LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7.

Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions.IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

Characterization and Complete Genome Analysis of Pseudomonas aeruginosa Bacteriophage vB_PaeP_LP14 Belonging to Genus Litunavirus.

A lytic Pseudomonas aeruginosa phage vB_PaeP_LP14 belonging to the family Podoviridae was isolated from infected mink. The microbiological characterization revealed that LP14 was stable at 40 to 50 °C and stable over a broad range of pH (5 to 12). The latent period was 5 min, and the burst size was 785 pfu/infected cell. The whole-genome sequencing showed that LP14 was a dsDNA virus and has a genome of 73,080 bp. The genome contained 93 predicted open reading frames (ORFs), 17 of which have known functions including DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. No tRNA genes were identified. BLASTn analysis revealed that phage LP14 had a high-sequence identity (96%) with P. aeruginosa phage YH6. Both morphological characterization and genome annotation indicate that phage LP14 is a memberof the family Podoviridae genus Litunavirus. The study of phage LP14 will provide basic information for further research on treatment of P. aeruginosa infections.

Characterization and genome analysis of a novel bacteriophage vB_SpuP_Spp16 that infects Salmonella enterica serovar pullorum.

A novel virulent bacteriophage vB_SpuP_Spp16 (hereafter designated Spp16) that infects Salmonella enterica serovar pullorum was isolated. Transmission electron microscopy showed that Spp16 possessed an isometric polyhedral head (60 nm in diameter) and a short tail (10 nm in length) belonging to the family Podoviridae. Its complete genome was determined to be 41,832 bp, with a 39.46% GC content by next-generation sequencing. The genome contains 53 proposed open reading frames that are involved in DNA replication and modification, transcriptional regulation, phage structural and packaging proteins and bacterial lysis. No transfer RNA genes were identified. The termini of genome were determined using our previously proposed termini identification method, which suggests that this phage has redundant termini with 421 bp direct terminal repeats. BLASTn analysis revealed the highest sequence similarity with Yersinia phage phi80-18, with a genome coverage of 33% and highest sequence identity of 69%. The phylogenetic analysis indicated that Spp16 forms a distinct branch of the subfamily Autographivirinae. Comparative genomics analysis showed that the phage Spp16 should be regarded as a new subcluster within the GAP227-like cluster in the Autographivirinae subfamily. The phage Spp16 has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that Spp16 may be a proper candidate to control diseases caused by Salmonella enterica serovar pullorum.

Characterization of a lytic Pseudomonas aeruginosa phage vB_PaeP_ASP23 and functional analysis of its lysin LysASP and holin HolASP.

In this study, we isolated a lytic Pseudomonas aeruginosa phage (vB_PaeP_ASP23) from the sewage of a mink farm, characterized its complete genome and analyzed the function of its putative lysin and holin. Morphological characterization and genome annotation showed that phage ASP23 belonged to the Krylovirinae family genus Phikmvvirus, and it had a latent period of 10 min and a burst size of 140 pfu/infected cell. In minks challenged with P. aeruginosa, phage ASP23 significantly reduced bacterial counts in the liver, lung, and blood. The whole-genome sequencing showed that its genome was a 42,735-bp linear and double-stranded DNA (dsDNA), with a G + C content of 62.15%. Its genome contained 54 predicted open reading frames (ORFs), 25 of which had known functions. The lysin of phage ASP23 (LysASP), in combination with EDTA, showed high lytic activity against P. aeruginosa L64. The holin of phage ASP23 was synthesized by M13 phage display technology, to produce recombinant phages (HolASP). Though HolASP exhibited a narrow lytic spectrum, it was effective against Staphylococcus aureus and Bacillus subtilis. However, these two bacteria were insensitive to LysASP. The findings highlight the potential of phage ASP23 to be used in the development of new antibacterial agents.

The Immunoprotection of OmpH Gene Deletion Mutation of Pasteurella multocida on Hemorrhagic Sepsis in Qinghai Yak.

OmpH is among the most important virulence factors of Pasteurella multocida, which mediates septicemia in yaks (Bos grunniens I) after infection with the bacteria. In the present study, yaks were infected with wild-type (WT) (P0910) and OmpH-deficient (ΔOmpH) P. multocida strains. The mutant strain was generated through the reverse genetic operation system of pathogens and proteomics technology. The live-cell bacterial count and clinical manifestations of P. multocida infection in Qinghai yak tissues (thymus, lung, spleen, lymph node, liver, kidney, and heart) were analyzed. The expression of differential proteins in the yak spleen under different treatments was analyzed using the marker-free method. We found that compared with the mutant strain, the titer of wild-type strains was significantly higher in tissues. Additionally, compared with other organs, the bacteria titer was significantly higher in the spleen. Compared with the WT p0910 strain, the mutant strain generated milder pathological changes in the tissues of yak. Proteomics analysis revealed that 57 of the 773 proteins expressed in P. multocida were significantly differentially expressed between the ΔOmpH and P0910 groups. Of the 57, 14 were over-expressed, whereas 43 were under-expressed. The differentially expressed proteins in the ΔompH group regulated the ABC transporter (ATP-powered translocation of many substrates across membranes) system, the two-component system, RNA degradation, RNA transcription, glycolysis/gluconeogenesis, biosynthesis of ubiquinone and other terpenoid-quinones, oxidative phosphorylation (citrate cycle) as well as fructose and mannose metabolism. The relationship among 54 significantly regulated proteins was analyzed using STRING. We found that WT P0910 and ΔOmpH of P. multocida infection activated the expression of ropE, HSPBP1, FERH, ATP10A, ABCA13, RRP7A, IL-10, IFN-γ, IL-17A, EGFR, and dnaJ. Overall, deletion of the OmpH gene weakened the virulence but maintained the immunogenicity of P. multocida in yak. The findings of this study provide a strong foundation for the pathogenesis of P. multocida and the management of related septicemia in yaks.

Characterization and Genomic Analysis of a Novel Jumbo Bacteriophage vB_StaM_SA1 Infecting Staphylococcus aureus With Two Lysins.

The development of new antimicrobial agents is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. Phages have been widely considered as effective alternatives to antibiotics. A novel phage vB_StaM_SA1 (hereinafter as SA1) that can infect multiple Staphylococcus strains was isolated from untreated sewage of a pig farm, which belonged to Myoviridae family. At MOI of 0.1, the latent period of phage SA1 was 55 min, and the final titer reached about 109 PFU/mL. The genome of phage SA1 was 260,727 bp, indicating that it can be classified as a jumbo phage. The genome of SA1 had 258 ORFs and a serine tRNA, while only 53 ORFs were annotated with functions. Phage SA1 contained a group of core genes that was characterized by multiple RNA polymerase subunits and also found in phiKZ-related jumbo phages. The phylogenetic tree showed that phage SA1 was a phiKZ-related phage and was closer to jumbo phages compared with Staphylococcus phages with small genome. Three proteins (lys4, lys210, and lys211) were predicted to be associated with lysins, and two proteins with lytic function were verified by recombinant expression and bacterial survival test. Both lys210 and lys211 possessed efficient bactericidal ability, and lys210 could lyse all test strains. The results show that phage SA1 and lys210/lys211 could be potentially used as antibiotic agents to treat Staphylococcus infection.

Complete genome sequencing of a Tequintavirus bacteriophage with a broad host range against Salmonella Abortus equi isolates from donkeys.

Salmonella enterica subspecies enterica serovar abortus equi (S. Abortus equi) is the most common cause of abortion in mares. It has recently been found to cause abortion in donkeys more frequently in China. A novel virulent bacteriophage vB_SabS_Sds2 (hereafter designated as Sds2) was isolated from the feces of donkeys using a S. Abortus equi strain as a host. Phage Sds2 had an isometric polyhedral head and an uncontracted long tail, belonging to the Tequintavirus, Markadamsvirinae, Demerecviridae, Caudovirales. The genome of phage Sds2 was 114,770 bp, with a GC content of 40.26%. The genome contained 160 open reading frames (ORFs), and no ORFs were associated with pathogenicity, drug resistance, or lysogenization by sequence analysis. Both genome annotation and phylogenetic analysis indicated that phage Sds2 was highly similar to T5-like bacteriophages. Phage Sds2 could lyse 100% (30/30) of S. Abortus equi strains, 25.3% (24/95) of other serotypes of Salmonella strains, and 27.6% (8/29) of Escherichia coli strains using the double-layer agar plate method. The in vitro test showed that phage Sds2 had high bactericidal activity against S. Abortus equi at a wide range of MOIs. The in vivo test indicated that phage Sds2 had an inhibitory effect on abortion in mice challenged with S. Abortus equi. In general, phage Sds2 is a novel lytic phage with a wide host range and has the potential to prevent abortion caused by S. Abortus equi.

Characterization and complete genome analysis of a bacteriophage vB_EcoM_DE7 infecting donkey-derived Escherichia coli.

A lytic bacteriophage vB_EcoM_DE7 (hereafter designated DE7) that could infect donkey-derived Escherichia coli was isolated. The bacteriophage was examined by transmission electron microscopy, and the result showed that DE7 belonged to the family Myoviridae. The microbiological characterization revealed that DE7 was stable over a broad range of pHs (3 ∼10) at 40-50 °C. The latent period was 10 min, and the burst size was 43 PFUs/infected cell. The whole-genome sequencing showed that DE7 was a dsDNA virus and had a genome of 86,130 bp. The genome contained 124 predicted open reading frames (ORFs), 35 of which had known functions, including DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. Twenty tRNA genes were identified, but no genes associated with bacterial pathogenicity, lysogeny and drug resistance were identified. BLASTN analysis revealed that phage DE7 had a high sequence identity (96%) with Salmonella phage vB_SPuM_SP116, but it could not lyse any Salmonella strain tested in this study. DE7 was classified as a Felix O1-like virus based on its general characterization and genomic information. Since phage DE7 exhibited high efficacy in lysing E. coli and lacked genes associated with bacterial virulence, antimicrobial resistance and lysogeny, it could be potentially used to control foal diarrhoea caused by E. coli.

Isolation and characterization of Vibrio parahaemolyticus bacteriophage vB_VpaS_PG07.

A novel bacteriophage vB_VpaS_PG07 (hereafter designated PG07) that infects Vibrio parahaemolyticus was isolated. The bacteriophage was examined by transmission electron microscopy, and the result showed that PG07 belonged to family Siphoviridae, with an isometric polyhedral head (80 nm in diameter) and a long tail (175 nm in length). The one-step growth curve showed that the latent period and burst size were 10 min and 60 PFUs/infected cell, respectively. PG07 had double-stranded DNA genome of 112, 106 bp with 43.65 % G+C content. A total of 158 putative open reading frames (ORFs) were identified in the genome of PG07, including functional genes associated with integration, nucleotide metabolism and replication, structure and packaging and bacterial lysis. Sixteen tRNA genes were discovered, and no genes associated with pathogenicity and virulence were identified. The genome of PG07 showed very low similarity to phage genomes deposited in public databases (77.65 % nucleotide identity and 9 % query coverage). The newly sequenced PG07 could be considered as a novel T5-like virus. PG07 significantly reduced the mortality of shrimps challenged with V. parahaemolyticus, a bacterium causing acute hepatopancreatic necrosis disease (AHPND). The findings highlight the potential of PG07 as an effective antibacterial agent for phage prophylaxis and phage therapy in aquaculture.

Isolation and characterization of the Staphylococcus aureus bacteriophage vB_SauS_SA2.

A novel bacteriophage vB_SauS_SA2 (hereafter designated SA2) that infects Staphylococcus aureus was isolated. At a multiplicity of infection (MOI) of 0.1, phage SA2 had a latent period of about 10 min with a burst size of 293 PFUs/infected cell (PFU, plaque forming unit). Phage SA2 had a double-stranded DNA genome with a length of 89,055 bp and a G + C content of 31.9%. The genome contained 130 open reading frames (ORFs), 28 of which had assigned functions, and 18 were unique. One tRNA gene (tRNAAsn ) was discovered, and no virulence genes were identified. Its genome showed very low similarity with phage genomes deposited in public databases (75% nucleotide identity and 7% query coverage). The unique characteristics of phage SA2 led to the proposal of a new Siphoviridae genus named 'SA2likevirus'.

Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.

The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.