Identification And Characterization Of Holin Gene From Streptococcus Suis Phage

Streptococcus suis（S. suis） type2is an important zoonotic pathogen that leads to economic losses and severe invasive diseases in humans, and the increasing prevalence of antibiotics-resistant SS2strains became one of the greatest challenges worldwide. Bacteriophage has been examined as potential agent for SS2control, but its biochemistry and bacterial lysis mechanism is still open. Holin-lysin two-step lysis system typically existing in the double-stranded DNA phage is effective and specific. By accumulating and forming lesions in the cytoplasmic membrane, holins control the access of phage-encoded endolysins to the peptidoglycan and thereby trigger the lysis of the host cell at a precise time point. S. suis serotype2（SS2） lytic bacteriophage SMP is a double-stranded DNA phage. LySMP was identified to be the lysin gene and a putative holin-lysin lysis system was supposed to exist in SMP. In this work, a putative holin gene, HolSMP, was predicted and identified. The relationship of gene sequence and its biological function was analyzed. Moreover, the antibacterial activity of holin from SMP and the synergistic antimicrobial activity of holin and lysin against S suis were tested.To find and locate the putative holin gene of phage SMP, DNA and protein sequence homology alignments were performed using BLAST tools on NCBI. TMHMM, SOSUI and PredictProtein servers were used to predict the transmembrane helices. The results showed that HolSMP, which is located upstream of LySMP gene, was a putative holin gene of SMP. HolSMP is429bp long and shows similarity to the sequence of holin gene from Streptococcus phage. HolSMP（142）, a putative Class I holin with three transmambrane domains encoded by HolSMP, shows similarity to holin protein of Streptococcus phage and exhibits the characteristics of phage-holin₄superfamily.To characterize the transcription and expression of the HolSMP gene in SMP-infected S. suis, the protocols for phage collection and membrane sample extraction were optimized. Based on one-step growth curve of SMP, the total RNA samples and membrane samples were prepared at a series time points during phage infection. The results of reverse transcript PCR, real-time fluorescence quantitative PCR and western blotting assay revealed that HolSMP was transcribed and expressed at15-20min. Accumulation of HolSMP in the cytoplasmic membrane was found before60min. With a reduction of HolSMP transcripts and destruction of host cells, the amount of HolSMP in the membrane also reduced. All the results show that HolSMP is a membrane protein and HolSMP is a late stage gene in conformity with holin gene.To determine the function of HolSMP gene, it is crucial to develop an evaluating system. Holin-dependent membrane lesions are non-specific, and this enables testing of HolSMP in Escherichia coli（E. coli）. Plasmid pEXH1harboring gene HolSMP was transformed into E. coli. Expression of HolSMP inhibited growth of BL21（DE3） and resulted in collapse and death of BL21（DE3）pLysS. Production of HolSMP accumulated in membrane is sensitive to energy toxin potassium cyanide, which is a very important character to identify a new holin family member. Significant cell lysis in the presence of LySMP suggested their putative synergistic effect in physiological situation. Gene HolSMP complemented the defective λ S allele in a non-suppressing E. coli strain to produce phage plaques. These results indicated that HolSMP was the holin gene of SMP. To understand the correlation of structure and function of HolSMP, the start codons of the gene were analyzed. There are ten methionines in HolSMP（142）. These production of sequences started from different "ATG" resulted in various lysis phenotypes. The sequence HolSMP（387） leaded by the fifth "ATG" showed an early-dominance phenotype, starting at about20min ahead of normal lysis time point, while a mutant HolSMP at the fifth "ATG", HolSMPa43c, exhibited an delayed-onset lytic profile, at about55min after induction. These results supported an inference that a dual started model of holin gene Sfrom lambda phage probably existed in gene HolSMP. HolSMP（142） encoded by HolSMP might be the antiholin, while the HolSMP（128） encoded by HolSMP（387） might be the real holin protein. The avirulence protein HolSMP（103） was expressed for preparation of polyclonal antibody against HolSMP.It is also found that the activity of HolSMP is closely related to the encoding sequence, especially to that of N-terminal sequence and integrity of the first transmembrane domain. The addition of an oligohistidine tag at one terminus fundamentally changed the character of HolSMP, altered the triggering time and weakened its lesion-forming activity. Insertion of an oligohistidine tag after codon139yielded HolSMP that showed essentially normal timing and lysis profiles, which might be helpful for preparation and application of HolSMP in the future.To prepare the holin for the purpose in large scale, the expression systems were constructed and characterized. Compared with Lactococcus lactis, mammalian cells and yeast, E. coli strain BL21（DE3）pLysS was the optimum expression system. The expressed HolSMP139t exerted efficient activity at37℃, pH5.2, with addition of0.8%β-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or mixture of HolSMP and LySMP were investigated. The results showed that HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and B. subtilis, which were insensitive to LySMP. However, the simultaneous administration of HolSMP and LySMP has a synergistic effect when applied exogenously.Taken together, this work focused on identification and characterization of holin gene from phage SMP, and a holin-lysin two step lysis system was determined as well. The combined application of HolSMP and LySMP proved that holin and lysin had a synergistic effect when administered exogenously. Further investigation of the topological structure and function of HolSMP and a candidate combined strategy for solution of drug resistance is now warranted.