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Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.
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Proliferação de Células , Hidroxiureia , Taenia , Hidroxiureia/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Taenia/efeitos dos fármacos , Taenia/genética , Taenia/crescimento & desenvolvimento , Taenia/metabolismo , Proteômica/métodos , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteoma/metabolismo , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismoRESUMO
Chilhuacle negro chili (Capsicum annuum L.) is an ancient Mexican landrace that is deeply linked to the culinary heritage of the country. Because of the high profitability and uniqueness of this crop, the Universidad Autónoma del Estado de Morelos is exploring its production in controlled environments. In the crop cycles of 2018 to 2019, the production of chilhuacle negro plants was seriously affected by an unidentified pathogen causing fruit rot, which reduced its quality, yield, and market value. Therefore, the main objective of this work was to study and characterize the fruit microbiota, which could help reveal the causal agent of this disease. Using DNA metabarcoding coupled with Illumina and nanopore sequencing technologies, we collected and analyzed both healthy and infected chili fruit, along with greenhouse bioaerosols. We also explored the bacterial and fungal microbiota by using microbiological techniques to isolate some of the culturable bacterial and fungal species. Our results suggest that the seedborne fungus Alternaria alternata is activated during the maturation stage of chilhuacle negro fruit, triggering a microbiome imbalance, which may in turn enable the establishment of other opportunistic pathogenic fungi during fruit decay, such as Mucor sp. To our knowledge, this is the first study of the chilhuacle negro chili microbiome, which can shed some light on our understanding of one of the main diseases that affect this valuable crop.
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Capsicum , Micobioma , Negro ou Afro-Americano , Frutas , HumanosRESUMO
Summary: Operon-mapper is a web server that accurately, easily and directly predicts the operons of any bacterial or archaeal genome sequence. The operon predictions are based on the intergenic distance of neighboring genes as well as the functional relationships of their protein-coding products. To this end, Operon-mapper finds all the ORFs within a given nucleotide sequence, along with their genomic coordinates, orthology groups and functional relationships. We believe that Operon-mapper, due to its accuracy, simplicity and speed, as well as the relevant information that it generates, will be a useful tool for annotating and characterizing genomic sequences. Availability and implementation: http://biocomputo.ibt.unam.mx/operon_mapper/.
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Genoma Arqueal , Genoma Bacteriano , Genômica , Óperon , Software , Biologia Computacional , Internet , Fases de Leitura AbertaRESUMO
Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.
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Adaptação Fisiológica/genética , Cestoides/genética , Genoma Helmíntico/genética , Parasitos/genética , Animais , Evolução Biológica , Cestoides/efeitos dos fármacos , Cestoides/fisiologia , Infecções por Cestoides/tratamento farmacológico , Infecções por Cestoides/metabolismo , Sequência Conservada/genética , Echinococcus granulosus/genética , Echinococcus multilocularis/efeitos dos fármacos , Echinococcus multilocularis/genética , Echinococcus multilocularis/metabolismo , Genes de Helmintos/genética , Genes Homeobox/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Hymenolepis/genética , Redes e Vias Metabólicas/genética , Terapia de Alvo Molecular , Parasitos/efeitos dos fármacos , Parasitos/fisiologia , Proteoma/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Taenia solium/genéticaRESUMO
Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines.
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Antígenos de Helmintos/imunologia , Taenia solium/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Clonagem Molecular , Cisticercose/imunologia , Cisticercose/prevenção & controle , Cisticercose/veterinária , Mapeamento de Epitopos , Escherichia coli/genética , Expressão Gênica , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Linfócitos T/imunologia , Taenia solium/genéticaRESUMO
Translation initiation in prokaryotes is mainly defined, although not exclusively, by the interaction between the anti-Shine-Dalgarno sequence (antiSD), located at the 3'-terminus of the 16S ribosomal RNA, and a complementary sequence, the ribosome binding site, or Shine-Dalgarno (SD), located upstream of the start codon in prokaryotic mRNAs. The antiSD has a conserved 5'-CCUCC-3' core, but inter-species variations have been found regarding the participation of flanking bases in binding. These variations have been described for certain bacteria and, to a lesser extent, for some archaea. To further analyze these variations, we conducted binding-energy prediction analyses on over 6,400 genomic sequences from both domains. We identified 15 groups of antiSD variants that could be associated with the organisms' phylogenetic origin. Additionally, our findings revealed that certain organisms exhibit variations in the core itself. Importantly, an unaltered core is not necessarily required for the interaction between the 3'-terminus of the rRNA and the region preceding the AUG of the mRNA. In our study, we classified organisms into four distinct categories: i) those possessing a conserved core and demonstrating binding; ii) those with a conserved core but lacking evidence of binding; iii) those exhibiting binding in the absence of a conserved core; and iv) those lacking both a conserved core and evidence of binding. Our results demonstrate the flexibility of organisms in evolving different sequences involved in translation initiation beyond the traditional Shine-Dalgarno sequence. These findings are discussed in terms of the evolution of translation initiation in prokaryotic organisms.
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Iniciação Traducional da Cadeia Peptídica , Células Procarióticas , Iniciação Traducional da Cadeia Peptídica/genética , Filogenia , Células Procarióticas/metabolismo , Códon de Iniciação/genética , Bactérias/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Biossíntese de ProteínasRESUMO
Taenia crassiceps has been used for decades as an experimental model for the study of human and porcine cysticercosis. Even though, its life cycle, tissue organization, ultrastructure and immune response elicited in the host, have been extensively described, there are many other biological questions remaining to be addressed. In the present study we revisited the muscle and neural architecture of cysticerci in two of the most frequently used strains (WFU and ORF), using conventional staining and confocal microscopy imaging, aiming to assemble an updated anatomy. Differences between both strains, including polarization processes during development of the young budding larvae, are emphasized. We also performed a search for genes that have been related to peptidergic neural processes in other related flatworms. These findings can help to understand the anatomical and molecular consequences of the scolex presence or absence in both strains.
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Cysticercus , Larva , Músculos , Taenia , Animais , Cysticercus/imunologia , Músculos/parasitologia , Taenia/fisiologia , Microscopia Confocal , Cisticercose/parasitologia , Suínos , Humanos , Sistema NervosoRESUMO
Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates.
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AbstractBabesiosis, caused by protozoan parasites of the genus Babesia, is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of new Babesia species underscores the ongoing risk of zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental changes. One such pathogen, Babesia MO1, previously implicated in severe cases of human babesiosis in the United States, was initially considered a subspecies of B. divergens, the predominant agent of human babesiosis in Europe. Here we report comparative multiomics analyses of B. divergens and B. MO1 that offer insight into their biology and evolution. Our analysis shows that despite their highly similar genomic sequences, substantial genetic and genomic divergence occurred throughout their evolution resulting in major differences in gene functions, expression and regulation, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens, and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.
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Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.
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Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.
Assuntos
Babesia , Babesiose , Carrapatos , Animais , Humanos , Camundongos , Babesia/genética , Babesiose/tratamento farmacológico , Multiômica , Eritrócitos/parasitologiaRESUMO
Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.
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Human cysticercosis by Taenia solium is the major cause of neurological illness in countries of Africa, Southeast Asia, and the Americas. Publication of four cestode genomes (T. solium, Echinococcus multilocularis, E. granulosus and Hymenolepis microstoma) in the last decade, marked the advent of novel approaches on the study of the host-parasite molecular crosstalk for cestode parasites of importance for human and animal health. Taenia crassiceps is another cestode parasite, closely related to T. solium, which has been used in numerous studies as an animal model for human cysticercosis. Therefore, characterization of the T. crassiceps genome will also contribute to the understanding of the human infection. Here, we report the genome of T. crassiceps WFU strain, reconstructed to a noncontiguous finished resolution and performed a genomic and differential expression comparison analysis against ORF strain. Both strain genomes were sequenced using Oxford Nanopore (MinION) and Illumina technologies, achieving high quality assemblies of about 107 Mb for both strains. Dotplot comparison between WFU and ORF demonstrated that both genomes were extremely similar. Additionally, karyotyping results for both strains failed to demonstrate a difference in chromosome composition. Therefore, our results strongly support the concept that the absence of scolex in the ORF strain of T. crassiceps was not the result of a chromosomal loss as proposed elsewhere. Instead, it appears to be the result of subtle and extensive differences in the regulation of gene expression. Analysis of variants between the two strains identified 2,487 sites with changes distributed in 31 of 65 scaffolds. The differential expression analysis revealed that genes related to development and morphogenesis in the ORF strain might be involved in the lack of scolex formation.
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Cisticercose , Taenia solium , África , Animais , Cisticercose/veterinária , Modelos Animais de Doenças , Genômica , Humanos , Taenia solium/genéticaRESUMO
AIM: To evaluate the taxonomic profile of the gut microbiota using metagenomics and the association with diet-dependent childhood obesity. METHODS: A cross-sectional study of a subsample of 46 children was conducted. The children were classified as normal-weight, overweight, and obese according to their age and sex and the World Health Organization (WHO) guidelines. Dietary patterns were determined through principal component analysis. The profile of the human gut microbiota was determined by bioinformatic analysis using whole metagenome shotgun sequencing. The association of gut microbiota and z-BMI, waist circumference and hip circumference, and the possible modifying effect of diet were analyzed using multiple regression models. RESULTS: Children with an abundance of Holdemania spp. and high protein and complex carbohydrate consumption had a lower z-BMI (ß -19.06, p = 0.011), waist circumference (ß -171.92, p = 0.003), and hip circumference (ß -157.57, p = 0.004). In contrast, observed a positive association between Coprococcus catus and the low intake of this dietary pattern with hip circumference (ß 147.87, p = 0.025). Furthermore, the presence of Bilophila spp. and Paraprevotella xylaniphila with high saturated fat and simple carbohydrate consumption we observed a positive association between z-BMI (ß 47.5, p = 0.002), hip circumference (ß 44.54, p = 0.025), and waist circumference (ß 44.34, p = 0.004). CONCLUSION: We suggest that the synergism between diet and the profile of children's gut microbiota can be a factor that could be associated with the development of obesity and its complications in childhood.
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Microbioma Gastrointestinal , Obesidade Infantil , Índice de Massa Corporal , Carboidratos , Criança , Estudos Transversais , Dieta , Humanos , Obesidade Infantil/epidemiologia , Obesidade Infantil/etiologiaRESUMO
One of the major families of host defense peptides (HDPs) in vertebrates are ß-defensins. They constitute important components of innate immunity and have remained an interesting topic of research for more than two decades. While many ß-defensin sequences in mammals and birds have been identified and their properties and functions characterized, ß-defensin peptides from other groups of vertebrates, particularly reptiles, are still largely unexplored. In this review, we focus on reptilian ß-defensins and summarize different aspects of their biology, such as their genomic organization, evolution, structure, and biological activities. Reptilian ß-defensin genes exhibit similar genomic organization to birds and their number and gene structure are variable among different species. During the evolution of reptiles, several gene duplication and deletion events have occurred and the functional diversification of ß-defensins has been mainly driven by positive selection. These peptides display broad antimicrobial activity in vitro, but a deeper understanding of their mechanisms of action in vivo, including their role as immunomodulators, is still lacking. Reptilian ß-defensins constitute unique polypeptide sequences to expand our current understanding of innate immunity in these animals and elucidate core biological functions of this family of HDPs across amniotes.
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Jacarés e Crocodilos/genética , Evolução Molecular , Peptídeos/genética , beta-Defensinas/genética , Animais , Genoma/genética , Répteis/genéticaRESUMO
ß-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, ß-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian ß-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human ß-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian ß-defensin in general.
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Jacarés e Crocodilos , Antibacterianos/farmacologia , beta-Defensinas/química , beta-Defensinas/farmacologia , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Linhagem Celular , Células Epiteliais , Humanos , Agentes de Imunomodulação/química , Agentes de Imunomodulação/farmacologia , Testes de Sensibilidade Microbiana , Dobramento de Proteína , beta-Defensinas/síntese químicaRESUMO
Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.
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GMP Cíclico/análogos & derivados , Genoma Bacteriano , Pectobacterium/genética , Pectobacterium/fisiologia , Polissacarídeos Bacterianos/biossíntese , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Genômica , México , Movimento , Opuntia/microbiologia , Fenótipo , Filogenia , Nicotiana/microbiologiaRESUMO
In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.
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Caryophyllaceae/genética , Genoma de Cloroplastos , Polimorfismo Genético , Loci Gênicos , Repetições de MicrossatélitesRESUMO
The complete sequence of chloroplast genome (cpDNA) has been documented for single large columnar species of Cactaceae, lacking inverted repeats (IRs). We sequenced cpDNA for seven species of the short-globose cacti of Mammillaria and de novo assembly revealed three novel structures in land plants. These structures have a large single copy (LSC) that is 2.5 to 10 times larger than the small single copy (SSC), and two IRs that contain strong differences in length and gene composition. Structure 1 is distinguished by short IRs of <1 kb composed by rpl23-trnI-CAU-ycf2; with a total length of 110,189 bp and 113 genes. In structure 2, each IR is approximately 7.2 kb and is composed of 11 genes and one Intergenic Spacer-(psbK-trnQ)-trnQ-UUG-rps16-trnK-UUU-matK-trnK-UUU-psbA-trnH-GUG-rpl2-rpl23-trnI-CAU-ycf2; with a total size of 116,175 bp and 120 genes. Structure 3 has divergent IRs of approximately 14.1 kb, where IRA is composed of 20 genes: psbA-trnH-GUG-rpl23-trnI-CAU-ycf2-ndhB-rps7-rps12-trnV-GAC-rrn16-ycf68-trnI-GAU-trnA-AGC-rrn23-rrn4.5-rrn5-trnR-ACG-trnN-GUU-ndhF-rpl32; and IRB is identical to the IRA, but lacks rpl23. This structure has 131 genes and, by pseudogenization, it is shown to have the shortest cpDNA, of just 107,343 bp. Our findings show that Mammillaria bears an unusual structural diversity of cpDNA, which supports the elucidation of the evolutionary processes involved in cacti lineages.
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Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.