The lysogenization of the non-O157 Escherichia coli strains by stx-converting bacteriophage phi24B is associated with the O antigen loss and reduced fitness

The ability of the Shiga-toxigenic E. coli (STEC) to produce the toxin depends on the lysogenic conversion by stx-bacteriophages. The canonical stx-phage phi24B can lysogenize a wide variety of E. coli strains. In vivo the secondary lysogenization of symbiotic E. coli strains by the phages released by infecting STEC populations may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. However, in our experiment all the phi24B lysogens obtained from the environmental E. coli isolates had compromised O-antigen (Oag) biosynthesis. These lysogenic strains gained the sensitivity to the T5-like bacteriophages and featured increased sensitivity to the bactericidal activity of the horse serum. We conclude that in most of E. coli strains the Oag effectively restricts phi24B infection. The lysogenic clones predominantly rise from the Oag deficient mutants and therefore they have reduced fitness compared to the parental strain.


Introduction
The verotoxigenic (VTEC) and shigatoxigenic (STEC) Escherichia coli strains are associated with multiple foodborne diseases causing morbidity and mortality in humans 1-3 . VTEC and STEC are zoonotic pathogens usually transmitted in the agricultural loop 4 . The majority of STEC strains belong to the O157:H7 serotype 5 , although non-O157 STEC strains have been identified and currently gain increased importance 3 as, for example, the so-called "Big Six" -O26, O45, O1; O111,O121 and O145 6 , as well as the O104:H4 serotype that caused the well-known 2011 outbreak in Germany 7 .
STEC strains possess a number of pathogenicity factors, the foremost being Shiga toxin production 5,8 .
Although the Stx-converting bacteriophages are quite divergent genetically and morphologically, from the genome organization perspective all of them belong to a broad group of lambdoid bacteriophages 9,10 . In these phages, the toxin gene stx is located downstream of the conserved gene Q encoding the antiterminator of the late gene region 9 . Toxin expression is repressed in normally growing lysogenic bacterial cells, and takes place only upon the prophage induction. Toxin molecules lacking the signal leading peptide for secretion are released upon cell lysis. The lysogeny in stxconverting phages is less stable compared to stx-lambdoid phages [11][12][13][14] resulting in higher rate of spontaneous induction and in increased sensitivity to environmental factors. Many antibiotics also increase the induction rate of Stx-converting prophages thus enhancing the toxin production. Therefore, the use of antibiotics to treat STEC infections remains controversial 15 .
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 infection 16 .
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. coli strains 17 .
About 70% of Stx-converting bacteriophages are podoviruses related to the bacteriophage vb_EcoP_24B, also known as phage φ24B 14,18 . The phage φ24B lysogenization host range was reported to be much broader than its range of hosts that support plaque formation 17 . The same observations were also made for some other stx phages 19,20 . 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 lineages 3,19 .
The secondary (terminal) receptor of bacteriophage φ24B has been identified as BamA protein, previously referred to as YaeT 21 , responsible for insertion of the newly synthesized beta-barrel outer membrane proteins into the bacterial outer membrane 22 .
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 lysogenized in vivo and thus get involved in STEC evolution and/or pathogenesis of the STEC-induced diseases.
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 specifically [23][24][25][26] . 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, gp61 14

E. coli and bacteriophage strains and their cultivation
The E. coli strain MG1655 lysogenized for phage φ24B:cat was a kind gift of Prof. G.
Wegrzyn, University of Gdansk, Poland. Phage T5 was a gift of Dr. V. Ksenzenko (Institute of protein research RAS, Puschino-na-Oke, Russia). We previously described T5-like bacteriophages of DT57C species and their LTF mutants 24 . These include: phage DT57C, phage DT571/2, DT571/2 ltfAmutant lacking the LTFs (hereafter FimX) and DT571/2 mutant ABF that carries LTF non-branched LTF with only one receptor-binding domain (instead of two such domains on the branched LTFs of the phages DT57C or DT571/2). Bacteriophage 9g, a siphovirus representing the type strain of the genus Nonagvirus 27 . Gostya9 is a T5-like bacteriophage that was shown to recognize a different secondary receptor distinct from the receptors of the phages T5, DT57C and 9g 28 . Bacteriophage G7C, a N4-related podovirus specifically recognizing O antigen of E. coli 4s strain was isolated and characterized by us previously 29,30 . We isolated all the above-mentioned phages except for T5 and engineered phage mutants from horse feces as it described in the corresponding publications cited above.
All the E. coli strains were cultured on LB medium (trypton 10 g, yeast extract 5 g, NaCl -10 g, distilled H 2 O -up to 1 l). This medium was supplemented with 15 g of bacto-agar per 1 l for plates or with 6 g of bacto-agar for top agar.
Bacteriophage FimX was propagated on E. coli 4sR and enumerated using the conventional double-layer plating technique.

Lysogenization of the E. coli strains
This procedure was performed as described in James et al. 17 with minor modifications.
Briefly, a mid-log liquid culture of an appropriate strain was grown in LB medium, the phage was added at a multiplicity of 5 pfu/host cfu, and the mixture was incubated at 37 o C for 30 min. After the incubation, the cells were spun down in a table-top centrifuge (10000 × g, 1 min), the cells were resuspended in LB, washed twice with LB to remove non bound phage and plated on plates supplemented with 34 μ g/ml of chloramphenicol for lysogen selection.

LPS profiling
by SDS-PAGE electrophoresis was performed as recently described 25 .

Serum bactericidal activity (SBA)
against different strains was measured as follows. The blood samples collected from

Results
We decided to use these two approaches to evaluate the O antigen production status of the φ24B lysogens generated in environmental E. coli isolates. To do so, liquid cultures of the O antigen producing strains 4s, HS1/2, HS3-104, F5, F17, UP1 and UP11 and of the rough strains 4sR and C600 were challenged with phage φ24B:cat as described by James 17 . The lysogens were then selected by plating the mixture on LB plates supplemented with 34 μg/ml of chloramphenicol. The lysogens were obtained for strains 4s, HS1/2, HS3-104, F5 and F17. No lysogens were observed on strain UP11.
The lysogenization frequency was about 10 -4 lysogen cfu/ phage pfu for the rough strains and about 10 -7 -10 -6 in O antigen-producing strains. The latter value is comparable to the level of spontaneous mutations in E. coli inactivating a medium-sized gene (e.g. phage-resistant mutants).
We selected 3 lysogen clones per strain and confirmed the φ24B prophage presence using PCR for gene 61 (the tailspike protein gene). For E. coli 4s lysogens we also performed mitomycin C induction followed by transmission electron microscopy that confirmed that a phage morphologically identical to φ24B was produced.
LPS profiling of the lysogens obtained indicated that in all cases these strains did not produce O-antigen at all or the O-chain synthesis was greatly decreased compared to the parental strains (Fig. 1).
We tested the ability of phage FimX to grow on the lawns of the lysogens obtained. The other T5-like phages (DT57C, DT571/2, ABF and Gostya9) as well as the siphovirus 9g demonstrated the gain of the infectivity on the lysogens derivatives of some strains that were initially resistant to these phages. Phage G7C that is dependent on the specific O antigen recognition for infection of E. coli 4s cells 30 was not able to infect E. coli 4s (φ24B:cat) lysogenic strains in good agreement with O antigen production loss detected by the LPS profiling (Fig. 1).
Since the O antigen synthesis compromised strains are believed to be more vulnerable to immunity factors, we decided to measure the susceptibility of the lysogens obtained to the bactericidal activity of the horse serum (SBA). All the wild type strains were resistant to SBA in our conditions. Their cultures grew in presence of the serum as well or even slightly more rapidly than in the control experiment. In the absence of the serum the lysogenic strains showed the growth rates close to their cognate wild type strains. At the same time the growth of the lysogens was almost completely abolished in the presence of the serum (Fig. 2). Only one of the lysogenic clones tested, the derivative of the strain HS3-104, was able to grow significantly in presence of the horse serum, though the rise of the optical density was delayed and the growth rate was significantly lower than in the parental strain (Fig.2). This result can be explained by the fact that in HS3-104 lysogens the O antigen synthesis was strongly decreased but not completely abolished (Fig.1). So, the actual synthesis of O-polysaccharide could be upregulated in this particular clone in the conditions of the experiment.

Discussion
The results obtained allow us to conclude that lysogenization by the phage φ24B of diverse E. coli strains producing O antigens was not due to an unusual ability of this virus to penetrate the O antigen shield, but was mediated by spontaneous formation of bacterial rough mutants or of mutants with significantly compromised O antigen biosynthesis. It is not clear why the lysogenization was not effective for some strains.
The activity of antiviral systems, such as restriction-modification, avoiding the lysogenization at stages after the viral DNA penetration into the cell 38  We also should note that the lysogenization by φ24B:cat appears to be a simple and efficient procedure for selection for mutants with compromised or completely abolished O antigen synthesis. This procedure may be particularly valuable for the researchers working with field isolates of E. coli for which the genomic sequences are not yet available and/or in which other rapid techniques such as recombination with PCR fragments for genes knockout 42 are frequently less effective than in laboratory E. coli.

Disclosure of the potential conflicts of interests
The manuscript has not been published elsewhere and has not been submitted simultaneously for publication elsewhere.   growth to the horse serum bactericidal activity. Black lines -the wild type strain, grey lines -three lysogenic clones tested for each original strain.