Table_1_Molecular Cloning and Docking of speB Gene Encoding Cysteine Protease With Antibiotic Interaction in Streptococcus pyogenes NBMKU12 From the Clinical Isolates.DOCX

<p>Streptococcus pyogenes causes a variety of diseases ranging from mild diseases to severe invasive infections which result in significant morbidity and mortality. This study focuses on the antibiotic resistance of S. pyogenes and their interaction with cysteine protease. Around 36 beta-hemolytic isolates were collected from the clinical lab, of which seven isolates (19.4%) were identified as Streptococcus pyogenes. One of the seven isolates was collected from a urinary tract infection, which was identified by antibody agglutination and MALTI-TOF-MS, and it is designated as S. pyogenes NBMKU12. Around 8.3 to 66.6 % of the isolates were found to be resistant to one or more antimicrobial agents, especially, penicillin-G resistance was exhibited by 29.1% of the isolates. In the NBMKU12 isolate, the beta lactem (TEM) gene was detected among the 13 antibiotic genes for which it was tested. Furthermore, when analysis for presence of 13 virulence genes were carried out in NBMKU12 isolate, only speJ and speB were detected. The speB (streptococcal pyrogenic exotoxin B) encoding cysteine protease gene was cloned. This was followed by performing DNA sequencing to understand the putative cysteine protease interaction with antibiotics, inhibitors, and substrate. The speB gene consists of 1197 nucleotides and encodes a protein with multiple domains, including a signal peptide (aa 1–22), an inhibitor region (aa 27–156), and a catalytic cysteine domain (aa 160–367). The signal peptide cleavage site is predicted between Ala22 and Asn23. The putative 398 amino acid residues were found to have a theoretical pI of 8.76 and a molecular mass of 43,204.36 Da. The tested culture supernatants of NBMKU12 isolate exhibited the proteolytic activity against casein, papaya and pineapple used as substrates. The proteolytic activity suggests the expression of speB gene. Molecular docking analysis of cysteine protease showed that erythromycin (bond length 2.41 Å), followed by chloramphenicol (2.51 Å), exhibited a strong interaction; while penicillin-G (3.24 Å) exhibited a weak interaction, and this factor could be considered as a cause for penicillin-G resistance. The present study contributes to a better understanding of speB gene encoding cysteine protease, antibiotic resistance, and their interaction in the isolate, S. pyogenes NBMKU12. The antibiotics and cysteine protease interaction study confirms the resistance or sensitivity of S. pyogenes. Hence, it could be hypothesized that the isolate NBMKU12 is resistant to most of the tested antibiotics, and this resistance might be a cause for mutation.</p

A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

<div><p><i>Steinernema carpocapsae</i> is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (<i>sc-srp-6</i>) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in <i>Escherichia coli</i> had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with <i>Ki</i> of 0.42×10⁻⁷M and 1.22×10⁻⁷ M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction.</p></div

Sc-SRP-6 impairs the formation of hardened clots.

<p><b>A1:</b> In the drop clot assay, melanin aggregates were easily recognised in controls by optical microscopy, but they were not visible in plasma treated with Sc-SRP-6 a few minutes after the plasma was stimulated. <b>A2:</b> Approximately 30 min later, material adherent to lamina treated with Sc-SRP-6 remains clear, indicating that melanin was not incorporated into the clot. <b>A3:</b> The clot supernatant formed in plasma treated with Sc-SRP-6 remains dark whereas the control supernatant is clear. <b>B:</b> Scanning electron micrographs show that beads in treated and untreated plasma were attached to clot filaments forming aggregates, but full encapsulation was inhibited in the presence of Sc-SRP-6.</p

Sc-SRP-6 shares RCL signatures and has phylogenetic relationships with parasitic nematode serpins.

<p><b>A:</b> Multiple alignment of the C-terminus of serpins from different species highlight a conserved GVTA motif (346–349), the serpin signature INADRP (373–378) and a predicted P1–P1′ cleavage site (Met360–Sep361). High variability is observed in the region immediately proximal to P1 position of the RSL. <b>B:</b> Phylogenetic tree reconstructed by Maximum Likelihood using PhyML with robustness assessed by the bootstrap method (1000 pseudoreplicates). Human serpin was used as an outgroup to root the phylogeny. Alignments were performed with the amino-acid sequences of the following serpins: <i>Caenorhabditis briggsae</i> Cbr-SRP-3 (XP_002647307); <i>C. elegans</i> Cel-SRP-3 (NP_503528); <i>Brugia malayi</i> Bml-SRP-1 (AAB65745), BML-SRP-2 (XP_001893428) and BML-SRP-3 (XP_001896647); <i>Trichinella sp</i>. Tr-SRP (ABI32311); <i>Ascaris suum</i> Asn-SRP-1 (ADY44079); and <i>Anisakis simplex</i> Asm-SRP-1 (CBX25525) and Human Hsa-SRP-1 (ABV21360).</p

Sc-SRP-6 formed SDS-stable complexes with <i>G. mellonella</i> plasma proteins.

<p><b>A and B:</b> Complex formation between Sc-SRP-6 and plasma proteins at serpin/plasma proteins ratios of 4 (well 1), 15 (well 2) and 14 (well 3). <b>C:</b> Plasma proteins incubated with Sc-SRP-6 in the presence of 1.0 mM PMSF. After incubation for 30 min at 37°C, reactions were heated to 90°C under non-reducing conditions for 10 min and resolved in a 10% SDS-PAGE gel. The mobility of the serpin-protease complex (asterisk), serpin (black arrow) and hydrolysed serpin (white arrows) are indicated on the side of each gel. <b>D:</b> Immune-detected bands were analysed by MALDI-TOF/TOF. (a): For serpin complexes, masses originating from Sc-SRP-6 were excluded from the peptide mass list used for database searching to ensure the highest confidence scores for identified plasma proteins. (b): The protein score probability limit (where P<0.05) was 86.</p

Sc-SRP-6 is upregulated in invasive parasites.

<p><b>A</b>: The <i>S. carpocapsae</i> parasitic process. Infective juveniles (IJs) exposed to <i>G. mellonella</i> larvae recover and enter the insect mid-gut (L3-gut) to invade the intestinal barrier to become established in the hemocoel (L3-hem) where they molt (L4) and resume the life cycle in the insect carcass (adults, L1 and L2). <b>B:</b> Sc-SRP-6 relative expression was determined by real-time PCR using 18S as a control. Nematodes in each phase were collected from parasitised insects: L3-gut – third juveniles inside gut lumen; L3-hem – third juveniles in the hemocoel; L4– forth juveniles; and Adults – male and female; L1/L2– a pool of first and second juveniles. The bars represent standard deviations from three independent replicates. The different letters indicate significant differences (p<0.05).</p

Sc-SRP-6 inhibits insect midgut enzymes.

<p><b>A:</b> Inhibitory activity of Sc-SRP-6 against trypsin and chymotrypsin in insect digestive juices. The juices were partially fractionated by gel filtration, and the inhibitory activity tested using BApNA and AAPPpNA substrates for trypsin and chymotrypsin. <b>B: </b><i>P. unipuncta</i> larvae fed a diet incorporated with albumin-bromphenol blue show faeces with no traces of colour (control), and larvae fed on the same diet supplemented with 0.2% Sc-SRP-6 (w/v) retained the coloured compound. <b>C:</b> Incremental weight of larvae treated with 0.2% Sc-SRP-6 (w/v) compared with untreated controls. The bars represent standard deviations from three independent replicates. The different letters indicate significant differences (p<0.05).</p

Sc-SRP-6 inhibits chymotrypsin-like enzymes by a classical mechanism involving RCL.

<p><b>A–B:</b> Lineweaver-Burk double reciprocal plots showing that Sc-SRP-6 is a competitive inhibitor of trypsin and α-chymotrypsin. C – D: Formation of SDS-stable complexes of Sc-SRP-6/trypsin and Sc-SRP-6/α-chymotrypsin. An increased SI for Sc-SRP-6 was used (5, 10 and 15 in wells 1, 2 and 3). A serpin/protease complex (asterisk), a serpin (black arrow), a hydrolysed serpin (white arrows) and a degraded serpin (grey arrow) are indicated at specific sites in each gel.</p

Sc-SRP-6 affects insect responses to injury.

<p><b>A:</b> A melanised plug was identified in the wound site of untreated larvae shortly after injury (control), but no plug was formed in larvae treated with Sc-SRP-6. The larvae were punctured in the ventral midline using a 0.5 mm steel syringe in 3–4 intersegments. Control larvae were treated with 15 µl of buffer. <b>B:</b> Concavities in the treated larvae (such as the wound site) accumulates darkened hemolymph (arrowheads), providing evidence of melanisation.</p