LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi

In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and the luxS mutant. However, the deletion of luxS decreased the secretion of extracellular protease, while increased the ability of swimming and swarming. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented the defect. Since motility is flagella dependent, 16 of V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly up-regulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phase.

Ozoroa insignis reticulata (Baker f.) R. Fern. & A. Fern. Root Extract Inhibits the Production of Extracellular Proteases by Staphylococcus aureus

Treatment of infections caused by S. aureus has become a challenge due to the emergency of resistant strains. Ozoroa reticulata root extracts have been used in traditional medicine to treat throat and chest pains in Zimbabwe. The objective of the study was to determine the effects of O. reticulata root bark extracts on the production of extracellular proteases by S. aureus. The root barks were collected, dried, and crushed into powder. To obtain different phytoconstituents, plant extractions were performed. Extractions were carried out using two solvent mixtures: ethanol : water (50 : 50 v/v) and dichloromethane : methanol (50 : 50 v/v). Serial exhaustive extractions were also performed using methanol, ethanol, dichloromethane, acetone, ethyl acetate, hexane, and water. The broth microdilution assays were used to assess the antibacterial effects of the Ozoroa reticulata root bark extracts against S. aureus. Ciprofloxacin was used as a positive control. Qualitative screening for extracellular protease production by S. aureus on BCG-skim milk agar plates using the most potent extract was carried out. The proteolytic zones were measured and expressed as the ratio of the diameter of the colony to the total diameter of the colony plus the zone of hydrolysis ( P z values). The ethyl acetate extract was found to be the most potent inhibitor of the growth of S. aureus with 99% inhibition and a minimum inhibitory concentration (MIC) of 100 µg/mL. Inhibition of extracellular protease production was directly proportional to the concentration of the extract. At 100 µg/mL, the ethyl acetate extract had a P z value of 0.84, indicative of mild proteolytic activity. A P z value of 0.94 was observed at a concentration of 200 µg/mL and signified weak proteolytic activity. In conclusion, the extract inhibited the production of extracellular proteases in S. aureus. Further work on the isolation and purification of bioactive compounds responsible for inhibiting the production of extracellular proteases is of importance in the discovery of agents with antivirulent effects on S. aureus.

The protease SplB of Staphylococcus aureus targets host complement components and inhibits complement-mediated bacterial opsonophagocytosis

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune-evasion proteins, including the six serine protease-like proteins SplA-SplF. Spl proteins are expressed by most clinical isolates of S. aureus , but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. By contrast, the important soluble human complement regulators, Factor H and C4BP, as well as C1q, were left intact. Thereby SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered as a novel staphylococcal complement-evasion protein. Importance Success of bacterial pathogens in immunocompetent humans depends on control and inactivation of host immunity. S aureus , like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus -encoded orphan proteases SplA-SplD, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three-complement activation pathways as well as the lytic terminal complement pathway. This blocks opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8 C9 as well as Factor B, but not the complement inhibitors Factor H and C4BP. Thus we identified the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement-evasion protein.

Expression of degQ gene and its effect on lipopeptide production as well as the formation of secretory proteases in Bacillus subtilis strains

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T, surfactin, and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T, which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T, while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T. While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a 6-fold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by 3-fold but decreased plipastatin production by 5-fold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Production of an extracellular neutral protease by Bacillus aerius UB02 endophytic to carnivorous plant Utricularia stellaris

Aim: Endophytic bacteria indigenous to carnivorous plants have been explored for production of novel bioactive metabolites including extracellular enzymes. Bacillus aerius UB02, an extracellular neutral protease producing isolate endophytic to bladder of Utricularia stellaris L. f. was used in this study. Methodology: The bacterial isolate UB02 was identified following morpholological, physiological, biochemical and 16S rRNA gene sequence analyses. The media as well as the cultural conditions for production of protease were optimized. The extracellular protease was isolated and purified from the cell-free culture filtrate by ammonium sulphate precipitation, dialysis and DEAE Sephadex ion exchange column chromatography and the optimum conditions for its activity were determined. Results: The isolate Bacillus aerius UB02 (GenBank accession no. MK 696417, MCC accession no. 4132), produced significant amount of extracellular protease (38.29 U mg-1 protein) during growth in casein supplemented synthetic medium. However, peptone yeast extract glucose medium appeared to be the best for the synthesis of enzyme. Production of enzyme was enhanced by the inoculum density of 1.5% (v/v), culture volume: flask volume (CVF) ratio of 1:10, substrate concentration of 2.5% (w/v) with temperature and pH adjusted at 37°C and 7.4, respectively. Glucose (2.2%, w/v) and ammonium chloride (1.2 g/L) as carbon and nitrogen sources also favoured the enzyme production. The neutral protease with a molecular weight of approximately 35 kDa showed maximum activity at 40°C, pH 7.8 with 2% (w/v) casein. The enzyme exhibited Km and Vmax values of 6.81 mg ml-1 and 62.5 U mg-1 of protein, respectively, and was moderately thermostable. The protease activity was inhibited by Pb and Cd as well as 1,10-phenanthroline and β-mercaptoethanol. Interpretation: These findings will help not only in understanding the role of endophytic bacteria and the enzymes produced by them in the digestion of prey by carnivorous plant but could also be explored for application in the field of biotechnology.

Probiotic Bacillus Affect Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from cCF10 without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30-50 kDa extracellular protease present in B. subtilis natto. Nattokinase of B. subtilis natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into “LVTL + VFV” fragments. Moreover, the cleavage product “LVTL” (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. Importance Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.