In silico assessment of missense point mutations on human cathelicidin LL-37.

Cathelicidin antimicrobial peptides are a diverse family of cationic amphipathic peptides with multiple activities. In humans, cathelicidin LL-37 is one of the main host defense peptides with a remarkable medical and biotechnological potential. Deregulation of LL-37 expression has been associated with inflammatory diseases. However the effects of point mutations driven by single nucleotide polymorphisms (SNPs) on LL-37 are unknown. Here we applied an array of computational tools to investigate the effects of such mutations on LL-37 structure and activity. Due to the fact that, on cathelicidins, the prodomain is more conserved than the mature peptide, the SNP effect predictions were biased and, overall, resulted in neutral effects; and due to the slight changes in physicochemical properties, the antimicrobial predictions indicated the maintenance of such activity. Nonetheless, R07P, R07W, R29Q, R29W mutations reduced the peptide net charge, which in turn could result in less active LL-37 variants. Molecular dynamics data indicated that R07Q and N30Y mutations altered the LL-37 structure, leading to potential deleterious effects. In addition, the helix dipole is altered in G03A, R07P, R07W and L31P mutations, which could also alter the antimicrobial activity. Our results indicated that despite the mutations did not alter the residues from LL-37 active core, they could influence the antimicrobial activity and consequently, could be involved in inflammatory diseases.

Use of next generation sequencing and bioinformatics for profiling freshwater eukaryotic microalgae in a novel peatland integrated multi-trophic aquaculture (IMTA) system: Case study from the Republic of Ireland.

Development of integrated multi-trophic aquaculture (IMTA) systems constitutes a step change in the sustainable production of freshwater fish to meet emerging needs for high-protein foods globally. Recently, there has been a paradigm shift away from harvesting peat as a fuel towards the development of wettable peatland innovation (termed 'paludiculture'), such as aquaculture. Such eco-innovations support carbon sequestration and align with a balanced environmental approach to protecting biodiversity. This novel peatland-based IMTA process in the Irish midlands relies upon natural microalgae for waste treatment, recirculation and water quality where there is no use of pesticides or antibiotics. This novel IMTA system is powered with a wind turbine and the process has 'organic status'; moreover, it does not discharge aquaculture effluent to receiving water. However, there is a significant lack of understanding as to diversity of microalgae in this 'paludiculture'-based IMTA processes. This constitutes the first case study to use conventional microscopy combined with next-generation sequencing and bioinformatics to profile microalgae occurring in this novel IMTA system from pooled samples over a 12 month period in 2020. Conventional microscopy combined with classic identification revealed twenty genera of algae; with Chlorophyta and Charophyta being the most common present. However, algal DNA isolation, 16 s sequencing and bioinformatics revealed a combined total of 982 species from 341 genera across nine phyla from the same IMTA system, which emphasized a significant underestimation in the number and diversity of beneficial or potentially harmful algae in the IMTA-microbiome. These new methods also yield rich data that can be used by digital technologies to transform future monitoring and performance of the IMTA system for sustainability. The findings of this study align with many sustainability development goals of the United Nations including no poverty, zero hunger, good health and well-being, responsible consumption and production, climate change, and life below water.

Genomic insights into the diversity, virulence and resistance of Klebsiella pneumoniae extensively drug resistant clinical isolates.

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. ß-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different ß-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D ß-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

Structural effects driven by rare point mutations in amylin hormone, the type II diabetes-associated peptide.

BACKGROUND: Amylin is a 37-amino-acid peptide hormone co-secreted with insulin, which participates in glucose homeostasis. This hormone is able to aggregate in a ß-sheet conformation and deposit in islet amyloids, a hallmark in type II diabetes. Since amylin is a gene-encoded hormone, this peptide has variants caused by point mutations that can impact its functions. METHODS: Here, we analyzed the structural effects caused by S20G and G33R point mutations which, according to the 1000 Genomes Project, have frequency in East Asian and European populations, respectively. The analyses were performed by means of aggrescan server, SNP functional effect predictors, and molecular dynamics. RESULTS: We found that both mutations have aggregation potential and cause changes in the monomeric forms when compared with wild-type amylin. Furthermore, comparative analyses with pramlintide, an amylin drug analogue, allowed us to infer that second α-helix maintenance may be related to the aggregation potential. CONCLUSIONS: The S20G mutation has been described as pathologically related, which is in agreement with our findings. In addition, our data suggest that the G33R mutation might have a deleterious effect. The data presented here also provide new therapy opportunities, whether for creating more effective drugs for diabetes or implementing specific treatment for patients with these mutations. GENERAL SIGNIFICANCE: Our data could help to better understand the impact of mutations on the wild-type amylin sequence, as a starting point for the evaluation and characterization of other variations. Moreover, these findings could improve the health of patients with type II diabetes.

Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains.

Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.

Genomic Comparison among Lethal Invasive Strains of Streptococcus pyogenes Serotype M1.

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS). A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades.

In silico analyses of deleterious missense SNPs of human apolipoprotein E3.

ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer's disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.

Theoretical structural characterization of lymphoguanylin: A potential candidate for the development of drugs to treat gastrointestinal disorders.

Guanylin peptides (GPs) are small cysteine-rich peptide hormones involved in salt absorption, regulation of fluids and electrolyte homeostasis. This family presents four members: guanylin (GN), uroguanylin (UGN), lymphoguanylin (LGN) and renoguanylin (RGN). GPs have been used as templates for the development of drugs for the treatment of gastrointestinal disorders. Currently, LGN is the only GP with only one disulfide bridge, making it a remarkable member of this family and a potential drug template; however, there is no structural information about this peptide. In fact, LGN is predicted to be highly disordered and flexible, making it difficult to obtain structural information using in vitro methods. Therefore, this study applied a series of 1µs molecular dynamics simulations in order to understand the structural behavior of LGN, comparing it to the C115Y variant of GN, which shows the same Cys to Tyr modification. LGN showed to be more flexible than GN C115Y. While the negatively charged N-terminal, despite its repellent behavior, seems to be involved mainly in pH-dependent activity, the hydrophobic core showed to be the determinant factor in LGN's flexibility, which could be essential in its activity. These findings may be determinant in the development of new medicines to help in the treatment of gastrointestinal disorders. Moreover, our investigation of LGN structure clarified some issues in the structure-activity relationship of this peptide, providing new knowledge of guanylin peptides and clarifying the differences between GN C115Y and LGN.

Structural impact analysis of missense SNPs present in the uroguanylin gene by long-term molecular dynamics simulations.

The guanylate cyclase activator 2B, also known as uroguanylin, is part of the guanylin peptide family, which includes peptides such as guanylin and lymphoguanylin. The guanylin peptides could be related to sodium absorption inhibition and water secretion induction and their dysfunction may be related to various pathologies such as chronic renal failure, congestive heart failure and nephrotic syndrome. Besides, uroguanylin point mutations have been associated with essential hypertension. However, currently there are no studies on the impact of missense SNPs on uroguanylin structure. This study applied in silico SNP impact prediction tools to evaluate the impact of uroguanylin missense SNPs and to filter those considered as convergent deleterious, which were then further analyzed through long-term molecular dynamics simulations of 1µs of duration. The simulations suggested that all missense SNPs considered as convergent deleterious caused some kind of structural change to the uroguanylin peptide. Additionally, four of these SNPs were also shown to cause modifications in peptide flexibility, possibly resulting in functional changes.

Prediction of the impact of coding missense and nonsense single nucleotide polymorphisms on HD5 and HBD1 antibacterial activity against Escherichia coli.

Defensins confer host defense against microorganisms and are important for human health. Single nucleotide polymorphisms (SNPs) in defensin gene-coding regions could lead to less active variants. Using SNP data available at the dbSNP database and frequency information from the 1000 Genomes Project, two DEFA5 (L26I and R13H) and eight DEFB1 (C35S, K31T, K33R, R29G, V06I, C12Y, Y28* and C05*) missense and nonsense SNPs that are located within mature regions of the coded defensins were retrieved. Such SNPs are rare and population restricted. In order to assess their antibacterial activity against Escherichia coli, two linear regression models were used from a previous work, which models the antibacterial activity as a function of solvation potential energy, using molecular dynamics data. Regarding only the antibacterial predictions, for HD5, no biological differences between wild-type and its variants were observed; while for HBD1, the results suggest that the R29G, K31T, Y28* and C05* variants could be less active than the wild-type one. The data here reported could lead to a substantial improvement in knowledge about the impact of missense SNPs in human defensins and their world distribution. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 633-644, 2016.

HD5 and HBD1 variants' solvation potential energy correlates with their antibacterial activity against Escherichia coli.

The structure-activity relationship of defensins is not clear. It is known that point mutations in HD5 and HBD1 could modify their activities; however, these mutations do not seem to alter their three-dimensional structures. Here, applying molecular dynamics simulations, this relationship was studied in depth. There are modifications in flexibility, solvent accessible surface area and radius of gyration, but these properties are not reflected in the activity. Only alterations in the solvation potential energy were correlated to antibacterial activity against Escherichia coli. Data here reported could lead to a better understanding of structural and functional aspects of α- and ß-defensins.

Computational analyses and prediction of guanylin deleterious SNPs.

Human guanylin, coded by the GUCA2A gene, is a member of a peptide family that activates intestinal membrane guanylate cyclase, regulating electrolyte and water transport in intestinal and renal epithelia. Deregulation of guanylin peptide activity has been associated with colon adenocarcinoma, adenoma and intestinal polyps. Besides, it is known that mutations on guanylin receptors could be involved in meconium ileus. However, there are no previous works regarding the alterations driven by single nucleotide polymorphisms in guanylin peptides. A comprehensive in silico analysis of missense SNPs present in the GUCA2A gene was performed taking into account 16 prediction tools in order to select the deleterious variations for further evaluation by molecular dynamics simulations (50 ns). Molecular dynamics data suggest that the three out of five variants (Cys104Arg, Cys112Ser and Cys115Tyr) have undergone structural modifications in terms of flexibility, volume and/or solvation. In addition, two nonsense SNPs were identified, both preventing the formation of disulfide bonds and resulting in the synthesis of truncated proteins. In summary the structural analysis of missense SNPs is important to decrease the number of potential mutations to be in vitro evaluated for associating them with some genetic diseases. In addition, data reported here could lead to a better understanding of structural and functional aspects of guanylin peptides.