Novel antimicrobial peptides identified in legume plant, Medicago truncatula.

One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

Identification of Gut Microbiota Profile Associated with Colorectal Cancer in Saudi Population.

Colorectal cancer (CRC) is a significant global health concern. Microbial dysbiosis and associated metabolites have been associated with CRC occurrence and progression. This study aims to analyze the gut microbiota composition and the enriched metabolic pathways in patients with late-stage CRC. In this study, a cohort of 25 CRC patients diagnosed at late stage III and IV and 25 healthy participants were enrolled. The fecal bacterial composition was investigated using V3-V4 ribosomal RNA gene sequencing, followed by clustering and linear discriminant analysis (LDA) effect size (LEfSe) analyses. A cluster of ortholog genes' (COG) functional annotations and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to identify enrichment pathways between the two groups. The findings showed that the fecal microbiota between the two groups varied significantly in alpha and beta diversities. CRC patients' fecal samples had significantly enriched populations of Streptococcus salivarius, S. parasanguins, S. anginosus, Lactobacillus mucosae, L. gasseri, Peptostreptococcus, Eubacterium, Aerococcus, Family XIII_AD3001 Group, Erysipelatoclostridium, Escherichia-Shigella, Klebsiella, Enterobacter, Alistipes, Ralstonia, and Pseudomonas (Q < 0.05). The enriched pathways identified in the CRC group were amino acid transport, signaling and metabolism, membrane biogenesis, DNA replication and mismatch repair system, and protease activity (Q < 0.05). These results suggested that the imbalance between intestinal bacteria and the elevated level of the predicated functions and pathways may contribute to the development of advanced CRC tumors. Further research is warranted to elucidate the exact role of the gut microbiome in CRC and its potential implications for use in diagnostic, prevention, and treatment strategies.

Mining the nanotube-forming Bacillus amyloliquefaciens MR14M3 genome for determining anti-Candida auris and anti-Candida albicans potential by pathogenicity and comparative genomics analysis.

There is a global health concern associated with the emergence of the multidrug-resistant (MDR) fungus Candida auris, which has significant mortality rates. Finding innovative and distinctive anti-Candida compounds is essential for treating infections caused by MDR C. auris. A bacterial strain with anti-Candida activity was isolated and identified using 16 S rRNA gene sequencing. The whole genome was sequenced to identify biosynthesis-related gene clusters. The pathogenicity and cytotoxicity of the isolate were analyzed in Candida and HFF-1 cell lines, respectively. This study set out to show that whole-genome sequencing, cytotoxicity testing, and pathogenicity analysis combined with genome mining and comparative genomics can successfully identify biosynthesis-related gene clusters in native bacterial isolates that encode antifungal natural compounds active against Candida albicans and C. auris. The native isolate MR14M3 has the ability to inhibit C. auris (zone of inhibition 25 mm) and C. albicans (zone of inhibition 25 mm). The 16 S rRNA gene sequence of MR14M3 aligned with Bacillus amyloliquefaciens with similarity (100%). Bacillus amyloliquefaciens MR14M3 establishes bridges of intercellular nanotubes (L 258.56 ± 35.83 nm; W 25.32 ± 6.09 nm) connecting neighboring cells. Candida cell size was reduced significantly, and crushed phenotypes were observed upon treatment with the defused metabolites of B. amyloliquefaciens MR14M3. Furthermore, the pathogenicity of B. amyloliquefaciens MR14M3 on Candida cells was observed through cell membrane disruption and lysed yeast cells. The whole-genome alignment of the MR14M3 genome (3981,643 bp) using 100 genes confirmed its affiliation with Bacillus amyloliquefaciens. Genome mining analysis revealed that MR14M3-coded secondary metabolites are involved in the biosynthesis of polyketides (PKs) and nonribosomal peptide synthases (NRPSs), including 11 biosynthesis-related gene clusters with one hundred percent similarity. Highly conserved biosynthesis-related gene clusters with anti-C. albicans and anti-C. auris potentials and cytotoxic-free activity of B. amyloliquefaciens MR14M3 proposes the utilization of Bacillus amyloliquefaciens MR14M3 as a biofactory for an anti-Candida auris and anti-C. albicans compound synthesizer.