Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B (original) (raw)
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Background: Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT)TM and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. Results: Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. Conclusion: Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.
2000
Shiga-toxin-2 (stx 2 )-encoding bacteriophages were isolated from Norwegian Escherichia coli O157 :H7 isolates of cattle and human origin. The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx 2 region of the phages. The stx 2 -phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay. The results indicate that the population of stx 2 -carrying phages is heterogeneous, although the phages from epidemiologically linked E. coli O157 :H7 isolates were similar. There appears to have been frequent recombination of stx 2 phages with other lambdoid phages.
Investigation on the Evolution of Shiga Toxin-Converting Phages Based on Whole Genome Sequencing
Frontiers in Microbiology, 2020
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.
Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction
Frontiers in Cellular and Infection Microbiology, 2015
Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage φ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the φ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli φ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H 2 O 2 treatment, 8 out of 13 commensal lysogens produced more φ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H 2 O 2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.
Infection and Immunity, 2003
The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx 1 and/or stx 2 ) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx 1 or stx 2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage , and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.
Gene, 2003
Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210-3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2f-I, and compared it with three recently reported Stx2-phage genomes. Stx2f-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I-VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2f-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I -IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2f-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area. q
Comparative genomics of Shiga toxin encoding bacteriophages
Background: Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as ð24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of ð24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results: The genome of the Stx2 encoding phage, ð24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however ð24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The ð24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions: The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.
Comparative genomics of Shiga toxin encoding bacteriophages (2012)
Background: Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24 B , commonly referred to as Ф24 B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24 B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results: The genome of the Stx2 encoding phage, Ф24 B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24 B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24 B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions: The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.
The ea22 gene of lambdoid phages: preserved prolysogenic function despite of high sequence diversity
Virus Genes, 2020
The exo-xis region of lambdoid phages contains open reading frames and genes that appear to be evolutionarily important. However, this region has received little attention up to now. In this study, we provided evidence that ea22, the largest gene of this region, favors the lysogenic pathway over the lytic pathway in contrast to other characterized exo-xis region genes including ea8.5, orf61, orf60a, and orf63. Our assays also suggest some functional analogies between Ea22 and the phage integrase protein (Int). While it is unsurprising that Ea22 operates similarly in both λ and Stx phages, we have observed some distinctions that may arise from considerable sequence dissimilarity at the carboxy termini of each protein.
Zoonoses and Public Health, 2019
Shiga toxin-producing Escherichia coli (STEC) can cause diarrhoea and severe diseases in humans, such as haemolytic uraemic syndrome. STEC virulence is considered to correlate with the amount of Shiga toxins (Stx) produced, especially Stx2, whose subtype Stx2a is most frequently associated with high virulence. Stx are encoded in prophages, which play an important role in STEC pathogenesis. The aim of this study was to evaluate stx 2a expression levels and Stx2a phage production using qPCR and the double-agar-layer method in 29 STEC strains, corresponding to serotypes O26:H11 (6), O91:H21 (1), O145:H-(11) and O157:H7 (11), isolated from cattle and humans. Results were then tested for possible associations with serotype, origin or some genetic features. We observed heterogeneous levels of stx 2a expression and Stx2a phage production. However, statistical comparisons identified a higher stx 2a expression in response to mitomycin C in strains isolated from cattle than in those from humans. At the same time, compared to stx 2a /stx 2c strains, stx 2a strains showed a higher increase in phage production under induced conditions. Notably, most of the strains studied, regardless of serotype and origin, carried inducible Stx2a phages and evidenced expression of stx 2a that increased along with phage production levels under induced conditions.