Discovery of antimicrobial peptides clostrisin and cellulosin from Clostridium: insights into their structures, co-localized biosynthetic gene clusters, and antibiotic activity.

Antimicrobial resistance presents a substantial threat to global public health, demanding urgent attention and action. This study focuses on lanthipeptides, ribosomally encoded peptides that display significant structural diversity and hold promising potential as antibiotics. Genome mining was employed to locate biosynthetic gene clusters (BGCs) containing class II lanthipeptide synthetases encoded by lanM genes. A phylogenetic study analyzing homologous sequences of functional LanM sequences revealed a unique evolutionary clade of 17 LanM proteins associated with 12 Clostridium bacterial genomes. In silico exploration identified nine complete BGCs, including one super-cluster containing two co-localized operons from Clostridium cellulovorans 743B, that encode for two new peptides named clostrisin and cellulosin. Each operon was heterologously expressed in Escherichia coli. Molecular weights associated with the expected post-translational modifications of the purified lanthipeptide were confirmed by MS-MS/MS analysis for cellulosin, while clostrisin was not post-translationally modified. Both peptides demonstrated antimicrobial activity against multidrug-resistant bacteria, such as a clinical strain of Staphylococcus epidermidis MIQ43 and Pseudomonas aeruginosa PA14. This is the first report of lanthipeptides from the Clostridium genus produced with its native biosynthetic machinery, as well as chemically and biologically characterized. This study showcases the immense potential of genome mining in identifying new RiPP synthetases and associated bioactive peptides.

Biological Dark Matter Exploration using Data Mining for the Discovery of Antimicrobial Natural Products.

The discovery of novel antimicrobials has significantly slowed down over the last three decades. At the same time, humans rely increasingly on antimicrobials because of the progressive antimicrobial resistance in medical practices, human communities, and the environment. Data mining is currently considered a promising option in the discovery of new antibiotics. Some of the advantages of data mining are the ability to predict chemical structures from sequence data, anticipation of the presence of novel metabolites, the understanding of gene evolution, and the corroboration of data from multiple omics technologies. This review analyzes the state-of-the-art for data mining in the fields of bacteria, fungi, and plant genomic data, as well as metabologenomics. It also summarizes some of the most recent research accomplishments in the field, all pinpointing to innovation through uncovering and implementing the next generation of antimicrobials.

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling.

Lactobacillus rhamnosus is a bacterial species commonly colonizing the gastrointestinal (GI) tract of humans and also frequently used in food products. While some strains have been studied extensively, physiological variability among isolates of the species found in healthy humans or their diet is largely unexplored. The aim of this study was to characterize the diversity of carbohydrate utilization capabilities of human isolates and food-derived strains of L. rhamnosus in relation to their niche of isolation and genotype. We investigated the genotypic and phenotypic diversity of 25 out of 65 L. rhamnosus strains from various niches, mainly human feces and fermented dairy products. Genetic fingerprinting of the strains by amplified fragment length polymorphism (AFLP) identified 11 distinct subgroups at 70% similarity and suggested niche enrichment within particular genetic clades. High-resolution carbohydrate utilization profiling (OmniLog) identified 14 carbon sources that could be used by all of the strains tested for growth, while the utilization of 58 carbon sources differed significantly between strains, enabling the stratification of L. rhamnosus strains into three metabolic clusters that partially correlate with the genotypic clades but appear uncorrelated with the strain's origin of isolation. Draft genome sequences of 8 strains were generated and employed in a gene-trait matching (GTM) analysis together with the publicly available genomes of L. rhamnosus GG (ATCC 53103) and HN001 for several carbohydrates that were distinct for the different metabolic clusters: l-rhamnose, cellobiose, l-sorbose, and α-methyl-d-glucoside. From the analysis, candidate genes were identified that correlate with l-sorbose and α-methyl-d-glucoside utilization, and the proposed function of these genes could be confirmed by heterologous expression in a strain lacking the genes. This study expands our insight into the phenotypic and genotypic diversity of the species L. rhamnosus and explores the relationships between specific carbohydrate utilization capacities and genotype and/or niche adaptation of this species.

Exoprotease exploitation and social cheating in a Pseudomonas aeruginosa environmental lysogenic strain with a noncanonical quorum sensing system.

Social cheating is the exploitation of public goods that are costly metabolites, like exoproteases. Exoprotease exploitation in Pseudomonas aeruginosa has been studied in reference strains. Experimental evolution with reference strains during continuous growth in casein has demonstrated that nonexoprotease producers that are lasR mutants are selected while they behave as social cheaters. However, noncanonical quorum-sensing systems exist in P. aeruginosa strains, which are diverse. In this work, the exploitation of exoproteases in the environmental strain ID4365 was evaluated; ID4365 has a nonsense mutation that precludes expression of LasR. ID4365 produces exoproteases under the control of RhlR, and harbors an inducible prophage. As expected, rhlR mutants of ID4365 behave as social cheaters, and exoprotease-deficient individuals accumulate upon continuous growth in casein. Moreover, in all continuous cultures, population collapses occur. However, this also sometimes happens before cheaters dominate. Interestingly, during growth in casein, ID4565's native prophage is induced, suggesting that the metabolic costs imposed by social cheating may increase its induction, promoting population collapses. Accordingly, lysogenization of the PAO1 lasR mutant with this prophage accelerated its collapse. These findings highlight the influence of temperate phages in social cheating.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

Impact of ∼omics in the detection and validation of potential anti-infective drugs.

New anti-infective drugs are an unmet necessity of modern medicine. The use of ∼omics technologies has exponentially increased the knowledge on active anti-infective structures, where to search for them and their mechanisms of action. Research involving extreme and unique environments (such as endophytes) revealed their potential for many yet unknown active molecules. This work intends to review a recent research involving discovery of secondary metabolites with an established anti-infective action which was mediated by one of the ∼omics sciences: genomics, proteomics, transcriptomics, metabolomics, glycomics or their combinations, as well as the software at the base of these discoveries.

Genome mining of Streptomyces scabrisporus NF3 reveals symbiotic features including genes related to plant interactions.

Endophytic bacteria are wide-spread and associated with plant physiological benefits, yet their genomes and secondary metabolites remain largely unidentified. In this study, we explored the genome of the endophyte Streptomyces scabrisporus NF3 for discovery of potential novel molecules as well as genes and metabolites involved in host interactions. The complete genomes of seven Streptomyces and three other more distantly related bacteria were used to define the functional landscape of this unique microbe. The S. scabrisporus NF3 genome is larger than the average Streptomyces genome and not structured for an obligate endosymbiotic lifestyle; this and the fact that can grow in R2YE media implies that it could include a soil-living stage. The genome displays an enrichment of genes associated with amino acid production, protein secretion, secondary metabolite and antioxidants production and xenobiotic degradation, indicating that S. scabrisporus NF3 could contribute to the metabolic enrichment of soil microbial communities and of its hosts. Importantly, besides its metabolic advantages, the genome showed evidence for differential functional specificity and diversification of plant interaction molecules, including genes for the production of plant hormones, stress resistance molecules, chitinases, antibiotics and siderophores. Given the diversity of S. scabrisporus mechanisms for host upkeep, we propose that these strategies were necessary for its adaptation to plant hosts and to face changes in environmental conditions.

Synthetic biology era: Improving antibiotic's world.

The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge.

Draft Genome Sequence of Streptomyces scabrisporus NF3, an Endophyte Isolated from Amphipterygium adstringens.

We report the draft genome sequence of Streptomyces scabrisporus NF3, an endophyte isolated from Amphipterygium adstringens in Chiapas, Mexico. This strain produces a new modified linaridin peptide. The genome harbors at least 50 gene clusters for synthases of polyketide and nonribosomal peptides, suggesting a prospective production of various secondary metabolites.

The Variable Regions of Lactobacillus rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires.

Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes (core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer (variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total (the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides (EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat (CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core (shared) proteins matched with the clade distribution obtained from the presence-absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains' core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.

Influence of fermented milk products, prebiotics and probiotics on microbiota composition and health.

The gut microbiota is a highly diverse and relative stabile ecosystem increasingly recognized for its impact on human health. The homeostasis of microbes and the host is also referred to as eubiosis. In contrast, deviation from the normal composition, defined as dysbiosis, is often associated with localized diseases such as inflammatory bowel disease or colonic cancer, but also with systemic diseases like metabolic syndrome and allergic diseases. Modulating a gut microbiota dysbiosis with nutritional concepts may contribute to improving health status, reducing diseases or disease symptoms or supporting already established treatments. The gut microbiota can be modulated by different nutritional concepts, varying from specific food ingredients to complex diets or by the ingestion of particular live microorganisms. To underpin the importance of bacteria in the gut, we describe molecular mechanisms involved in the crosstalk between gut bacteria and the human host, and review the impact of different nutritional concepts such as pre-, pro- and synbiotics on the gastrointestinal ecosystem and their potential health benefits. The aim of this review is to provide examples of potential nutritional concepts that target the gut microbiota to support human physiology and potentially health outcomes.

Adapted response of the antioxidant defense system to oxidative stress induced by deoxynivalenol in Hek-293 cells.

The mycotoxin deoxynivalenol (DON), a contaminant of certain foods and feeds, is cytotoxic and genotoxic to mammalians cells. Exposure of human embryonic kidney (Hek-293) cells to DON led to a dose- and time-dependent decrease in cell viability, with an IC(50) about 7.6 µM. The DON effects on Hek-293 morphology, reactive oxygen species, lipid peroxidation and antioxidative system and caspase 3 and bcl-2 expression were studied. Cells became round and in some are progressive loss of cell attachment appeared. These biochemical parameters were assessed after 6, 12 and 24 h of treatment with 2.5 and 5 µM DON. An increase in superoxide dismutase activity within the interval 6-12 h and almost complete recovery by the end of experiment for both concentrations was observed, whereas the profile of catalase activity was the same with the superoxide dismutase one for 2.5 µM and decreased in a time-dependent manner for 5 µM. A temporary activation of glutathione peroxidase and glutathione reductase was recorded at 12 h post-exposure, while the glutathione-S-transferase activity was unchanged for both concentrations. The NADP(+)-dependent isocitrate dehydrogenase activity showed a transient increase at the 12 h post-exposure. The caspase 3 expression remained unchanged and the bcl-2 one decreased after 24 h of exposure for the two concentrations. Our results showed the dose- and time specific changes in the antioxidants system of Hek-293 cells, which could not counteract efficiently the effects DON exposure. The different types of cell death which could be activated by this DON induced changes are mentioned.