O antigen restricts lysogenization of non-O157 Escherichia coli strains by Stx-converting bacteriophage phi24B

Acquisition of new prophages that are able to increase the bacterial fitness by the lysogenic conversion is believed to be an important strategy of bacterial adaptation to the changing environment. However, in contrast to the factors determining the range of bacteriophage lytic activity, little is known about the factors that define the lysogenization host range. Bacteriophage phi24B is the paradigmal model of Stx-converting phages, encoding the toxins of the Shiga-toxigenic E. coli (STEC). This virus has been shown to lysogenize a wide range of E. coli strains that is much broader than the range of the strains supporting its lytic growth. Therefore, phages produced by the STEC population colonizing the small or large intestine are potentially able to lysogenize symbiotic E. coli in the hindgut, and these secondary lysogens may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. We demonstrate, however, that O antigen effectively limit the lysogenization of the wild E. coli strains by phi24B phage. The lysogens are formed from the spontaneous rough mutants and therefore have increased sensitivity to other bacteriophages and to the bactericidal activity of the serum if compared to their respective parental strains.

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.

Prophage-Encoded Peroxidase in ‘Candidatus Liberibacter asiaticus’ Is a Secreted Effector That Suppresses Plant Defenses

‘Candidatus Liberibacter asiaticus’ is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic ‘Ca. L. asiaticus’ strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high-copy excision plasmid encoding a reactive oxygen species–scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus and was suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into Escherichia coli and Liberibacter crescens, a culturable proxy for ‘Ca. L. asiaticus’. Transformed L. crescens cells showed 20 to 25% enhanced resistance to H2O2 on agar plates, 47% greater enzymatic activity, and enhanced growth in liquid cultures. A nonclassical secretion potential was predicted for SC2_gp095 and secretion from L. crescens was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta resulted in strong transcriptional downregulation of RbohB, the key gatekeeper of the H2O2-mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in ‘Ca. L. asiaticus’–infected citrus. ‘Ca. L. asiaticus’ peroxidase is likely a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens.

BaeSR, Involved in Envelope Stress Response, Protects against Lysogenic Conversion by Shiga Toxin 2-Encoding Phages

Infection and lysogenic conversion with Shiga toxin-encoding bacteriophages (Stx phages) drive the emergence of new Shiga toxin-producingEscherichia colistrains. Phage attachment to the bacterial surface is the first stage of phage infection. Envelope perturbation causes activation of envelope stress responses in bacterial cells. Although many external factors are known to activate envelope stress responses, the role of these responses in the phage-bacterium interaction remains unexplored. Here, we investigate the link between three envelope signaling systems inE. coli(RcsBC, CpxAR, and BaeSR) and Stx2 phage infection by determining the success of bacterial lysogenic conversion. For this purpose,E. coliDH5α wild-type (WT) and mutant strains lacking RcsBC, CpxAR, or BaeSR signaling systems were incubated with a recombinant Stx2 phage (933W). Notably, the number of lysogens obtained with the BaeSR mutant was 5 log10units higher than with the WT, and the same differences were observed when using 7 different Stx2 phages. To assess whether the membrane receptor used by Stx phages, BamA, was involved in the differences observed,bamAgene expression was monitored by reverse transcription-quantitative PCR (RT-qPCR) in all host strains. A 4-fold-higherbamAexpression level was observed in the BaeSR mutant than in the WT strain, suggesting that differential expression of the receptor used by Stx phages accounted for the increase in the number of lysogenization events. Establishing the link between the role of stress responses and phage infection has important implications for understanding the factors affecting lysogenic conversion, which drives the emergence of new pathogenic clones.