Subtilisin-like Protease Research Articles

Molecular docking analysis of subtilisin-like alkaline serine protease (SLASP) and laccase with natural biopolymers

Abstract Alkaline serine proteases (ASPs) and laccases (Lacs) have been reported to possess several industrial applications, particularly in the food, cosmetic, and leather industries. Thus, in the present study, eighteen natural biopolymers, including agar, agarose, alginate, kappa carrageenan, cellulose, chitosan pentamer, chitosan oligosaccharide, chondroitin sulfate, dextran, fucoidan, heparin, hyaluronan, lignin, mannan, pectin, phytic acid, pullulan and starch soluble, were studied for their docking behavior, such as subtilisin-like alkaline serine protease (SLASP) KP-43 and Bacillus subtilis laccase (Bs Lac), using the SwissDock method. Additionally, the toxicity toward honey bees and human liver was determined by utilizing the free Bee-Tox and pkCSM web servers, respectively. Bee-Tox analysis demonstrated that four ligands (namely, agar, cellulose, lignin, and pullulan) exhibited acute oral toxicity toward honey bees (Apis mellifera). The docking study revealed that chitosan pentamer and lignin exhibited maximum binding energies of −9.67 and −11.37 kcal/mol against the target proteins SLASP (KP-43) and Lac (Bs Lac), respectively. Interestingly, in the present study, agarose was shown to interact with the His68 and Ser255 amino acid residues of SLASP (KP-43) from KSM-KP43 Bacillus sp. Thus, the current investigation showed the potential of eighteen natural biopolymers as immobilizing agents to prepare ASP and Lac nanoparticles for biomedical applications.

Genomic insights and Taguchi-based optimization of culture conditions for enhanced alkaline protease production in Streptomyces barkulensis RC1831

This study presents a genome analysis identifying various alkaline protease genes and culture condition optimization to enhance the production of the enzyme in Streptomyces barkulensis RC1831. The analysis of genes using bioinformatics tools such as RAST, KEGG, KAAS, and BLASTx revealed the presence of 10 genes that are responsible for alkaline protease production belonging to different families. Multiple sequence alignment showed a high sequence identity with M24 family serine protease followed by subtilisin-like serine protease. Optimal conditions for enhanced protease production were determined to be at 37°C, pH 11, casein1 % (W/V), dextrose 0.5 % (W/V), urea 0.5 % (W/V), tryptophan 1 % (W/V), 1 mM Mn+2, 1 % (V/V) Tween-80 in LB medium and an incubation time of 72 hours. Out of the 10 alkaline protease genes, 2 genes expressed significantly according to the activity observed during optimization processes i.e., M24 family, Mn+2 dependent metalloprotease and subtilisin-like serine, ca+2 dependent metalloprotease. Furthermore, the AP enzyme was remarkably stable in the presence of various cofactors (Metal ions) and surfactants, indicating its potential for industrial applications under diverse conditions.

In silico and experimental characterization of a new polyextremophilic subtilisin-like protease from Microbacterium metallidurans and its application as a laundry detergent additive.

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60°C and 1M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75min), temperature (30-60°C), MmSP enzyme concentration (5-10U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5U/mL of MmSP during 30min at 32°C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.

Peptidic Boronic Acid Plasmodium falciparum SUB1 Inhibitors with Improved Selectivity over Human Proteasome.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Integrated analysis of transcriptomics and defense-related phytohormones to discover hub genes conferring maize Gibberella ear rot caused by Fusarium Graminearum

BackgroundGibberella ear rot (GER) is one of the most devastating diseases in maize growing areas, which directly reduces grain yield and quality. However, the underlying defense response of maize to pathogens infection is largely unknown.ResultsTo gain a comprehensive understanding of the defense response in GER resistance, two contrasting inbred lines ‘Nov-82’ and ‘H10’ were used to explore transcriptomic profiles and defense-related phytohormonal alterations during Fusarium graminearum infection. Transcriptomic analysis revealed 4,417 and 4,313 differentially expressed genes (DEGs) from the Nov-82 and H10, respectively, and 647 common DEGs between the two lines. More DEGs were obviously enriched in phenylpropanoid biosynthesis, secondary metabolites biosynthesis, metabolic process and defense-related pathways. In addition, the concentration of the defense-related phytohormones, jasmonates (JAs) and salicylates (SAs), was greatly induced after the pathogen infection. The level of JAs in H10 was more higher than in Nov-82, whereas an opposite pattern for the SA between the both lines. Integrated analysis of the DEGs and the phytohormones revealed five vital modules based on co-expression network analysis according to their correlation. A total of 12 hub genes encoding fatty acid desaturase, subtilisin-like protease, ethylene-responsive transcription factor, 1-aminocyclopropane-1-carboxylate oxidase, and sugar transport protein were captured from the key modules, indicating that these genes might play unique roles in response to pathogen infection,ConclusionsOverall, our results indicate that large number DEGs related to plant disease resistance and different alteration of defensive phytohormones were activated during F. graminearum infection, providing new insight into the defense response against pathogen invasion, in addition to the identified hub genes that can be further investigated for enhancing maize GER resistance.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain

BackgroundThe malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. MethodsHere we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. ResultsWe show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. ConclusionsOur results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. General significanceOur findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

Thermostable Bacterial Collagenolytic Proteases: A Review.

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the β-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.

Cotton leaf curl Multan virus subverts the processing of hydroxyproline-rich systemin to suppress tobacco defenses against insect vectors.

Insect vector-virus-plant interactions have important ecological and evolutionary implications. The constant struggle of plants against viruses and insect vectors has driven the evolution of multiple defense strategies in the host as well as counter-defense strategies in the viruses and insect vectors. Cotton leaf curl Multan virus (CLCuMuV) is a major causal agent of cotton leaf curl disease in Asia and is exclusively transmitted by the whitefly Bemisia tabaci. Here, we report that plants infected with CLCuMuV and its betasatellite CLCuMuB enhance the performance of the B. tabaci vector, and βC1 encoded by CLCuMuB plays an important role in begomovirus-whitefly-tobacco tripartite interactions. We showed that CLCuMuB βC1 suppresses the jasmonic acid signaling pathway by interacting with the subtilisin-like protease 1.7 (NtSBT1.7) protein, thereby enhancing whitefly performance on tobacco plants. Further studies revealed that in wild-type plants, NtSBT1.7 could process tobacco preprohydroxyproline-rich systemin B (NtpreproHypSysB). After CLCuMuB infection, CLCuMuB βC1 could interfere with the processing of NtpreproHypSysB by NtSBT1.7, thereby impairing plant defenses against whitefly. These results contribute to our understanding of tripartite interactions among virus, plant, and whitefly, thus offering ecological insights into the spread of vector insect populations and the prevalence of viral diseases.

Nattokinase, a Subtilisin-like Alkaline-Serine Protease, Reduces Mutacin Activity by Inactivating the Competence-Stimulating Peptide in Streptococcus mutans.

Streptococcus mutans is a major cariogenic organism because of its ability to form biofilms on tooth surfaces. Bacteriocins produced by S. mutans (known as mutacins) are indirect pathogenic factors that play a role in the persistence of this microbe in the oral environment. Nattokinase, a subtilisin-like alkaline serine protease, potently inhibits biofilm formation without affecting S. mutans growth. However, effective strategies utilizing nattokinase to control mutacin production by S. mutans are lacking. In this study, we evaluated the effect of nattokinase on mutacin activity in 46 strains of S. mutans with different mutacin genotypes isolated from the dental plaques of pediatric patients with caries. Nattokinase reduced the activity of mutacin against oral streptococci at a concentration of 1 mg/mL in all clinical isolates. Furthermore, nattokinase reduced the expression of non-lantibiotic mutacin structural genes (nlmABCD) and inactivated the extracellular competence-stimulating peptide involved in comDE activation, which regulates non-lantibiotic mutacin gene expression. These results suggest that nattokinase may reduce the virulence of S. mutans and could potentially be used as a new caries-preventive agent as an alternative to conventional drug treatments.

Subtilisin-like proteases from Fusarium graminearum induce plant cell death and contribute to virulence.

Fusarium head blight (FHB), caused by Fusarium graminearum, causes huge annual economic losses in cereal production. To successfully colonize host plants, pathogens secrete hundreds of effectors that interfere with plant immunity and facilitate infection. However, the roles of most secreted effectors of F. graminearum in pathogenesis remain unclear. We analyzed the secreted proteins of F. graminearum and identified 255 candidate effector proteins by liquid chromatography-mass spectrometry (LC-MS). Five subtilisin-like family proteases (FgSLPs) were identified that can induce cell death in Nicotiana benthamiana leaves. Further experiments showed that these FgSLPs induced cell death in cotton (Gossypium barbadense) and Arabidopsis (Arabidopsis thaliana). A signal peptide and light were not essential for the cell death-inducing activity of FgSLPs. The I9 inhibitor domain and the entire C-terminus of FgSLPs were indispensable for their self-processing and cell death-inducing activity. FgSLP-induced cell death occurred independent of the plant signal transduction components BRI-ASSOCIATED KINASE 1 (BAK1), SUPPRESSOR OF BIR1 1 (SOBIR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4). Reduced virulence was observed when FgSLP1 and FgSLP2 were simultaneously knocked out. This study reveals a class of secreted toxic proteins essential for F. graminearum virulence.

Transcriptome analysis of the synergistic mechanisms between two strains of potato virus Y in Solanum tuberosum L.

Many viruses employ a process known as superinfection exclusion (SIE) to block subsequent entry or replication of the same or closely related viruses in the cells they occupy. SIE is also referred to as Cross-protection refers to the situation where a host plant infected by a mild strain of a virus or viroid gains immunity against a more severe strain closely related to the initial infectant. The mechanisms underlying cross-protection are not fully understood. In this study, we performed a comparative transcriptomic analysis of potato (Solanum tuberosum L.) leaves. The strains PVYN−Wi-HLJ-BDH-2 and PVYNTN−NW-INM-W-369-12 are henceforth designated as BDH and 369, respectively. In total, 806 differentially expressed genes (DEGs) were detected between the Control and JZ (preinfected with BDH and challenge with 369) treatment. Gene Ontology (GO) analysis showed that the response to external biological stimulation, signal transduction, kinase, immunity, redox pathways were significantly enriched. Among these pathways, we identified numerous differentially expressed metabolites related to virus infection. Moreover, our data also identified a small set of genes that likely play important roles in the establishment of cross-protection. Specifically, we observed significant differential expression of the A1-II gamma-like gene, elongation factor 1-alpha-like gene, and subtilisin-like protease StSBT1.7 gene, with StSBT1.7 being the most significant in our transcriptome data. These genes can stimulate the expression of defense plant genes, induce plant chemical defense, and participate in the induction of trauma and pathogenic bacteria. Our findings provided insights into the mechanisms underlying the ability of mild viruses to protect host plants against subsequent closely related virus infection in Solanum tuberosum L.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Cilia Provide a Platform for the Generation, Regulated Secretion, and Reception of Peptidergic Signals.

Cilia are microtubule-based cellular projections that act as motile, sensory, and secretory organelles. These structures receive information from the environment and transmit downstream signals to the cell body. Cilia also release vesicular ectosomes that bud from the ciliary membrane and carry an array of bioactive enzymes and peptide products. Peptidergic signals represent an ancient mode of intercellular communication, and in metazoans are involved in the maintenance of cellular homeostasis and various other physiological processes and responses. Numerous peptide receptors, subtilisin-like proteases, the peptide-amidating enzyme, and bioactive amidated peptide products have been localized to these organelles. In this review, we detail how cilia serve as specialized signaling organelles and act as a platform for the regulated processing and secretion of peptidergic signals. We especially focus on the processing and trafficking pathways by which a peptide precursor from the green alga Chlamydomonas reinhardtii is converted into an amidated bioactive product-a chemotactic modulator-and released from cilia in ectosomes. Biochemical dissection of this complex ciliary secretory pathway provides a paradigm for understanding cilia-based peptidergic signaling in mammals and other eukaryotes.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis.In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent.In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Transcription factor LBD16 targets cell wall modification/ion transport genes in peach lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.

Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection

ABSTRACT Streptococcus suis (S. suis), a significant zoonotic bacterial pathogen impacting swine and human, is associated with severe systemic diseases such as streptococcal toxic shock-like syndrome, meningitis, septicaemia, and abrupt fatality. The multifaceted roles of complement components C5a and C3a extend to orchestrating inflammatory cells recruitment, oxidative burst induction, and cytokines release. Despite the pivotal role of subtilisin-like serine proteases in S. suis pathogenicity, their involvement in immune evasion remains underexplored. In the present study, we identify two cell wall-anchored subtilisin-like serine proteases in S. suis, SspA-1 and SspA-2, as binding partners for C3a and C5a. Through Co-Immunoprecipitation, Enzyme-Linked Immunosorbent and Far-Western Blotting Assays, we validate their interactions with the aforementioned components. However, SspA-1 and SspA-2 have no cleavage activity against complement C3a and C5a performed by Cleavage assay. Chemotaxis assays reveal that recombinant SspA-1 and SspA-2 effectively attenuate monocyte chemotaxis towards C3a and C5a. Notably, the ΔsspA-1, ΔsspA-1, and ΔsspA-1/2 mutant strains exhibit compromised survival in blood, and resistance of opsonophagocytosis, alongside impaired survival in blood and in vivo colonization compared to the parental strain SC-19. Critical insights from the murine and Galleria mellonella larva infection models further underscore the significance of sspA-1 in altering mortality rates. Collectively, our findings indicate that SspA-1 and SspA-2 are novel binding proteins for C3a and C5a, thereby shedding light on their pivotal roles in S. suis immune evasion and the pathogenesis.

Purification of Phytaspases Using a Biotinylated Peptide Inhibitor.

Phytaspases are plant subtilisin-like proteases (subtilases) that possess a rarely occurring substrate cleavage specificity. These proteolytic enzymes hydrolyze their substrates strictly after an aspartate residue preceded by a characteristic, though degenerate, tripeptide amino acid motif. Having been initially discovered as proteases involved in the accomplishment of programmed cell death in plants, phytaspases were also demonstrated to be instrumental in specific processing of precursor proteins of several plant peptide hormones, thus generating biologically active peptides. Here we provide a protocol for isolation of active phytaspases from leaves, which was shown to be efficient for a wide range of plant species. The key element of the proposed scheme is the use of a specific and reversible biotinylated peptide aldehyde inhibitor of phytaspases for purification of the enzymes by means of affinity chromatography. We also discuss nuances, pitfalls, and possible alternatives for successful isolation of proteolytically active phytaspases.

Dermatophytes and mammalian hair: aspects of the evolution of Arthrodermataceae

AbstractDermatophytes and other members of Onygenales are unique in their ability to degrade keratin, affecting hair and nails, and in the case of human hosts, causing skin infections. Subtillisins are essential proteases in keratin assimilation, and subtilisin-like protease 1 (SUB1) and SUB3–7 are specific for dermatophytes. eIF2α kinases are serine-threonine kinases that perform essential functions in response to infection, proteotoxicity, and nutrient scavenging. The relatively conserved nature of EIF2AK4 among fungi makes them potential evolutionary markers, which may contribute to a deeper understanding of dermatophyte taxonomy and evolution. This study aimed to assess the phylogeny of dermatophytes by examining the EIF2AK4 and SUB1 genes compared to the ITS gene marker. The phylogenetic trees generated from the EIF2AK4 and SUB1 genes exhibited a similar topology, which differed from that observed in the ITS tree. Our preliminary findings with a limited dataset suggest that the EIF2AK4 and SUB1 genes provide a reasonably correct reflection of the evolution of Arthrodermataceae. In addition, the study analyzed in vitro keratinolytic responses of 19 dermatophyte species using hairs of a broad range of mammals, including ancestral as well as derived species, as substrates. Trichophyton mentagrophytes and Nannizzia gypsea were the most active in degrading hair, while Trichophyton verrucosum, Trichophyton tonsurans and Epidermophyton floccosum showed low response. Hairs of Hyracoidea and Rodentia were most affected of all mammal hairs, while in contrast, bat hairs were difficult to degrade by nearly all tested dermatophyte species. Zoophilic species showed more activity than anthropophilic dermatophytes, but hair degradation profiles were not diagnostic for particular dermatophyte species.

Diverse biological activities and secondary metabolites profile of Penicillium brevicompactum HE19ct isolated from the high-Andean medicinal plant Perezia coerulescens

Endophytic fungi produced attractive primary and secondary metabolites for industries, pharmacology, and biotechnology. The bioactive potential of HE19ct, identified as Penicillium brevicompactum according to ITS-BenA-caM, was addressed. Antimicrobial and antioxidant activities and secondary metabolite contents using four culture media in Agar-plate (ApF) and Submerged (SmF) fermentation were evaluated. Some plant growth-promoting (PGP) traits and their related genes were tested. HE19ct exhibited antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Candida albicans, C. tropicalis, Fusarium sp., Geotrichum candidum, and Alternaria sp. All cultures showed DPPH scavenging activity and phenolic compounds, where ethyl acetate extract of SmF with malt extract showed higher activity and SmF/ApF with potato-dextrose exhibited higher yield, respectively. HE19ct solubilized tricalcium-phosphate and produced siderophore, endoglucanase, proteinase, and amylase. It enhanced the alfalfa's germination at 15 °C, root development, and phenols production at 15 and 24 °C. Phenols, tannins, anthraquinones, triterpenoids/steroids, and alkaloids production were detected depending on culture media. Polyketide synthase type I gene (PksI), subtilisin-like protease prb 1 (Pbr), and siderophore D (sidD) were PCR-amplified. Finally, HE19CT could be a promising source of interesting bioactive compounds for pharmacology and agriculture mainly in extreme conditions, then metabolomic and functional genetic research must be performed to support their appropriate application.