and their whole genomes have been sequenced, analyzed and compared to those of other Stx2 phages available in the public domain. The phages' regions containing the stx2 genes have been analyzed in depth allowing to make inference on the possible mechanisms of selection and maintenance of certain Stx2 phages in the reservoir. The "stx regions" of different stx2 gene subtypes grouped into three different evolutionary lines in the comparative analysis, reflecting the frequency with which these subtypes are found in different animal niches, suggesting that the colonization of specific reservoir by STEC strains could be influenced by the Stx phage that they carry. Noteworthy, we could identify the presence of nanS-p gene exclusively in the "stx regions" of the phages identified in STEC strains commonly found in cattle. As a matter of fact, this gene encodes an esterase capable of metabolizing sialic acids produced by submaxillary glands of bovines and present in great quantities in their gastrointestinal tract.

Bacteriophage-Derived Depolymerases against Bacterial Biofilm

“…This option might be beneficial if there are difficulties in supplying materials directly to biofilms, e.g., during treatment of clinical biofilm infections, as phages may allow for more effective delivery of these enzymes to the target place [ 105 ]. On the other hand, phages can transfer genes coding for toxins and antibiotic resistance proteins between bacterial cells, which causes safety issues [ 106 , 107 , 108 ]. The development of bacteriophage resistance among bacteria is another problem [ 109 , 110 ].…”

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity

“…In addition to stx , many additional genes have been described in Stx prophage genomes, which may contribute to pathogenicity and virulence, but also to the competitiveness with other gut bacteria in the human host. There are a number of reviews and book chapters that have described the role of some genes in the Stx phages that contribute to regulating pathogenicity in STEC [ 9 , 10 , 46 , 71 , 95 ], and therefore, their structure, function, and roles in pathogenicity will not be reviewed here.…”

“…For this reason, it is difficult to distinguish between Stx and non-Stx phages in the STEC complete genomes because these similar sequences confound the software used for contigs assembly, producing false chimeras. This problem is overcome when using sequencing platforms that generate longer reads [ 47 ], or by inducing and isolating the prophages before sequencing [ 10 ].…”

“…Whole genome sequencing yielded the sequence data of hundreds of pathogenic STEC genomes in the National Center for Biotechnology Information (NCBI) database ( , accessed on 26 March 2021), which confirmed the heterogeneity of Stx phage genomes. These differences, in turn, influence the bacterial genome structure and its functionality [ 10 ]. Furthermore, the prophage sequences demonstrate that all Stx phages conserve a basic lambdoid structure that is discussed below.…”

Phages in the Gut Ecosystem

“…Such mutualism is exemplified by virions carrying genes that have no direct impact on the phage life cycle but can enhance the fitness of the bacterial host, termed “morons” ( Cumby et al., 2012 ) in a phenomenon called lysogenic conversion. Common morons include, for example, bacterial virulence ( Wagner and Waldor, 2002 ) or metabolic genes ( Breitbart et al., 2018 ; Zuppi et al., 2020 ). Alternatively, morons may provide resistance from other infecting virions, in the superinfection exclusion phenomenon ( Bondy-Denomy et al., 2016 ).…”