解淀粉欧文氏菌噬菌体Kuerle的分离、基因组测定及其裂解功能分析

【背景】解淀粉欧文氏菌(Erwiniaamylovora)是仁果类果树火疫病的病原体,对全球苹果和梨的生产构成严重威胁。随着抗生素耐药菌株的出现,火疫病的防治面临巨大的挑战。【目的】分离一种新的裂解解淀粉欧文氏菌的噬菌体,并分析该噬菌体裂解相关基因的功能,为火疫病的防治提供新的选择。【方法】以解淀粉欧文氏菌Ea102为宿主菌,采用双层平板法从流行火疫病的果园土壤中分离噬菌体。通过噬菌斑和透射电镜观察其形态。用PacBio测序技术测定噬菌体基因组,SPAdes组装序列,RAST注释基因组。通过大肠杆菌原核表达系统分析噬菌体Kuerle的裂解机制。【结果】分离纯化出一株裂解解淀粉欧文氏菌的噬菌体,命名为Kuerle。Kuerle由二十面体衣壳的头部和短尾组成,潜伏期约为50 min,裂解量约为240 pfu/cell。噬菌体基因组全长75599 bp,GC含量48.0%,末端有393 bp的重复序列,未发现与溶原调控相关的基因。共预测到85个开放阅读框(open reading frame,ORF),其中33个已知功能蛋白中包含一个由3550 aa组成的巨大病毒颗粒相关的RNA聚合酶(virion-associated RNA polymerase,vRNAP),该vRNAP是Schitoviridae科噬菌体的主要特征之一。病毒粒子和基因结构表明噬菌体Kuerle属于有尾噬菌体类的Schitoviridae科病毒。聚集在DNA晚期表达区的3个裂解相关基因holin、endolysin和spanin组成了“裂解盒”。在大肠杆菌中Kuerle-holin的表达会抑制细胞的生长,而Kuerle-endolysin的表达可裂解细胞。二者共表达时Kuerle-holin会加速Kuerle-endolysin引起的细胞裂解。用叠氮化钠抑制细菌的一般分泌系统(Sec)或Kuerle-endolysin N端信号序列的缺失均会导致endolysin裂解功能丧失。上述结果表明噬菌体Kuerle具有pinholin-SAR endolysin裂解系统。Kuerle-holin(pinholin)使细胞质膜去极化以激活分泌的Kuerle-endolysin(SAR endolysin)降解肽聚糖层。【结论】Kuerle是一株烈性噬菌体并且Kuerle-endolysin抑菌效果显著,为后续火疫病生防试剂的制备提供了理论依据和研究材料。

Phage therapy: An alternative to antibiotics in the age of multi-drug resistance

The practice of phage therapy, which uses bacterial viruses(phages) to treat bacterial infections, has been around for almost a century. The universal decline in the effectiveness of antibiotics has generated renewed interest in revisiting this practice. Conventionally, phage therapy relies on the use of naturally-occurring phages to infect and lyse bacteria at the site of infection. Biotechnological advances have further expanded the repertoire of potential phage therapeutics to include novel strategies using bioengineered phages and purified phage lytic proteins. Current research on the use of phages and their lytic proteins against multidrug-resistant bacterial infections, suggests phage therapy has the potential to be used as either an alternative or a supplement to antibiotic treatments. Antibacterial therapies, whether phage-or antibiotic-based, each have relative advantages and disadvantages; accordingly, many considerations must be taken into account when designing novel therapeutic approaches for preventing and treating bacterial infection. Although much about phages and human health is still being discovered, the time to take phage therapy serious again seems to be rapidly approaching.

Specific inhibition of Streptococcus bovis by endolysin LyJH307 supplementation shifts the rumen microbiota and metabolic pathways related to carbohydrate metabolism

Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S.bovis and to explore changes in rumen fermentation and microbiota in an in vitro system.Two treatments were used:1)control,corn grain without LyJH307;and 2)LyJH307,corn grain with LyJH307(4 U/mL).An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers(450±30 kg)and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time.In vitro dry matter digestibility,pH,volatile fatty acids,and lactate concentration were estimated at 12 h,and the gas production was measured at 6,9,and 12 h.The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing.Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S.bovis(approximately 70% compared to control,P=0.0289)and increased ruminal pH(P=0.0335)at the 12 h incubation.The acetate proportion(P=0.0362)was significantly increased after LyJH307 addition,whereas propionate(P=0.0379)was decreased.LyJH307 supplementation increased D-lactate(P=0.0340)without any change in L-lactate concentration(P>0.10).There were no significant differences in Shannon’s index,Simpson’s index,Chao1 estimates,and evenness(P>0.10).Based on Bray-Curtis dissimilarity matrices,the LyJH307 affected the overall shift in microbiota(P=0.097).LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium,WCHB1-41,unclassified genus Selenomonadaceae,Paraprevotella,vadinBE97,Ruminococcus gauvreauii group,Lactobacillus,Anaerorhabdus furcosa group,Victivallaceae,Desulfuromonadaceae,and Sediminispirochaeta.The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism.Conclusions:LyJH307 caused a reduction of S.bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism.This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.

The bacteriophage mu lysis system–A new mechanism of host lysis?

Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection,i.e.,lysis or lysogeny.Phage lysis is one of the conventional biological processes required to spread infection from one bacterium to another.Our analysis suggests that in the paradigm bacteriophage Mu,six proteins might be involved in host cell lysis.Mu has a broad host range,and Mu-like phages were found in both Gram-negative and Gram-positive bacteria.An analysis of the genomes of Mu and Mu-like phages could be useful in elucidating the lysis mechanism in this group of phages.A detailed review of the various mechanisms of phage lysis and different proteins associated with the process will help researchers understand the phage biology and their life cycle in different bacteria.The recent increase in the number of multidrug-resistant(MDR)strains of bacteria and the usual long-term nature of new drug development has encouraged scientists to look for alternative strategies like phage therapy and the discovery of new lysis mechanisms.Understanding the lysis mechanism in the Mu-like phages could be exploited to develop alternative therapeutics to kill drug-resistant pathogenic bacteria.In this review article,we have analyzed the phage Mu-mediated host lysis system,which is unknown till now,and our analysis indicates a possibility of the existence of a new lysis mechanism operating in Mu.

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 p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-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.