Structural and functional analysis of Cyanovirin-N homologs: Carbohydrate binding affinities and antiviral potential of cyanobacterial peptides.

Cyanobacteria, a group of photosynthetic prokaryotes, can sinthesize several substances due to their secondary metabolism, with notable properties, such as Cyanovirin-N(CVN), a carbohydrate-binding lectin, that exhibits antiviral activity against several pathogens, due to its ability to bind viral surface carbohydrates such as mannose, thus interfering with the viral entry on the cell. CVN has been described in several cyanobacterial strains and shows biotechnological potential for the development of drugs of pharmaceutical interest. This study focuses on the genomic exploration and characterization of Cyanovirin-N homologs to assess the conservation of carbohydrate-binding affinity within the group. The analysis of their antiviral properties was carried out using bioinformatics tools to study protein models through an in silico pipeline, following the steps of genomic prospection on public databases, homology modeling, docking, molecular dynamics and energetic analysis. Mannose served as the reference ligand, and the lectins' binding affinity with mannose was assessed across Cyanovirin-N homologs. Genomic mining identified 33 cyanobacterial lectin sequences, which underwent structural and functional characterization. The results obtained from this work indicate strong carbohydrate affinity on several homologs, pointing to the conservation of antiviral properties alongside the group. However, this affinity was not uniformly distributed among sequences, exhibiting significant heterogeneity in binding site residues, suggesting potential multi-ligand binding capabilities on the Cyanovirin-N homologs group. Studies focused on the properties involved in these molecules and the investigation of the genetic diversity of Cyanovirin-N homologs could provide valuable insights into the discovery of new drug candidates, harvesting the potential of bioinformatics for large-scale functional and structural analysis.

First molecular detection of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felis catus) from an urban area in eastern Amazon.

Background: Domestic cats have been implicated as accidental hosts of Leishmania sp. However, in recent years, the recurrent description of new cases in endemic and nonendemic areas draw attention to the potential epidemiological role of cats as reservoir hosts. Although dogs are considered urban reservoirs, cats could act as a secondary natural reservoirs in these areas. Thus, feline leishmaniasis has become an emerging disease in several countries worldwide. Case presentation: This study aimed to describe the first case of feline leishmaniasis in a stray animal that presented lesions compatible with the disease in Belém, Pará, Brazil, an important urban area in eastern Amazon. Serological tests for Leishmania infantum (ELISA and IFA) were nonreactive, whereas histopathological examination indicated infectious dermatitis caused by Leishmania spp. or Toxoplasma gondii. Cytopathological study of lesion aspirate confirmed the presence of Leishmania sp. amastigotes within macrophages. Finally, molecular analyses revealed that the feline infection was caused by Leishmania (Leishmania) infantum chagasi. Conclusion: To the best of the authors' knowledge, this study reports the first case of natural infection by Leishmania (Leishmania) infantum chagasi in a feline from eastern Amazon. These findings suggest domestic cats as potential secondary reservoir hosts of Leishmania spp. in Belém, which reinforces the importance of further epidemiological investigation of feline leishmaniasis, especially in urban areas with human cases.

First molecular detection of canine herpesvirus 1 (CaHV-1) in the Eastern Brazilian Amazon.

BACKGROUND: Canine herpesvirus type 1 (CaHV-1) infects dogs and is associated with neonatal deaths and reproductive, ocular, neurological, and respiratory problems. In Brazil, reports of CaHV-1 have been restricted to the southeast and south regions, particularly in municipalities in the state of Rio Grande do Sul. OBJECTIVES: To assess the presence and variability of CaHV-1 in canine populations in the state of Pará, North Brazil. METHODS: Biological samples from 159 dogs from 4 municipalities in the State of Pará were evaluated using polymerase chain reaction and phylogenetic analyses, with the target being the viral enzyme, thymidine kinase. RESULTS: CaHV-1 was detected in 13 dogs (8.2%), with 2 animals being from the municipality of Santa Bárbara do Pará, 8 from Algodoal Island, 2 from Salinópolis, and one from Capanema. The study sequences revealed 100% identity among themselves and 64% to 100% identity with the other nucleotide sequences from Australia, Brazil, United Kingdom, and United States, including 100% identity with the 2002 isolate from Australia. The 1996 isolate from France was grouped in a branch that was different from the sequence of this study. CONCLUSIONS: This study presents the first molecular detection of CaHV-1 in dogs from the Amazon region in northern Brazil. The nucleotide identity between the strains and cytosine insertion in the sequences isolated in this study suggests at least 2 strains of CaHV-1 circulating in Brazil (Pará and BTU-1).

In silico improvement of the cyanobacterial lectin microvirin and mannose interaction.

Lectins that bind to HIV envelope glycoprotein can inhibit virus-cell fusion and be used for rational drug design. This paper presents the results of an in silico approach to improve affinity interaction between the cyanobacterial lectin microvirin and its ligand Manα(1-2)Man. Comparative modeling and molecular dynamics tools were used. Additionally, the alanine scanning webserver was used to study the importance of protein residues in the binding site and to guide mutant production. The model obtained presented two homologous domains designated as domains A and B, each consisting of a single strand with triple and antiparallel ß-sheets of (ß1-ß3 and ß6-ß8). Disulfide bonds between the cysteines (Cys60-Cys80, Cys63-Cys78 and Cys8-Cys24) were also found. The highly conserved binding site, including residues Asn44, Ile45, Asp46, Gln54, Asn55, Glu58, Thr59, Gln81, Thr82 and Met83. The RMSD values of the di-mannose and the interaction site were very stable during the molecular dynamics. Calculations of the occupation time of the hydrogen bonds were made for the residues that showed interaction in the complex lectin and ligand. The residue that contributed most to the interaction with Manα(1-2)Man was Asn55. After validation, the model generated remained stable during the entire simulation. Despite its structural similarity with the template we used, our mutant (Thr82Arg) showed a higher affinity interaction with Manα(1-2)Man. Communicated by Ramaswamy H. Sarma.

Correction: Tissue expression of antigens of ABH blood groups in species of New World Monkeys (Aotus infulatus, Callithrix jacchus, Sapajus apella and Saimiri sciureus).

[This corrects the article DOI: 10.1371/journal.pone.0241487.].

Genomic screening and molecular dynamics simulations of cyanovirin-N homologs from cyanobacteria phylum.

The phylum cyanobacteria are one of the most ancient groups of organisms on the planet and are well recognized due to its wide distribution, ecological role, and biotechnological potential. Cyanobacterial lectins are being extensively explored due to their antiviral activity, mainly because of their capacity of inhibiting HIV strains from infecting human cells by gp120 and gp41 binding. Cianovirin-N from Nostoc ellipsosporum was the first lectin isolated with this property. Since then, various homologs have been discovered and characterized. In this article, we present results of a genomic screening to find cyanovirin-N homologs (CVNH) in all cyanobacteria genomes available in the GenBank, resulting in 155 CVNH proteins with 63 presenting significant identity differences of cyanovirin-N. Homology modeling and molecular dynamics were employed to characterize 18 unexplored models and their functional capacity of binding to Manα(1-2)Man. Results presented here support the hypothesis of multiple ligand recognition for the CVNH family and may help to understand the function of these lectins for the producer cyanobacteria. Additionally, the theoretical results observed here justify carrying out experimental investigations that can expand the therapeutic potential of cyanobacterial lectins.

First Report of Canine Infection by Leishmania (Viannia) guyanensis in the Brazilian Amazon.

The American cutaneous (CL) and visceral leishmaniasis (VL) are zooanthroponoses transmitted by sand flies. Brazil records thousands of human leishmaniasis cases annually. Dogs are reservoirs of Leishmania infantum, which causes VL, but their role in the transmission cycle of CL is debatable. Wild mammals are considered reservoirs of the aetiological agents of CL (Leishmania spp.). OBJECTIVE: To describe the aetiology of leishmaniasis in dogs in an endemic area for CL and VL in the Amazon, Brazil. METHODS: Clinical evaluation and blood collection of 40 dogs from the villages Ubim (20) and Socorro (20), city of Tomé-Açu, state of Pará, were carried out. The DNA extracted from the blood was used for PCR with Leishmania-specific primers targeting the hsp70-234 gene sequence. Products were sequenced (ABI3500XL), and the sequences were aligned, edited (BioEdit), and analyzed (Blastn). RESULTS: Of the 34 amplified samples, 21 were sequenced, namely Leishmania infantum (12), L. guyanensis (5), L. braziliensis (3), and Leishmania sp. (01). CONCLUSION: Given the diversity of circulating pathogens, elucidation of the role of the dog in the Leishmania spp. cycle in Amazonian villages is imperative to the surveillance of CL in the region. We present the first report in Brazil, confirmed by sequencing, of canine infection by L. guyanensis, a species highly resistant to treatment in humans, with the drug of first choice (Glucantime®).

Tissue expression of antigens of ABH blood groups in species of New World Monkeys (Aotus infulatus, Callithrix jacchus, Sapajus apella and Saimiri sciureus).

ABH antigens are histo-antigens, but were first described on the surface of human erythrocytes. They are found in those cells only in great apes and humans, while in more primitive animals they are found in tissues and body fluids. ABH antigens are mainly distributed in tissues that are in contact with the external environment and may serve as ligands for pathogens in tissues or block their connection. Description of the distribution of these molecules in non-human primate tissues is restricted to a few tissues and species. This paper describes the expression of human A, B and H type antigens in different organs from four species of New World Primates, obtained from the Centro Nacional de Primatas, as well as comparing that expression with what has been described for humans. In this study, although the tissue description of the antigens is similar to the genetic model for humans, some differences in expression between some organs from those species and those of humans were found. The differences occurred mainly in endodermal organs that have secretory functions and are probably under the control of the human-type FUT-2 enzyme. In the mesodermal-origin organs there was a reduction or absence of A and B antigen marking, particularly in the H precursor substance, indicating that those organs are under the control of the human-type FUT-1 enzyme. These findings have demonstrated that there is similar ABH antigen reactivity in tissue distribution between the species, although there are some species-specific cases.

Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics.

Currently only four genome sequences for Limnothrix spp. are publicly available, and information on the genetic properties of cyanobacteria belonging to this genus is limited. In this study, we report the draft genome of Limnothrix sp. CACIAM 69d, isolated from the reservoir of a hydroelectric dam located in the Amazon ecosystem, from where cyanobacterial genomic data are still scarce. Comparative genomic analysis of Limnothrix revealed the presence of key enzymes in the cyanobacterial central carbon metabolism and how it is well equipped for environmental sulfur and nitrogen acquisition. Additionally, this work covered the analysis of Limnothrix CRISPR-Cas systems, pathways related to biosynthesis of secondary metabolites and assembly of extracellular polymeric substances and their exportation. A trans-AT PKS gene cluster was identified in two strains, possibly related to the novel toxin Limnothrixin biosynthesis. Overall, the draft genome of Limnothrix sp. CACIAM 69d adds new data to the small Limnothrix genome library and contributes to a growing representativeness of cyanobacterial genomes from the Amazon region. The comparative genomic analysis of Limnothrix made it possible to highlight unique genes for each strain and understand the overall features of their metabolism.

Genomic screening of new putative antiviral lectins from Amazonian cyanobacteria based on a bioinformatics approach.

Lectins are proteins of nonimmune origin, which are capable of recognizing and binding to glycoconjugate moieties. Some of them can block the interaction of viral glycoproteins to the host cell receptors acting as antiviral agents. Although cyanobacterial lectins have presented broad biotechnological potential, little research has been directed to Amazonian Cyanobacterial diversity. In order to identify new antiviral lectins, we performed genomic analysis in seven cyanobacterial strains from Coleção Amazônica de Cianobactérias e Microalgas (CACIAM). We found 75 unique CDS presenting one or more lectin domains. Since almost all were annotated as hypothetical proteins, we used homology modeling and molecular dynamics simulations to evaluate the structural and functional properties of three CDS that were more similar to known antiviral lectins. Nostoc sp. CACIAM 19 as well as Tolypothrix sp. CACIAM 22 strains presented cyanovirin-N homologues whose function was confirmed by binding free energy calculations. Asn, Glu, Thr, Lys, Leu, and Gly, which were described as binding residues for cyanovirin, were also observed on those structures. As for other known cyanovirins, those residues in both our models also made favorable interactions with dimannose. Finally, Alkalinema sp. CACIAM 70d presented one CDS, which was identified as a seven-bladed beta-propeller structure with binding sites predicted for sialic acid and N-acetylglucosamine. Despite its singular structure, our analysis suggested this molecule as a new putative antiviral lectin. Overall, the identification and the characterization of new lectins and their homologues are a promising area in antiviral research, and Amazonian cyanobacteria present biotechnological potential to be explored in this regard.

In silico characterization of a cyanobacterial plant-type isoaspartyl aminopeptidase/asparaginase.

Asparaginases are found in a range of organisms, although those found in cyanobacteria have been little studied, in spite of their great potential for biotechnological application. This study therefore sought to characterize the molecular structure of an L-asparaginase from the cyanobacterium Limnothrix sp. CACIAM 69d, which was isolated from a freshwater Amazonian environment. After homology modeling, model validation was performed using a Ramachandran plot, VERIFY3D, and the RMSD. We also performed molecular docking and dynamics simulations based on binding free-energy analysis. Structural alignment revealed homology with the isoaspartyl peptidase/asparaginase (EcAIII) from Escherichia coli. When compared to the template, our model showed full conservation of the catalytic site. In silico simulations confirmed the interaction of cyanobacterial isoaspartyl peptidase/asparaginase with its substrate, ß-Asp-Leu dipeptide. We also observed that the residues Thr154, Thr187, Gly207, Asp218, and Gly237 were fundamental to protein-ligand complexation. Overall, our results suggest that L-asparaginase from Limnothrix sp. CACIAM 669d has similar properties to E. coli EcAIII asparaginase. Our study opens up new perspectives for the biotechnological exploitation of cyanobacterial asparaginases.

Genetic diversity of Hepatozoon spp. in Hydrochoerus hydrochaeris and Pecari tajacu from eastern Amazon.

This study aimed to identify and characterize genetically species of the genus Hepatozoon detected in Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) from two localities from the Eastern Amazon. Blood samples from 196 free-living H. hydrochaeris from Marajó Island and 109 P. tajacu kept in captivity in Belém, Pará, were collected and analyzed for the presence of Hepatozoon spp. Partial sequences of the 18S rRNA gene were obtained and analyzed in comparison to others available in the NCBI database. Our results demonstrated a high prevalence of Hepatozoon canis in both mammals and the existence of four haplotypes of Hepatozoon spp., three of Hepatozoon canis and one of Hepatozoon cuestensis, found only in H. hydrochaeris. In addition, these data increase the genetic diversity of H. canis from the Eastern Amazon, as well as reporting, for the first time, the infection of mammals by H. cuestensis and P. tajacu by H. canis.

Complete Genome Sequence of Porphyrobacter sp. Strain CACIAM 03H1, a Proteobacterium Obtained from a Nonaxenic Culture of Microcystis aeruginosa.

Here, we present a complete genome sequence and annotation of Porphyrobacter sp. strain CACIAM 03H1, which was obtained from a nonaxenic culture of Microcystis aeruginosa, a cyanobacterium isolated from an Amazonian freshwater environment.

Draft Genome Sequence of Alkalinema sp. Strain CACIAM 70d, a Cyanobacterium Isolated from an Amazonian Freshwater Environment.

In order to increase the genomic data of cyanobacterial strains isolated in Brazil, we hereby present the draft genome sequence of the Alkalinema sp. strain CACIAM 70d, isolated from an Amazonian freshwater environment. This report describes the first genome available for this genus.

Draft Genome Sequence of Microcystis aeruginosa CACIAM 03, a Cyanobacterium Isolated from an Amazonian Freshwater Environment.

Given its toxigenic potential, Microcystis aeruginosa is an important bloom-forming cyanobacterium. Here, we present a draft genome and annotation of the strain CACIAM 03, which was isolated from an Amazonian freshwater environment.

Molecular analysis reveals the diversity of Hepatozoon species naturally infecting domestic dogs in a northern region of Brazil.

This study aimed to optimize molecular methods for detecting DNA of Hepatozoon spp. as well as identify the phylogenetic relationships of Hepatozoon strains naturally infecting domestic dogs in Belém, Pará, northern Brazil. Blood samples were collected from 138 dogs, and screened for Hepatozoon spp. using a new nested PCR assay. Positive samples were subjected to genetic characterization based on amplification and sequencing of approximately 670bp of the Hepatozoon spp. 18S rRNA. Of the positive dogs, four shared the haplotype Belém 01, one dog presented the haplotype Belém 02 and two dogs shared the haplotype Belém 03. A Bayesian inference indicates that haplotypes Belém 01 and Belém 02 are phylogenetically related to H. canis, while Belém 03 is related to H. americanum. Overall, based on the first molecular evidence of H. americanum in Brazilian domestic dogs, the proposed protocol may improve the epidemiological investigation of canine hepatozoonosis.

Draft Genome Sequence of Flavihumibacter sp. Strain CACIAM 22H1, a Heterotrophic Bacterium Associated with Cyanobacteria.

Here, we present a draft genome and annotation of Flavihumibacter sp. CACIAM 22H1, isolated from Bolonha Lake, Brazil, which will provide further insight into the production of substances of biotechnological interest.

Optimization of a molecular method for the diagnosis of canine babesiosis.

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.

Optimization of a molecular method for the diagnosis of canine babesiosis / Otimização de método molecular para diagnóstico da babesiose canina

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.

A babesiose é uma doença hemolítica de ocorrência mundial, causada por protozoários do gênero Babesia (Apicomplexa), que são transmitidos por carrapatos a diversos mamíferos, incluindo o homem. O objetivo deste estudo foi otimizar um método molecular para a detecção de fragmento do 18S rDNA de Babesia canis, Babesia vogeli, Babesia rossi ou Babesia gibsoni com base em uma única semi-nested (snPCR), comparando sua eficiência com um protocolo de PCR simples. Para isso, 100 amostras de sangue de cães com suspeita de hemoparasitoses foram analisadas e, enquanto o protocolo de PCR simples indicou somente 5% (5/100) de amostras positivas, o protocolo de snPCR, com 22% (22/100) de amostras positivas, apresentou maior sensibilidade (p valor = 0,0000). Este resultado está de acordo com outros estudos que mostram a maior sensibilidade de detecção dos testes baseado em nested ou snPCR. Assim, como uma forma de prevenir resultados falso-negativos devido à baixa parasitemia, sugere-se que este protocolo seja preferencialmente usado nos estudos epidemiológicos de babesiose canina, em especial naqueles que tratam da sua prevalência.