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.
- This phage may exploit some uncharacterized molecular mechanism to
penetrate through diverse O antigens albeit with the efficiency
non-sufficient for plaque formation.
- 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.
- 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.