Exposing the Secrets of Two Well-Known Lactobacillus casei Phages,J-1 and PL-1, by Genomic and Structural Analysis

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using Lactobacillus casei strain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other Siphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically to Lactobacillus casei/paracasei cells, and the addition of l-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls of L. casei ATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into how Lactobacillus phages interact with their hosts at the first steps of infection.     

Differential Adherence and Expression of Virulence Traits by Candida albicans and Candida parapsilosis in Mono- and Dual-Species Cultures in Artificial Saliva

Aims

To evaluate specific virulence factors of Candida albicans and Candida parapsilosis clinical oral isolates in mono- and dual-species culture in the presence of artificial saliva.

Methods and Results

Two of the strains used in this study were isolated from co-infection (C. albicans AM and C. parapsilosis AM2), and the other two were isolated from single infection (C. albicans AC and C. parapsilosis AD). The number of adhered yeast cells was measured and their enzymatic activity was determined simultaneously. In mono-species culture, C. parapsilosis strains adhered to a higher extent to the surface in comparison with the C. albicans strains. In dual-species culture, the C. parapsilosis strains adhered more in the presence of C. albicans AM. Interestingly, C. albicans AM and C. parapsilosis AD adhered to a higher extent when compared with all other co-cultures. In dual-species culture, the enzymatic activity of C. parapsilosis strains in the presence of C. albicans AC was higher than in the presence of C. albicans AM.

Conclusions

The virulence factors of C. albicans and C. parapsilosis differ from strain to strain and are influenced by the presence of other species in culture.

Significance and Impact of the Study

To understand the expression of virulence factors in Candida dual-species systems.     

Staphylococcal Enterotoxins B and C

Abstract

The staphylococcal enterotoxins (SEs) are a subgroup of related protein exotoxins in the pyrogenic toxin (PT) family produced by Staphylococcus aureus and Streptococcus pyogenes (1). Like other members of the PT family, the SEs are superantigens and elaborate a set of biological activities linked to their ability to stimulate cells of the immune system (2). These activities contribute to their ability to induce toxic shock syndrome, immunosuppression, and probably other diseases (3). However, as is evident from the fact that they are designated as enterotoxins, the SEs are distinguishable from other members of the PT family by their ability to induce gastroenteritis when ingested. Hence, they are the causative agents in staphylococcal food poisoning (SFP), a very common form of food-associated gastroenteritis in the United States and worldwide (4).     

Isolation and Properties of Two New RNA Phages SP and FI

New RNA phages were isolated from feces of Siamang Gibbon and infants, and their characters were investigated. These phages, SP and FI were serologically unrelated to any of known RNA phages (group I, II and III) and were classified into groups IV and V. Several other characters, such as filtration and elution patterns through membrane filters, buoyant densities in CsCl, and UV sensitivities of SP and FI were similar to those of the group III phages, suggesting that a certain similarity between group III and IV or V might exist. Peculiar phage-host relations found in phages of FI group were also discussed. From these results, we propose new possible schemata for the grouping of RNA phages.     

Complete Genome Sequence Analysis of Two Pseudomonas plecoglossicida Phages,Potential Therapeutic Agents

Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416–1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33–39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.     

Capillary Tube Assay for Staphylococcal Enterotoxins A, B, and C

A miniaturized single-gel diffusion procedure for detection of staphylococcal enterotoxin is proposed. The technique effects substantial savings of reagents and is easy to perform.     

Production of Staphylococcal Enterotoxins A, B, and C in Various Media

The effect of initial pH and of length of incubation time at 37 C in four different growth media on the production of staphylococcal enterotoxins A, B, and C was determined. A starting pH of 6.8 gave higher yields of enterotoxins B and C than either pH 6.0 or 5.3. The production of enterotoxin A was, however, not materially affected by the low initial pH of 5.3. Prolonged incubation (48 to 72 hr) resulted only occasionally in higher yields of enterotoxin. The effect of the media on the amount of enterotoxin produced is considerable. Difco Brain Heart Infusion (BHI) was inferior to either Fisher BHI, 4% NZ Amine (NAK), or 3% NAK plus 3% protein hydrolysate powder at the three initial pH values, regardless of length of incubation time. The slight effect of the low starting pH on the production of enterotoxin A is being further investigated.     

Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions

Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken.     

Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to the Levivirus group and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found in Levivirus and Allolevivirus predate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making the Levivirus-like genome organization ancestral and the Allolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.     

A combination therapy of Phages and Antibiotics: Two is better than one

Emergence of antibiotic resistance presents a major setback to global health, and shortage of antibiotic pipelines has created an urgent need for development of alternative therapeutic strategies. Bacteriophage (phage) therapy is considered as a potential approach for treatment of the increasing number of antibiotic-resistant pathogens. Phage-antibiotic synergy (PAS) refers to sublethal concentrations of certain antibiotics that enhance release of progeny phages from bacterial cells. A combination of phages and antibiotics is a promising strategy to reduce the dose of antibiotics and the development of antibiotic resistance during treatment. In this review, we highlight the state-of-the-art advancements of PAS studies, including the analysis of bacterial-killing enhancement, bacterial resistance reduction, and anti-biofilm effect, at both in vitro and in vivo levels. A comprehensive review of the genetic and molecular mechanisms of phage antibiotic synergy is provided, and synthetic biology approaches used to engineer phages, and design novel therapies and diagnostic tools are discussed. In addition, the role of engineered phages in reducing pathogenicity of bacteria is explored.     

Thermal Inactivation of Staphylococcal Enterotoxins B and C

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.     

金黄色葡萄球菌肠毒素 C2 的基因克隆、 表达及其生物学活性

利用 PCR 技术从金黄色葡萄球菌的基因组 DNA 中克隆 SEC2 全长基因, PCR 产物与 pGEM-T 载体连接,经测序证实后进行亚克隆,构建其表达载体 pET-28a-SEC2 ,在大肠杆菌 BL21(DE3) 中表达成熟重组蛋白 (rSEC2) , 纯化 rSEC2 蛋白并对其生物学活性进行研究 . 结果表明：成功克隆了 SEC2 全长基因,测序证实该基因共 717 bp ,编码 239 个氨基酸,与 GenBank 中收录的 SEC2 成熟蛋白质序列完全一致, SEC2 基因登录 GenBank(Accession number ： AY450554) ; 构建了 SEC2 的表达载体 pET-28a-SEC2 ,并在大肠杆菌 BL21(DE3) 中得到高效可溶性表达,可溶性的 rSEC2 经 Ni²⁺ 亲和层析纯化达到电泳纯,纯化的 rSEC2 蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被 rSEC2 刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用 .     

金黄色葡萄球菌肠毒素A、B、C、D、E重组载体的构建及表达

金黄色葡萄球菌肠毒素（Staphylococcal enterotoxins,SEs）是一组结构毒力相似、血清型不同的可溶性小分子蛋白质,平均分子质量为26~30 kD,是引起细菌性食物中毒及肠胃炎的主要因素之一。为了制备金黄色葡萄球菌肠毒素的纯品,首先合成了SEA、SEB、SEC、SED和SEE的基因序列,然后构建了5种肠毒素的原核表达载体,分别转入BL21（DE3）细胞中进行诱导表达。通过SDS-PAGE和Western blot验证,5种肠毒素蛋白均被成功表达,并且在较低的诱导温度（16 ℃）获得一定量的可溶性蛋白。成功制备了5种金黄色葡萄球菌肠毒素的可溶性蛋白,为今后更好地解决因SEs引起的食品安全问题奠定了基础。     

Separation of Detoxin Complex and Characterization of Two Active Principles,Detoxin C1 and D1

Separation of the active principles of DX.C was worked out into eight groups with the use of an ion exchange chromatography. Further purification of main components, the isolation and characterization of two main active principles were described.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

Polyvalent Antiserum Agar System for the Detection of Staphylococcal Enterotoxins A, B, C, and E

A polyvalent antiserum agar system in capillary tubes was developed and evaluated for the detection of enterotoxins A, B, C, and/or E present in culture supernatant fluids.