Fig 1 - uploaded by Ashok k Sharma
Content may be subject to copyright.
Source publication
Background
The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for potential alternatives to antibiotics. In this scenario, peptidoglycan hydrolases can be used as alternate antibacterial agents due to their unique property of cleaving peptidogl...
Citations
... Peptidoglycan hydrolases (PGHs) represent a highly promising group of molecules with potent bactericidal effects. These enzymes cleave bonds within bacterial cell walls leading to instant death of bacteria (4). Peptidoglycan (PG) serves as the primary polymer in bacterial cell walls, comprising sugar moieties and amino acids that form a mesh-like structure encasing the cells. ...
Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.
IMPORTANCE
These studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins’ engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.
... Thus, it is important to explore the microbiome composition and genetic makeup of coastal marine ecological units. This diversity also holds substantial potential for biomedical applications 24,25 . ...
Ocean microbiome is crucial for global biogeochemical cycles and primary productivity. Despite numerous studies investigating the global ocean microbiomes, the microbiome composition of the Andaman region of the Indian Ocean remains largely unexplored. While this region harbors pristine biological diversity, the escalating anthropogenic activities along coastal habitats exert an influence on the microbial ecology and impact the aquatic ecosystems. We investigated the microbiome composition in the coastal waters of the Andaman Islands by 16S rRNA gene amplicon and metagenomic shotgun sequencing approaches and compared it with the Tara Oceans Consortium. In the coastal waters of the Andaman Islands, a significantly higher abundance and diversity of Synechococcus species was observed with a higher abundance of photosynthesis pigment-related genes to adapt to variable light conditions and nutrition. In contrast, Prochlorococcus species showed higher abundance in open ocean water samples of the Indian Ocean region, with a relatively limited functional diversity. A higher abundance of antibiotic-resistance genes was also noted in the coastal waters region. We also updated the ocean microbiome gene catalog with 93,172 unique genes from the Andaman coastal water microbiome. This study provides valuable insights into the Indian Ocean microbiome and supplements the global marine microbial ecosystem studies.
... The PG is formed by chains of alternate N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, bound by B-1,4 links and cross-linked by peptides formed out of L-and D-amino acids. Specifically, the bacteriolytic activity from N-acetylmuramoyl-L-alanine amidase originated due to the specific site of hydrolysis in the PG, which cleaves the amide link between MurNAc and the L-alanine of the peptide [47]. Furthermore, the efficiency of the N-acetylmuramoyl-L-alanine amidase activity identified can be attributed to the number of binding LysM modules, which recognize the GlcNAc scraps of the PG and contribute to their additive junction [48]. ...
Considering the objectives of “One Health” and the Sustainable development Goals “Good health and well-being” for the development of effective strategies to apply against bacterial resistance, food safety dangers, and zoonosis risks, this project explored the isolation and identification of Lactobacillus strains from the intestinal tract of recently weaned mice; as well as the assessment of antibacterial activity against clinical and zoonotic pathogens. For molecular identification, 16S rRNA gene-specific primers were used and, via BLAST-NCBI, 16 Ligilactobacillus murinus, one Ligilactobacillus animalis, and one Streptococcus salivarius strains were identified and registered in GenBank after the confirmation of their identity percentage and the phylogenetic analysis of the 16 Ligilactobacillus murinus strains and their association with Ligilactobacillus animalis. The 18 isolated strains showed antibacterial activity during agar diffusion tests against Listeria monocytogenes ATCC 15313, enteropathogenic Escherichia coli O103, and Campylobacter jejuni ATCC 49943. Electrophoretic and zymographic techniques confirmed the presence of bacteriolytic bands with a relative molecular mass of 107 kDa and another of 24 kDa in Ligilactobacillus murinus strains. UPLC-MS analysis allowed the identification of a 107 kDa lytic protein as an N-acetylmuramoyl-L-amidase involved in cytolysis and considered a bacteriolytic enzyme with antimicrobial activity. The 24 kDa band displayed similarity with a portion of protein with aminopeptidase function. It is expected that these findings will impact the search for new strains and their metabolites with antibacterial activity as an alternative strategy to inhibit pathogens associated with major health risks that help your solution.
... Considering the lack of knowledge regarding the repertoire of enzymes digesting PG in B. abortus, we first performed a bioinformatic analysis to identify and classify putative PG hydrolyzing enzymes encoded by the B. abortus genome. Predicted PG hydrolases were identified based on homology with A. tumefaciens and Caulobacter crescentus PG hydrolases and thanks to the "HyPe" tool, a software designed to identify PG hydrolases (Sharma et al., 2016). We compared the list of genes obtained with two sets of data acquired previously, the first being predicted essential identified by Tn-seq data analysis (Sternon et al., 2018) and the second being direct target genes of the cell division regulator CtrA identified by ChIP-seq analysis (Francis et al., 2017) (Table S1). ...
The pathogenic bacterium Brucella abortus invades and multiplies inside host cells. To grow inside host cells, B. abortus requires a functional histidine biosynthesis pathway. Here, we show that a B. abortus histidine auxotroph mutant also displays an unexpected chaining phenotype. The intensity of this phenotype varies according to the culture medium and is exacerbated inside host cells. Chains of bacteria consist of contiguous peptidoglycan, and likely result from the defective cleavage of peptidoglycan at septa. Genetic suppression of the chaining phenotype unearthed two essential genes with a role in B. abortus cell division, dipM and cdlP. Loss of function of dipM and cdlP generates swelling at the division site. While DipM is strictly localized at the division site, CdlP is localized at the growth pole and the division site. Altogether, the unexpected chaining phenotype of a hisB mutant allowed the discovery of new crucial actors in cell division in B. abortus. A Brucella abortus histidine auxotroph mutant forms chains inside host cells. Two previously uncharacterized cell division proteins, DipM and CdlP, are identified by suppression analysis.
... Therefore, lipoteichoic acid plays an important role in resisting bacterial invasion. In addition, the cell wall of S. aureus also contains about 6.7% of Staphylococcus protein A. Relevant studies have shown that peptidoglycan and lipoteichoic acid on the cell wall of gram-positive bacteria are the main attack targets of some antibacterial proteins [33][34][35][36]. Therefore, peptidoglycan, lipoteichoic acid, and staphylococcus protein A are the main components on the cell wall of S. aureus as competitive inhibitors of CB6-C targets, which analyzed the potential active site of antibacterial protein CB6-C on the cell wall of MRSA. ...
Given a serious threat of multidrug-resistant bacterial pathogens to global healthcare, there is an urgent need to find effective antibacterial compounds to treat drug-resistant bacterial infections. In our previous studies, Bacillus velezensis CB6 with broad-spectrum antibacterial activity was obtained from the soil of Changbaishan, China. In this study, with methicillin-resistant Staphylococcus aureus as an indicator bacterium, an antibacterial protein was purified by ammonium sulfate precipitation, Sephadex G-75 column, QAE-Sephadex A 25 column and RP-HPLC, which demonstrated a molecular weight of 31.405 kDa by SDS-PAGE. LC–MS/MS analysis indicated that the compound was an antibacterial protein CB6-C, which had 88.5% identity with chitosanase (Csn) produced by Bacillus subtilis 168. An antibacterial protein CB6-C showed an effective antimicrobial activity against gram-positive bacteria (in particular, the MIC for MRSA was 16 μg/mL), low toxicity, thermostability, stability in different organic reagents and pH values, and an additive effect with conventionally used antibiotics. Mechanistic studies showed that an antibacterial protein CB6-C exerted anti-MRSA activity through destruction of lipoteichoic acid (LTA) on the cell wall. In addition, an antibacterial protein CB6-C was efficient in preventing MRSA infections in in vivo models. In conclusion, this protein CB6-C is a newly discovered antibacterial protein and has the potential to become an effective antibacterial agent due to its high therapeutic index, safety, nontoxicity and great stability.
... The muropeptides released from the sacculus are recycled by the cell. The sacculus degradation and remodelling activities are catalysed by enzymes including glycosidases, amidases, endopeptidases and carboxypeptidases [4,5]. ...
Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.
... To generate relevant features for machine learning and deep learning models, three types of feature extraction techniques such as Amino-acid composition (AAC), Dipeptide composition (DPC) and PSSM-based features (PSSM) are used to develop prediction models for sirtuin family members. AAC (Sharma et al., 2016;Usmani et al., 2018;Manavalan et al., 2018) is a simple feature extraction technique to transform the original data into the amino acid information which has a vector of 20 dimensions. The AAC feature for each protein is calculated by the following equation: ...
Sirtuins are a family of proteins that play a key role in regulating a wide range of cellular processes including DNA regulation, metabolism, aging/longevity, cell survival, apoptosis, and stress resistance. Sirtuins are protein deacetylases and include in the class III family of histone deacetylase enzymes (HDACs). The class III HDACs contains seven members of the sirtuin family from SIRT1 to SIRT7. The seven members of the sirtuin family have various substrates and are present in nearly all subcellular localizations including the nucleus, cytoplasm, and mitochondria. In this study, a deep neural network approach using one-dimensional Convolutional Neural Networks (CNN) was proposed to build a prediction model that can accurately identify the outcome of the sirtuin protein by targeting their subcellular localizations. Therefore, the function and localization of sirtuin targets were analyzed and annotated to compartmentalize into distinct subcellular localizations. We further reduced the sequence similarity between protein sequences and three feature extraction methods were applied in datasets. Finally, the proposed method has been tested and compared with various machine-learning algorithms. The proposed method is validated on two independent datasets and showed an average of up to 85.77 % sensitivity, 97.32 % specificity, and 0.82 MCC for seven members of the sirtuin family of proteins.
... Here we present the computational analysis of peptidoglycan hydrolases from 41 completely sequenced genomes of Serratia marcescens and other Serratia species and analysis of their domain to find any conserved motif which could be used therapeutically. The peptidoglycan hydrolases can be developed into a new class of antibacterial agents to counteract the problem of multidrug resistant infections when used in conjunction with antibiotics (Sharma et al., 2016). ...
... Profile C belongs to endopeptidase which cleaves various peptide bonds from terminal amino acids in the peptidoglycan structure (Sharma et al., 2016). Among profile C, sequences are segregated on the basis of conserved domain and superfamily present. ...
... Due to their specificity, they are considered to be safe for natural microbiome. Also they possess additional advantages, i.e., low toxicity, immunogenicity and proven synergistic effect with antibiotics (Sharma et al., 2016). With the aid of computational tools, scientist can explore new horizons in different spheres of science. ...
... Digestive proteins for DNA/RNA were searched for using GO terms (GO:0004518). Digestive proteins for peptidoglycan were searched for using the HyPe web server 63 . ...
Phagocytosis is a key eukaryotic feature, conserved from unicellular protists to animals, that enabled eukaryotes to feed on other organisms. It could also be a driving force behind endosymbiosis, a process by which α-proteobacteria and cyanobacteria evolved into mito-chondria and plastids, respectively. Here we describe a planctomycete bacterium, 'Candidatus Uab amorphum', which is able to engulf other bacteria and small eukaryotic cells through a phagocytosis-like mechanism. Observations via light and electron microscopy suggest that this bacterium digests prey cells in specific compartments. With the possible exception of a gene encoding an actin-like protein, analysis of the 'Ca. Uab amorphum' genomic sequence does not reveal any genes homologous to eukaryotic phagocytosis genes, suggesting that cell engulfment in this microorganism is probably not homologous to eukaryotic phagocytosis. The discovery of this "phagotrophic" bacterium expands our understanding of the cellular complexity of prokaryotes, and may be relevant to the origin of eukaryotic cells.
... A continued and uncontrolled LT activity is detrimental to the cell as it could result in autolysis [75,76]. Others have pursued the use of peptidoglycan hydrolases as potential antibacterial strategy [75,77]. For example, the bacteriophage phi MR11-derived endolysin, MV-L was shown to completely lyse drug-resistant S. aureus strains such as MRSA and vancomycin-resistant S. aureus [78]. ...
Halogenated 4-hydroxybenzylidene indolinones have been shown to re-sensitize methicillin-resistant Staphylococcus aureus (MRSA)and vancomycin-resistant Enterococcus faecalis (VRE)to methicillin and vancomycin respectively. The mechanism of antibiotic re-sensitization was however not previously studied. Here, we probe the scope of antibiotic re-sensitization and present the global proteomics analysis of S. aureus treated with GW5074, a 4-hydroxybenzylidene indolinone compound. With a minimum inhibitory concentration (MIC)of 8 μg/mL against S. aureus, GW5074 synergized with beta-lactam antibiotics like ampicillin, carbenicillin and cloxacillin, the DNA synthesis inhibitor, ciprofloxacin, the protein synthesis inhibitor, gentamicin and the folate acid synthesis inhibitor, trimethoprim. Global proteomics analysis revealed that GW5074 treatment resulted in significant downregulation of enzymes involved in the purine biosynthesis. S. aureus proteins involved in amino acid metabolism and peptide transport were also observed to be downregulated. Interestingly, anti-virulence targets such as AgrC (a quorum sensing-related histidine kinase), AgrA (a quorum sensing-related response regulator)as well as downstream targets, such as hemolysins, lipases and proteases in S. aureus were also downregulated by GW5074. We observed that the peptidoglycan hydrolase, SceD was significantly upregulated. The activity of GW5074 on S. aureus suggests that the compound primes bacteria for the antibacterial action of ineffective antibiotics. Significance: Antibiotic resistance continues to present significant challenges to the treatment of bacterial infections. Given that antibiotic resistance is a natural phenomenon and that it has become increasingly difficult to discover novel antibiotics, efforts to improve the activity of existing agents are worth pursuing. A few small molecules that re-sensitize resistant bacteria to traditional antibiotics have been described but the molecular details that underpin how these compounds work to re-sensitize bacteria remain largely unknown. In this report, global label-free quantitative proteomics was used to identify changes in the proteome that occurs when GW5074, a compound that re-sensitize MRSA to methicillin, is administered to S. aureus. The identification of pathways that are impacted by GW5074 could help identify novel targets for antibiotic re-sensitization.
