Development of a dual fluorescent reporter system to identify inhibitors of Staphylococcus aureus virulence factors.

IMPORTANCE: Staphylococcus aureus is a formidable pathogen responsible for a wide range of infections, and the emergence of antibiotic-resistant strains has posed significant challenges in treating these infections. In this study, we have established a novel dual reporter system capable of concurrently monitoring the activities of two critical virulence regulators in S. aureus. By incorporating both reporters into a single screening platform, we provide a time- and cost-efficient approach for assessing the activity of compounds against two distinct targets in a single screening round. This innovative dual reporter system presents a promising strategy for the identification of molecules capable of modulating virulence gene expression in S. aureus, potentially expediting the development of antivirulence therapies.

Flavone inhibits Staphylococcus aureus virulence via inhibiting the sae two component system.

The rising prevalence of antibiotic resistance in Staphylococcus aureus calls for the development of innovative antimicrobial agents targeting novel pathways. S. aureus generates various virulence factors that compromise host defense mechanisms. Flavone, a core structure of flavonoids, has been shown to diminish the production of staphyloxanthin and alpha-hemolysin. Nonetheless, the influence of flavone on the majority of other virulence factors in S. aureus and its underlying molecular mechanism remain elusive. In this study, we examined the impact of flavone on the transcriptional profile of S. aureus using transcriptome sequencing. Our findings revealed that flavone substantially downregulated the expression of over 30 virulence factors implicated in immune evasion by the pathogen. Gene set enrichment analysis of the fold change-ranked gene list in relation to the Sae regulon indicated a robust association between flavone-induced downregulation and membership in the Sae regulon. Through the analysis of Sae target promoter-gfp fusion expression patterns, we observed a dose-dependent inhibition of Sae target promoter activity by flavone. Moreover, we discovered that flavone protected human neutrophils from S. aureus-mediated killing. Flavone also decreased the expression of alpha-hemolysin and other hemolytic toxins, resulting in a reduction in S. aureus' hemolytic capacity. Additionally, our data suggested that the inhibitory effect of flavone on the Sae system operates independently of its capacity to lower staphyloxanthin levels. In conclusion, our study proposes that flavone exhibits a broad inhibitory action on multiple virulence factors of S. aureus by targeting the Sae system, consequently diminishing the bacterium's pathogenicity.

Isolation and Characterization of a Ranavirus Associated with Disease Outbreaks in Cultured Hybrid Grouper (♀ Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper E. lanceolatus) in Guangxi, China.

An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24 h, and the effects increased at 48 h postinfection. The transmission electron microscopy results showed that viral particles that were about 200 nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1 h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1 h. The analyses showed that SGIV-Gx was stable for 1 h at 4°C and 25°C but was inactivated after 1 h at 40, 50, and 60°C.

Development of novel aptamer-based enzyme-linked apta-sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus.

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)-based enzyme-linked apta-sorbent assay (VA2-ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2-ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2-ELASA could specifically identify V. alginolyticus, but not other non-target bacterial strains. VA2-ELASA could detect V. alginolyticus at the concentration of 5 × 104 /ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2-ELASA in this study. It took less than one hour to accomplish the detection process by VA2-ELASA. The characteristics of specificity, sensitivity and easy operation make VA2-ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.

The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo.

Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well-known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers.

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus.

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single-stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem-loop structures, which could form the basis of aptamers' specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.

Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw.

A stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was conducted to enhance enzymatic hydrolysis of rice straw (RS). The results showed that the composition of RS changed significantly, the lignin and hemicellulose decreased while cellulose increased. The biological treatment with a bacterium Sphingobacterium sp. LD-1 achieved mild conditions for the sequential NU treatment, reducing the concentration of the NU solution from 7%/12% to 4%/6% and increasing the temperature from -20°C to -10°C. The saccharification of rice straw co-treated with bacterium Sphingobacterium sp. LD-1 and 4%/6% NU at -10°C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment.

Predicting Km values of beta-glucosidases using cellobiose as substrate.

The Michaelis-Menten constant Km is a very important parameter to relate enzyme with its substrate in enzymatic reaction. Although Km can be experimentally determined, the Km values are not easily available in literature. With rapid increase of newly designed enzymes, we face the shortage of parameters related to enzymatic reactions. The beta-glucosidase is a crucial enzyme for cellulose hydrolysis and cellobiose is one of its substrates. In this study, we attempt to develop models to predict Km with cellobiose as substrates using information about primary structure of beta-glucosidase. The results show that the 20-1 feedforward backpropagation neural network using the amino-acid distribution probability as predictor works best for prediction of Km values.