Introduction

Temperate bacteriophages affect many aspects of the life of lysogenic bacteria through multiple mechanisms including direct or indirect influence on the host genome expression1, gene transduction including the recently described highly effective lateral transduction2 and specific mobilization of some genomic islands3 and other mechanisms4, though the most known and, probably, the most ecologically significant mechanism of such influence is the lysogenic conversion1, 5 through expression in the lysogen of some prophage encoded genes, that confer the bacteria new features potentially increasing their fitness in particular habitats5, 6. Therefore, acquisition of new prophages is believed to be one of the important strategies of bacterial adaptation in nature4. Bacterial antiviral systems are often expressed in phase-variable manner7-9. The adaptive value of such variations may be in part due to the “opening of window” for acquisition of new potentially beneficial prophages.
However, the most important factor that determines the bacteriophage host range is not the activity of the intracellular antiviral systems but the specificity of the bacteriophage adsorption. The major determinants of adsorption specificity that define bacteriophage lytic activity host range are well characterized10. At the same time data on the factors determining the lysogenization host range are largely missing.
The verotoxigenic (VTEC) and shigatoxigenic (STEC) Escherichia coli strains are associated with multiple foodborne diseases causing morbidity and mortality in humans1, 4, 5. VTEC and STEC are zoonotic pathogens that may colonize livestock animals such as cattle without causing symptoms in them but making the agricultural environments and products dangerous for humans10. The majority of STEC strains belong to the O157:H7 serotype11, although non-O157 STEC strains have been identified and currently gain increased attention4 as, for example, the so-called “Big Six” - O26, O45, O1; O111,O121 and O14512, as well as the O104:H4 serotype that caused the well-known 2011 outbreak in Germany13.
STEC strains possess a number of pathogenicity factors, the foremost being Shiga toxin production11, 14.
Although the Stx-converting bacteriophages are quite divergent morphologically and contain genomic modules divergent by their sequences, however the genome organization of these viruses is similar to that of the bacteriophage λ, therefore the Stx phages are considered as lambdoid phages15, 16. In these phages, the toxin gene stx is located downstream of the conserved gene Q encoding the antiterminator of the late gene region15. Toxin expression is repressed in lysogenic bacterial cells, and takes place only upon the prophage induction. Toxin molecules lacking the signal peptide for secretion are released upon cell lysis. The lysogeny in stx-converting phages is less stable compared to stx- lambdoid phages17-20 resulting in higher rate of spontaneous induction and in increased sensitivity to environmental factors such as DNA-damaging agents, oxidative stress or increased salt concentrations. Many antibiotics also increase the induction rate of Stx-converting prophages thus enhancing the toxin production. The increase of the toxin production may worsen the patient’s conditions and provoke the haemolytic uremic syndrome often leading to fatal outcome1. Therefore, the use of antibiotics to treat STEC infections remains controversial21.
At the same time, the STEC infections are self-limiting, and the pathogen gets spontaneously eliminated in ca. 2 weeks. The standard for the treatment of these infections relies on supportive care (symptomatic treatment, plasma exchange, infusion therapy) aiming at stabilization of the patient condition during the time required for self–curing of the infection22.
Thus it is possible to speculate that the severity of the symptoms and the outcome of the disease may also depend on interaction of the stx phage released by the STEC population in the upper intestine with the resident E. coli population in the hindgut. In case of active phage multiplication in this site, the released toxin may contribute to the overall toxin load. However, stx phages are seldom able to form plaques in vitro on isolated symbiotic gut E. colistrains23.
About 70% of Stx-converting bacteriophages are podoviruses related to the bacteriophage vb_EcoP_24B, also known as phage φ24B20, 24. The phage φ24B lysogenization host range was reported to be much broader than its range of hosts that support plaque formation23. The same observations were also made for some other stx phages25, 26. The establishment of the lysogenic E. coli population in the patient’s hindgut may also represent a threat of inducible increased toxin load. The route of lateral toxin gene transmission to other (potentially) enteropathogenic E. coli strains adapted to gut environment may lead to emergence of new highly virulent STEC lineages4, 25.
The secondary (terminal) receptor of bacteriophage φ24B has been identified as BamA protein, previously referred to as YaeT27, responsible for insertion of the newly synthesized beta-barrel outer membrane proteins into the bacterial outer membrane28. BamA protein is essential for bacterial cell viability and is therefore highly conserved. This circumstance allows speculating that a large variety of the non-Stx-producing or even non-pathogenic E. coli strains can be potentially lysogenizedin vivo and thus get involved in STEC evolution and/or pathogenesis of the STEC-induced diseases 15.
The available data suggest that the presence of a suitable secondary receptor is not the only factor required for successful phage adsorption and DNA delivery into the host cell. For E. coli, it has been shown that many O-antigen types protect the cells nearly completely against the phages not able to recognize O-antigen specifically29-32. This is achieved by non-specific shielding of the intimate cell surface by this structure. It was unclear how phage φ24B and related viruses that encode only one potential tail spike protein, gp6120, may penetrate the O antigen shield in diverse E. coli strains belonging to different O-serotypes.
Obviously, the threshold of infection efficacy required for plaque formation is much higher than for lysogenization of a small fraction of the host population. Therefore, several hypotheses can be raised to explain wide lysogenization host range of φ24B.
  1. This phage may exploit some uncharacterized molecular mechanism to penetrate through diverse O antigens albeit with the efficiency non-sufficient for plaque formation.
  2. It is also possible that the phage takes an advantage of local or temporal breaks in the O-antigen shield. The existence of a temporary phenotypical sensitivity to bacteriophage in the population of the phage resistant derivative of E. coli O157:H7 strain has been demonstrated previously 33. In this system only small fraction (0,4% - 8%) of the cells were able to adsorb the bacteriophage but the progeny of such cells was resistant as the bulk of the population. Such phenotypical sensitivity window would be sufficient to form lysogens as it was described by James et al.23.
  3. Alternatively, it is possible to speculate that, in the experimental conditions used by James et al.23, when a massive amount of the phage is added to the host cell suspension, the phage may lysogenize the small fraction of the mutant cells depleted of the O-antigen biosynthesis that is normally present in bacterial cultures31.
In order to discriminate between these potential mechanisms we challenged with the phage φ24B:cat a series of environmental E. coli strains producing different types of O antigens. For the majority of these strains the effective non-specific protection of the cell surface by the O antigen was confirmed previously 29, 30, 34-39.We compared the status of the O antigen production the lysogens formed with their parental strains.