Immunization with a nontoxic naturally occurring Clostridium perfringens alpha toxin induces neutralizing antibodies in rabbits.

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin.

A MLST Clade 2 Clostridium difficile strain with a variant TcdB induces severe inflammatory and oxidative response associated with mucosal disruption.

The epidemiology of Clostridium difficile infections is highly dynamic as new strains continue to emerge worldwide. Here we present a detailed analysis of a new C. difficile strain (ICC-45) recovered from a cancer patient in Brazil that died from severe diarrhea. A polyphasic approach assigned a new PCR-ribotype and PFGE macrorestriction pattern to strain ICC-45, which is toxigenic (tcdA(+), tcdB(+) and ctdB(+)) and classified as ST41 from MLST Clade 2 and toxinotype IXb. Strain ICC-45 encodes for a variant TcdB that induces a distinct CPE in agreement with its toxinotype. Unlike epidemic NAP1/027 strains, which are also classified to MLST Clade 2, strain ICC-45 is susceptible to fluoroquinolones and does not overproduce toxins TcdA and TcdB. However, supernatants from strain ICC-45 and a NAP1/027 strain produced similar expression of pro-inflammatory cytokines, epithelial damage, and oxidative stress response in the mouse ileal loop model. These results highlight inflammation and oxidative stress as common features in the pathogenesis of C. difficile Clade 2 strains. Finally, this work contributes to the description of differences in virulence among various C. difficile strains.

Biological Response of Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae) Pupae After Submersion in Freshwater.

Forensic Entomology uses arthropods to aid in legal investigations. This study checked the biological response of Chrysomya putoria pupae to submersion in fresh water for up to 6 d, evaluating the critical submersion time, survival rate, and development time of the flies. Adults were collected using fish baits in two typical traps. Seven hundred and twenty fourth-generation pupae with 2 d of development were used and separated into submergence intervals: 24, 48, 72, 96, 120, and 144 h. An additional 120 pupae were used as a control. Each treatment was done in triplicate, consisting of 40 pupae distributed in four tulle-sealed test tubes containing 10 pupae each. All tubes of each treatment were co-adhered in test tube racks and were submerged in mineral water in a container with constant oxygenation, except those of the control group, which were not submerged. The tubes were removed from the water according to their respective submersion interval, until 144 h was completed. The control group had a survival rate of 90%, while the 24-h treatment had 85% and the 48-h treatment had 35.8%. The critical submersion time for pupae was 72 h, with 100% mortality by 144 h. The average development time for the control group was 3.2 d, while the 24- and 48-h treatments developed in 4.3 and 6.3 d, respectively. The longer the individuals were submerged, the lower the survival rate was, while the development time increased. The data obtained in this study have potential in applications to estimate the interval of submersion of a cadaver.

Analysis of NGS data from Peruvian Loxosceles laeta spider venom gland reveals toxin diversity.

Accidents involving spiders from the genus Loxosceles cause medical emergencies in several countries of South America. The species Loxosceles laeta is ubiquitously present in Peru and is responsible for severe accidents in this country. To further characterize L. laeta venom components and to unveil possible variations in the Peruvian population, we provide an overview of the toxins-related transcripts present in the venom gland of Peruvian L. laeta. A dataset from a cDNA library previously sequenced by MiSeq sequencer (Illumina) was re-analyzed and the obtained data was compared with available sequences from Loxosceles toxins. Phospholipase-D represent the majority (69,28 %) of the transcripts related to venom toxins, followed by metalloproteases (20,72 %), sicaritoxins (6,03 %), serine-proteases (2,28 %), hyaluronidases (1,80 %) and Translationally Controlled Tumor Protein (TCTP) (0,56 %). New sequences of phospholipases D,sicaritoxins, hyaluronidase, TCTP and serine proteinases were described. Differences between the here-described toxin sequences and others, previously identified in venom glands from other spiders, were visualized upon sequence alignments. In addition, an in vitro hyaluronidase activity assay was also performed to complement comparisons between Peruvian and Brazilian L. laeta venom enzymatic activities, revealing a superior activity in the venom from Brazilian specimens. These new data provide a molecular basis that can help to explain the difference in toxicity among L. laeta venoms from different countries in South America.

An Integrative View of the Phyllosphere Mycobiome of Native Rubber Trees in the Brazilian Amazon.

The rubber tree, Hevea brasiliensis, is a neotropical Amazonian species. Despite its high economic value and fungi associated with native individuals, in its original area in Brazil, it has been scarcely investigated and only using culture-dependent methods. Herein, we integrated in silico approaches with novel field/experimental approaches and a case study of shotgun metagenomics and small RNA metatranscriptomics of an adult individual. Scientific literature, host fungus, and DNA databases are biased to fungal taxa, and are mainly related to rubber tree diseases and in non-native ecosystems. Metabarcoding retrieved specific phyllospheric core fungal communities of all individuals, adults, plantlets, and leaves of the same plant, unravelling hierarchical structured core mycobiomes. Basidiomycotan yeast-like fungi that display the potential to produce antifungal compounds and a complex of non-invasive ectophytic parasites (Sooty Blotch and Flyspeck fungi) co-occurred in all samples, encompassing the strictest core mycobiome. The case study of the same adult tree (previously studied using culture-dependent approach) analyzed by amplicon, shotgun metagenomics, and small RNA transcriptomics revealed a high relative abundance of insect parasite-pathogens, anaerobic fungi and a high expression of Trichoderma (a fungal genus long reported as dominant in healthy wild rubber trees), respectively. Altogether, our study unravels new and intriguing information/hypotheses of the foliar mycobiome of native H. brasiliensis, which may also occur in other native Amazonian trees.

Exploring antibiotic resistance in environmental integron-cassettes through intI-attC amplicons deep sequencing.

INTRODUCTION: Freshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process. MATERIAL AND METHODS: In the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance. CONCLUSIONS: Overall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River.

Complete mitochondrial genome sequence of the high altitude Brazilian treefrog Pithecopus megacephalus (Anura, Phyllomedusidae).

The first complete mitochondrial genome (mtDNA) for the family Phyllomedusidae (genus Pithecopus) is presented. It is a circular molecule with 17713 pb including 13 protein coding genes, 22 tRNA genes, two rRNA genes, and a control region (D-loop). Pithecopus megacephalus was close to the only other phyllomedusid whose complete mtDNA sequence is available, but had the cytb gene 147 pb smaller.

Foliar mycoendophytome of an endemic plant of the Mediterranean biome (Myrtus communis) reveals the dominance of basidiomycete woody saprotrophs.

The true myrtle, Myrtus communis, is a small perennial evergreen tree that occurs in Europe, Africa, and Asia with a circum-Mediterranean geographic distribution. Unfortunately, the Mediterranean Forests, where M. communis occurs, are critically endangered and are currently restricted to small fragmented areas in protected conservation units. In the present work, we performed, for the first time, a metabarcoding study on the spatial variation of fungal community structure in the foliar endophytome of this endemic plant of the Mediterranean biome, using bipartite network analysis as a model. The local bipartite network of Myrtus communis individuals and their foliar endophytic fungi is very low connected, with low nestedness, and moderately high specialization and modularity. Similar network patterns were also retrieved in both culture-dependent and amplicon metagenomics of foliar endophytes in distinct arboreal hosts in varied biomes. Furthermore, the majority of putative fungal endophytes species were basidiomycete woody saprotrophs of the orders Polyporales, Agaricales, and Hymenochaetales. Altogether, these findings suggest a possible adaptation of these wood-decaying fungi to cope with moisture limitation and spatial scarcity of their primary substrate (dead wood), which are totally consistent with the predictions of the viaphytism hypothesis that wood-decomposing fungi inhabit the internal leaf tissue of forest trees in order to enhance dispersal to substrates on the forest floor, by using leaves as vectors and as refugia, during periods of environmental stress.

Mitochondrial sequences of Rhipicephalus and Coxiella endosymbiont reveal evidence of lineages co-cladogenesis.

Rhipicephalus ticks are competent vectors of several pathogens, such as Spotted Fever Group Rickettsiae (SFGR) and many Babesia species. Within this genus, different R. sanguineus s.l. lineages show an unequal vector competence and resistance regarding some pathogenic strains. Current literature supports that tick endosymbionts may play an essential role in the transmission ability of a vector. Indeed, the microbial community of Rhipicephalus seems to be dominated by Coxiella-like endosymbionts (CLE). Still, their co-evolutionary associations with the complicated phylogeny of Rhipicephalus lineages and their transmissible pathogens remain unclear. We performed a phylogenetic congruence analysis to address whether divergent R. sanguineus s.l. lineages had a different symbiont composition. For that, we applied a PCR based approach to screen part of the microbial community present in 279 Rhipicephalus ticks from the Iberian Peninsula and Africa. Our analyses detected several qPCR-positive signals for both SFGR and Babesia species, of which we suggest R. sanguineus-tropical lineage as a natural vector of Babesia vogeli and R. sanguineus-temperate lineage of SFGR. The acquisition of 190 CLE sequences allowed to evaluate co-phylogenetic associations between the tick and the symbiont. With this data, we observed a strong but incomplete co-cladogenesis between CLE strains and their Rhipicephalus tick lineages hosts.

Selected to survive and kill: Tityus serrulatus, the Brazilian yellow scorpion.

Annually, more than 1.2 million scorpion stings and more than 3,000 deaths occur worldwide. Tityus serrulatus Lutz and Mello, 1922 (Scorpiones, Buthidae) is the most medically relevant species in Brazil where it is spreading rapidly and causing over 90,000 cases of envenomation yearly. We monitored T. serrulatus longevity and ability to reproduce under conditions of food and/or water deprivation. We found that T. serrulatus is highly tolerant to food deprivation, with individuals enduring up to 400 days without food. On the other hand, access to water played a pivotal role in T. serrulatus survival. Food and water deprived scorpions showed weight reduction. Reproduction occurred throughout the year for food-deprived scorpions and controls, but not in the water-deprived groups. Remarkably, food-deprived animals were able to give birth after 209 days of starvation. Tityus serrulatus resistance to food and water deprivation is likely to be an additional factor underlying this species' geographic expansion and the difficulties encountered in controlling it.

Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization.

Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.

Mapping Alterations Induced by Long-Term Axenic Cultivation of Leishmania amazonensis Promastigotes With a Multiplatform Metabolomic Fingerprint Approach.

Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; ß-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

Complete mitochondrial genome sequence of Myrmecophaga tridactyla from Brazilian Savanna.

Myrmecophaga tridactyla, popularly known as giant anteater, is a member of Xenarthra magnorder which is under the threat of extinction. Herein, we describe the complete mitochondrial genome of M. tridactyla. The circular DNA molecule is 16,546 bp long, contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a non-coding Control Region of 1110 bp. All protein-coding genes are on the heavy strand, except for Nd6. Ten of the 13 PCGs contained an ATG start codon.

Changes in mouse gut bacterial community in response to different types of drinking water.

Gut microbiota exerts a fundamental role on host physiology, and how extrinsic perturbations influence its composition has been increasingly examined. However, the effect of drinking water on gut microbiota is still poorly understood. In this study, we explored the response of mouse gut bacterial community (fecal and mucosa-adhered) to the ingestion of different types of drinking water. The experimental cohort was divided according to different water sources into four groups of mice that consumed autoclaved tap water (control group), water collected directly from a drinking water treatment plant, tap water, and commercial bottled mineral water. Differences among groups were observed, especially related to control group, which exhibited the smallest intra-group variation, and the largest distance from test groups on the last experimental day. Clinically important taxa, such as Acinetobacter and Staphylococcus, increased in feces of mice that drank tap water and in mucosa-adhered samples of animals from disinfected and tap water groups. Furthermore, statistical analyses showed that both time elapsed between samplings and water type significantly influenced the variation observed in the samples. Our results reveal that drinking water potentially affects gut microbiota composition. Additionally, the increase of typical drinking water clinically relevant and antibiotic resistance-associated bacteria in gut microbiota is a cause of concern.

Insights into the Genome Sequence of Chromobacterium amazonense Isolated from a Tropical Freshwater Lake.

Members of the genus Chromobacterium have been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis of Chromobacterium amazonense strain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike other Chromobacterium species, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of the C. amazonense strain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involving Chromobacterium-related isolates and improves our understanding of the global genomic diversity of Chromobacterium species.

High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model.

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite loci for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite loci from the fish Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.

Insights into the skin microbiome dynamics of leprosy patients during multi-drug therapy and in healthy individuals from Brazil.

Leprosy is a chronic infectious peripheral neuropathy that is caused by Mycobacterium leprae, and the skin is one of its preferred target sites. However, the effects of this infection on the skin microbiome remain largely unexplored. Here, we characterize and compare the lesional and non-lesional skin microbiomes of leprosy patients and healthy individuals through the deep sequencing of 16 S rRNA genes. Additionally, a subset of patients was monitored throughout the multi-drug therapy to investigate its effect on the leprous skin microbiome. Firmicutes-associated OTUs (primarily Staphylococcus) prevailed in healthy individuals. By contrast, Firmicutes was underrepresented and Proteobacteria was enriched in the patients' skin, although a single dominant taxon has not been observed at a finer taxonomic resolution. These differences can be explained by the significant decrease in Staphylococcus and Streptococcus as well as the enrichment in Brevundimonas. The overrepresentation of Micrococcus in patients is also remarkable. Genus-level compositional profiles revealed no significant intrapersonal difference between lesional and non-lesional sites. Treatment-associated changes indicated a loss of diversity and a shift in the community composition, with stronger impacts on the OTUs that are considered indigenous bacteria. Therefore, the molecular signatures associated with leprosy identified herein might be of importance for early diagnostics.

CPP-Ts: a new intracellular calcium channel modulator and a promising tool for drug delivery in cancer cells.

Scorpion sting envenoming impacts millions of people worldwide, with cardiac effects being one of the main causes of death on victims. Here we describe the first Ca2+ channel toxin present in Tityus serrulatus (Ts) venom, a cell penetrating peptide (CPP) named CPP-Ts. We show that CPP-Ts increases intracellular Ca2+ release through the activation of nuclear InsP3R of cardiomyocytes, thereby causing an increase in the contraction frequency of these cells. Besides proposing a novel subfamily of Ca2+ active toxins, we investigated its potential use as a drug delivery system targeting cancer cell nucleus using CPP-Ts's nuclear-targeting property. To this end, we prepared a synthetic CPP-Ts sub peptide14-39 lacking pharmacological activity which was directed to the nucleus of specific cancer cell lines. This research identifies a novel subfamily of Ca2+ active toxins and provides new insights into biotechnological applications of animal venoms.

Complete mitochondrial genome sequence of neotropical fish Astyanax giton Eigenmann 1908 (Ostariophysi; Characidae).

Here we report, for the first time, the complete mitochondrial genome of Astyanax giton which, together with other species, are popularly known as tetras. The mitogenome's length is 16,643 bp, containing 13 protein-encoding genes (CDS), two ribosomal RNAs (rRNA), 22 RNA transfer (tRNA) and one control region (D-loop). As for other vertebrates, all genes are encoded on the heavy strand except for ND6 and eight tRNA genes. In the phylogenetic analyses, this species and other Astyanax were paraphyletic.