Suicin 90-1330 from a Nonvirulent Strain of Streptococcus suis: a Nisin-Related Lantibiotic Active on Gram-Positive Swine Pathogens

Streptococcus suis serotype 2 is known to cause severe infections (meningitis, endocarditis, and septicemia) in pigs and is considered an emerging zoonotic agent. Antibiotics have long been used in the swine industry for disease treatment/prevention and growth promoters. This pattern of utilization resulted in the spread of antibiotic resistance in S. suis worldwide. Interestingly, pigs may harbor S. suis in their tonsils without developing diseases, while North American strains belonging to the sequence type 28 (ST28) are nonvirulent in animal models. Consequently, the aim of this study was to purify and characterize a bacteriocin produced by a nonvirulent strain of S. suis serotype 2, with a view to a potential therapeutic and preventive application. S. suis 90-1330 belonging to ST28 and previously shown to be nonvirulent in an animal model exhibited antibacterial activity toward all S. suis pathogenic isolates tested. The bacteriocin produced by this strain was purified to homogeneity by cationic exchange and reversed-phase fast protein liquid chromatography. Given its properties (molecular mass of <4 kDa, heat, pH and protease stability, and the presence of modified amino acids), the bacteriocin, named suicin 90-1330, belongs to the lantibiotic class. Using a DNA-binding fluorophore, the bacteriocin was found to possess a membrane permeabilization activity. When tested on other swine pathogens, the suicin showed activity against Staphylococcus hyicus and Staphylococcus aureus, whereas it was inactive against all Gram-negative bacteria tested. Amino acid sequencing of the purified bacteriocin showed homology (90.9% identity) with nisin U produced by Streptococcus uberis. The putative gene cluster involved in suicin production was amplified by PCR and sequence analysis revealed the presence of 11 open reading frames, including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Further studies will evaluate the ability of suicin 90-1330 or the producing strain to prevent experimental S. suis infections in pigs.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Purification and Characterization of Suicin 65, a Novel Class I Type B Lantibiotic Produced by Streptococcus suis

Bacteriocins are antimicrobial peptides of bacterial origin that are considered as a promising alternative to the use of conventional antibiotics. Recently, our laboratory reported the purification and characterization of two lantibiotics, suicin 90–1330 and suicin 3908, produced by the swine pathogen and zoonotic agent Streptococcus suis (serotype 2). In this study, a novel bacteriocin produced by S. suis has been identified and characterized. The producing strain S. suis 65 (serotype 2) was found to belong to the sequence type 28, that includes strains known to be weakly or avirulent in a mouse model. The bacteriocin, whose production was only possible following growth on solid culture medium, was purified to homogeneity by cationic exchange and reversed-phase high-pressure liquid chromatography. The bacteriocin, named suicin 65, was heat, pH and protease resistant. Suicin 65 was active against all S. suis isolates tested, including antibiotic resistant strains. Amino acid sequencing of the purified bacteriocin by Edman degradation revealed the presence of modified amino acids suggesting a lantibiotic. Using the partial sequence obtained, a blast was performed against published genomes of S. suis and allowed to identify a putative lantibiotic locus in the genome of S. suis 89–1591. From this genome, primers were designed and the gene cluster involved in the production of suicin 65 by S. suis 65 was amplified by PCR. Sequence analysis revealed the presence of ten open reading frames, including a duplicate of the structural gene. The structural genes (sssA and sssA’) of suicin 65 encodes a 25-amino acid residue leader peptide and a 26-amino acid residue mature peptide yielding an active bacteriocin with a deducted molecular mass of 3,005 Da. Mature suicin 65 showed a high degree of identity with class I type B lantibiotics (globular structure) produced by Streptococcus pyogenes (streptococcin FF22; 84.6%), Streptococcus macedonicus (macedocin ACA-DC 198; 84.6%), and Lactococcus lactis subsp. lactis (lacticin 481; 74.1%). Further studies will evaluate the ability of suicin 65 or the producing strain to prevent experimental S. suis infections in pigs.     

Novel Variant Serotype of Streptococcus suis Isolated from Piglets with Meningitis

Streptococcus suis is an emerging zoonotic pathogen causing severe infections in pigs and humans. In previous studies, 33 serotypes of S. suis have been identified using serum agglutination. Here, we describe a novel S. suis strain, {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302, isolated from an outbreak of acute piglet meningitis in eastern China. Strong pathogenicity of meningitis caused by strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 was reproduced in the BALB/c mouse model. The strain showed a high fatality rate (8/10), higher than those for known virulent serotype 2 strains P1/7 (1/10) and 9801 (2/10). Cell adhesion assay results with bEnd.3 and HEp2 cells showed that {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 was significantly close to P1/7 and 9801. Both the agglutination test and its complementary test showed that strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 had no strong cross-reaction with the other 33 S. suis serotypes. The multiplex PCR assays revealed no specified bands for all four sets used to detect the other 33 serotypes. In addition, genetic analysis of the whole cps gene clusters of all serotypes was performed in this study. The results of comparative genomics showed that the cps gene cluster of {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302, which was not previously reported, showed no homology to the gene sequences of the other strains. Especially, the wzy, wzx, and acetyltransferase genes of strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 are phylogenetically distinct from strains of the other 33 serotypes. Therefore, this study suggested that strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 represents a novel variant serotype of S. suis (designated serotype Chz) which has a high potential to be virulent and associated with meningitis in animals.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

Genome Sequence of the Swine Pathogen Streptococcus suis Serotype 2 Strain S735

Streptococcus suis is a major swine pathogen responsible for significant, worldwide economic losses in the swine industry, in addition to being an emerging zoonotic agent. Strains of serotype 2 are the most commonly associated with infections causing meningitis, endocarditis, and septicemia. Here we present the genome sequence of S. suis serotype 2 strain S735.     

猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940的比较转录谱分析

目的:比较猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940毒力相关基因转录水平的差异,为进一步研究强毒株S.suis 05ZY毒力增强的原因提供实验基础。方法:分别提取S.suis 05ZY和S.suis 1940的RNA,反转录成cDNA并纯化,用Cy5或Cy3标记,与猪链球菌全基因组DNA芯片进行杂交,扫描芯片进行数据分析,比较二者在转录水平上的差异基因。结果:编码溶血素、精氨酸氨基肽酶的基因分别上调4.4和6.0倍,参与荚膜多糖合成的相关基因cps2H、cps2I、cps2J和一些可能的毒力相关基因ofs、dpr、SSU05₀196、SSU05₀272、SSU05₁408-1409均发生转录水平的上调。结论:溶血素、荚膜多糖、精氨酸氨基肽酶及一些可能的毒力因子在转录水平的上调很可能与S.suis 05ZY的毒力增强有关。     

致病性猪链球菌2型溶血素基因的克隆与表达

目的：克隆溶血素基因,为评价溶血素的毒力与抗原活性,构建猪链球菌疫苗提供依据。方法：从致病性猪链球菌II型四川资阳分离株基因组中分离溶血素基因,经中间T载体,将其克隆至改造的pGEX-6p1中,最后通过蛋白质电泳和溶血实验检验溶血素的表达及活性。结果：SDS-PAGE结果表明,重组溶血素在5h表达水平最高,10h次之,15h最弱;溶血实验重组菌周围形成明显的透明溶血环。结论：溶血素为分泌性早期表达,具有正常的β溶血活性。     

猪链球菌2型的致病机制

猪链球菌2型是重要的人兽共患传染病病原。通常认为该菌的毒力因子与荚膜多糖(CPS)、溶菌酶释放蛋白(MRP)、细胞外因子(EF)和溶血素(SLY)等有关。该文介绍2型猪链球菌的感染途径、侵袭和黏附机制,以及对机体产生细胞因子的影响等。     

Complete Genome Sequence of Actinobacillus suis H91-0380, a Virulent Serotype O2 Strain

Here, we report the first complete genome sequence of Actinobacillus suis, an important opportunistic pathogen of swine. By comparing the genome sequence of A. suis with those of other members of the family Pasteurellaceae, we hope to better understand the role of these organisms in health and disease in swine.     

2型猪链球菌中国强毒株05ZYH33 sao基因敲除突变株的构建及其生物学功能

构建2型猪链球菌(Streptococcus suis serotype 2,S.suis 2)中国强毒株05ZYH33的sao基因敲除突变株。构建中间为壮观霉素抗性基因,两侧为sao编码基因上下游同源序列的基因敲除载体,同源重组筛选sao基因敲除突变株。PCR、RT-PCR、Western Blot对疑似突变株进行验证,实验结果均证实sao基因完全被spc抗性基因替代,成功构建了突变株05ZYH33-sao。对野生型菌株和突变株进行菌落溶血活性、生长特性、小鼠致病性比较,结果表明sao基因的敲除并未使野生型菌株在以上三方面产生明显的变化。筛选获得的05ZYH33 sao基因突变株为进一步研究sao基因在05ZYH33致病过程中的作用奠定了基础。     

Construction and Characterization of a Streptococcus suis Serotype 2 Recombinant Expressing Enhanced Green Fluorescent Protein

Streptococcus suis serotype 2 (S. suis 2) is an important pathogen, responsible for diverse diseases in swine and humans. To obtain a S. suis 2 strain that can be tracked in vitro and in vivo, we constructed the Egfp-HA9801 recombinant S. suis 2 strain with egfp and spc(r) genes inserted via homologous recombination. To assess the effects of the egfp and spc(r) genes in HA9801, the biochemical characteristics, growth features and virulence in Balb/C mice were compared between the recombinant and the parent HA9801 strain. We detected the EGFP expression from Egfp-HA9801 by epifluorescence microscopy. The results showed that the biochemical characterization and growth features of the Egfp-HA9801 recombinant were highly similar to that of the parent HA9801. We did not find significant differences in lethality (50% lethal dose), morbidity and mortality between the two strains. Furthermore, the bacterial counts in each various tissues of Egfp-HA9801-infected mice displayed similar dynamic compared with the HA9801-infected mice. Our results also showed that the Egfp-HA9801 cells grown at 37°C for 36 h displayed greater green fluorescence signals than the cells grown at 28°C for 36 h and 37°C for 24 h. The fluorescence in the tissue cryosections of Egfp-HA9801-injected mice was also stronger than that of the HA9801 group. Together, these results indicate that the egfp and spc(r) insertions into the Egfp-HA9801 recombinant did not significantly change the virulence when compared with HA980, and this EGFP labeled strain can be used for future S. suis 2 pathogenesis research.     

Identification and Characterization of a Two-Peptide Class IIb Bacteriocin in Streptococcus pluranimalium Isolated from the Nasal Cavity of a Healthy Pig

Probiotics and Antimicrobial Proteins - In addition to be an important zoonotic agent, Streptococcus suis serotype 2 causes severe infections in pigs. In this study, we characterized a new...     

Complete Genome Sequence of Streptococcus mutans GS-5, a Serotype c Strain

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.     

猪链球菌2型菌毛骨架蛋白编码基因SSU2101敲除突变株的构建及其生物学功能

利用同源重组基因敲除方法构建猪链球菌2型(Streptococcus suis serotype2,S.suis2)中国强毒株05ZYH33菌毛骨架蛋白(Backbone protein,BP)编码基因SSU2101敲除突变株。采用引物特异性PCR分析、Southern杂交及RT-PCR等方法鉴定,证实成功构建了BP基因缺失突变株。生物学特性显示,突变株的菌落形态、溶血活性以及染色特性方面与野生株之间均无明显差异。小鼠致病性试验结果显示,突变株的毒力比野生株显著减弱。研究结果提示菌毛在S.suis2感染致病过程中起重要作用,为系统研究S.suis2菌毛分子装配机制及其生物学功能奠定了基础。     

Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484

Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions—Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys—-and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.