Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

Background Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. Results We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively. Conclusions In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

The effectiveness of Proteolytic bacteria in the leather and detergent industry isolated waste from the Modjo tannery

Protease also called proteinase or peptidase is a digestive enzyme that is categorized under proteolytic enzymes and it has great potential in industrial application. Extracellular proteases are used in a variety of industries because they exhibit practically all of the characteristics needed for biotech applications such as detergent, bioremediation, food, and leather processing. In the synthesis of all three major types of acidic, neutral, and alkaline proteases, microbial sources have dominated an unbeatable area. Alkaline proteases are a large group of industrial enzymes formed by a wide variety of species, including animals, fungi, and bacteria. The fermentation method serves to make bacteria, fungi, and yeast alkaline proteases. Proteases are produced in large quantities by Gram-positive bacteria, especially those belonging to the Bacillus genus. Following standard procedures, the bacterial isolates PMOJ-01 and PMOJ-05 with the prominent zone of clearance and efficient enzyme development were further characterized to the genus level. Moreover, the growth conditions for the highest protease production were optimized with different pH, temperatures, and NaCl concentrations, in the results of PMOJ-01 and PMOJ- 05 pH (7 and 8), temperatures 45oC, and 1% NaCl concentrations both cases respectively. The proteases activities from PMOJ-01, Pseudomonas aeruginosa, and PMOJ-05, Bacillus subtilis were most active at pH 7.0 and pH 8.0 and temperature at 35oC and 45oC, respectively. The enzyme activity and the total solid protease sample of the crude enzyme of Pseudomonas aeruginosa and Bacillus subtilis were 0.299 U/ mL and 0.289 U/ mL, 1.37±0.14 U/mg, and 1.199 U/mg respectively. The effect on dehairing, distaining, and scum removal revealed that the purified protease enzyme of PMOJ-01 and PMOJ-05 can be used in detergent and leather industries.

Characterization of the Biosynthetic Gene Cluster of Enterocin F4-9, a Glycosylated Bacteriocin

Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins.

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.

Croton cajucara Essential Oil Nanoemulsion and Its Antifungal Activities

The purpose of this study was to develop a stable nanoemulsion (NE) containing Croton cajucara 7-hydroxycalamenene-rich essential oil (NECC) with antifungal activity. The NECCs were prepared using an ultrasonic processor with Pluronic® F-127 as the aqueous phase. In order to evaluate the NECCs, the droplet size, polydispersity index (PdI), percentage of emulsification, and pH were determined along with a stability study. The NECC selected for the study had 15% surfactant, showed 100% emulsification, Pdl of 0.249, neutral pH, droplet diameters of about 40 nm, and remained stable over 150 days at room temperature. In addition, the NECC activity against some species of Zygomycetes and Candida, as well as the potential to inhibit fungal extracellular proteases, were assessed, and, finally, the hemolytic activity was evaluated. The best NECC antifungal activities were against Mucorramosissimus (Minimal inhibitory concentration (MIC) = 12.2 μg/mL) and Candida albicans (MIC = 25.6 μg/mL). The highest extracellular protease activities of M. ramosissimus and C. albicans were detected at pH 3 and 4, respectively, which were totally inhibited after NECC treatment. The NECC showed no hemolytic effect at the highest concentration tested (2 mg/mL).

Pro-IL-18 secreted by keratinocytes detects the group A streptococcal protease SpeB

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases and is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ that restrict GAS growth. Other major bacterial pathogens and microbiota of this skin did not have significant IL-18-maturing ability. Taken together, these results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

Extracellular Metalloproteinases in the Plasticity of Excitatory and Inhibitory Synapses

Long-term synaptic plasticity is shaped by the controlled reorganization of the synaptic proteome. A key component of this process is local proteolysis performed by the family of extracellular matrix metalloproteinases (MMPs). In recent years, considerable progress was achieved in identifying extracellular proteases involved in neuroplasticity phenomena and their protein substrates. Perisynaptic metalloproteinases regulate plastic changes at synapses through the processing of extracellular and membrane proteins. MMP9 was found to play a crucial role in excitatory synapses by controlling the NMDA-dependent LTP component. In addition, MMP3 regulates the L-type calcium channel-dependent form of LTP as well as the plasticity of neuronal excitability. Both MMP9 and MMP3 were implicated in memory and learning. Moreover, altered expression or mutations of different MMPs are associated with learning deficits and psychiatric disorders, including schizophrenia, addiction, or stress response. Contrary to excitatory drive, the investigation into the role of extracellular proteolysis in inhibitory synapses is only just beginning. Herein, we review the principal mechanisms of MMP involvement in the plasticity of excitatory transmission and the recently discovered role of proteolysis in inhibitory synapses. We discuss how different matrix metalloproteinases shape dynamics and turnover of synaptic adhesome and signal transduction pathways in neurons. Finally, we discuss future challenges in exploring synapse- and plasticity-specific functions of different metalloproteinases.