Serovar-dependent differences in Hfq-regulated phenotypes in actinobacillus pleuropneumoniae

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions, and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen A. pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 hour time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes

In silico analysis and functional characterization of small regulatory RNAs: targets and phenotypes involved in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (App) é o agente causal da pleuropneumonia suína, doença que causa uma pleuropneumonia fibrinosa, exsudativa, hemorrágica, necrosante, que afeta porcos de todas as idades, levando à morte súbita e grandes perdas econômicas em todo o mundo. Existem dois métodos básicos para limitar a infecção endêmica de App: antibióticos e vacinas, mas a segunda opção não é eficiente para controlar a pleuropneumonia porcina. A virulência de App é complexa e envolve diferentes fatores bacterianos que incluem exotoxinas, polissacarídeos capsulares e lipopolissacarídeos. Além disso, existem fatores adicionais de virulência que são up- ou down-regulados durante a infecção e pequenos RNAs reguladores (sRNAs) podem regulá-los. Recentemente, nosso grupo identificou 23 sRNAs em App, cuja transcrição foi validada por RNA-Seq. Neste trabalho selecionamos seis genes sRNA e produzimos mutantes de deleção a partir dos parentais selvagem (WT_ΔsRNA) e de seu mutante isogênico para a chaperona de sRNAs, Hfq (Δhfq_ΔsRNA). Além disso, propomos uma análise robusta in silico da estrutura, conservação e interação dos RNAs de App com seus alvos. Os fenótipos desses mutantes foram comparados aos da linhagem parental sob diferentes condições, como: curva de crescimento, crescimento sob diferentes condições de estresse, determinação da concentração inibitória mínima contra antibióticos, adesão em superfícies bióticas e abióticas, atividade hemolítica e virulência no modelo alternativo de infecção, Galleria mellonella. As linhagens ΔsRNA exibiram uma diferença fenotípica em relação às linhagens parentais sob pelo menos uma condição, revelando que os sRNAs de App estão envolvidos na regulação da virulência. Notavelmente, alguns mutantes de deleção simples mostraram um fenótipo de ganho de função, que ainda não foi descrito para nenhum outro mutante de deleção de gene de sRNA na família Pasteurellaceae. Além disso, um mutante mostrou uma forte atenuação de virulência, sendo um candidato para elaboração de uma vacina viva atenuada contra A. pleuropneumoniae. As análises in silico revelaram a presença de sRNAs de App em seis gêneros da família Pasteurellaceae. Vários alvos foram encontrados e categorizados em diferentes funções, evidenciando uma complexa rede de regulação gênica para esses RNAs. A partir desses resultados, propomos um modelo de ação via Arrc14 em que ele atua como regulador negativo no metabolismo de amino e nucleotídeo açúcares. Nossos resultados permitem concluir que os sRNAs aqui estudados interferem de forma distinta na regulação da virulência de Actinobacillus pleuropneumoniae sorotipo 8. Concluímos ainda que, com base nos dados obtidos para RNA01, a linhagem WT_Δrna01 possui potencial para ser utilizada no desenvolvimento de uma vacina atenuada viva para prevenir e controlar infecções por Actinobacillus pleuropneumoniae.Actinobacillus pleuropneumoniae (App), the porcine pleuropneumonia´s causative agent, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting pigs of all ages, leading to sudden death and great economic loss worldwide. There are two basic methods used to limit endemic infection of App: antibiotics and vaccines, but the second option is not efficient to control porcine pleuropneumonia. The App virulence is complex and involves different bacterial factors including exotoxins, capsular polysaccharides, and lipopolysaccharides. Beyond these, there are additional APP virulence factors that are down- or up-regulated during infection and regulatory small RNAs (sRNAs) can regulate them. Recently, our group identified 23 sRNAs in App, whose transcription was validated by RNA-Seq. In this work we selected six sRNA genes and produced deletion mutants from wild-type parental (WT_ΔsRNA) and its isogenic mutant for the sRNA chaperone, Hfq (Δhfq_ΔsRNA). In addition, we propose a robust in silico analysis of the structure, conservation and interaction of APP sRNAs with their targets. The phenotypes of these mutants were compared to that of the parental strain under different conditions, i.e. growth curve, growth under different stress conditions, determination of minimum inhibitory concentration against antibiotics, adhesion on biotic and abiotic surfaces, hemolytic activity and virulence in the alternative infection model Galleria mellonella. The sRNA strains exhibited a phenotype difference from the parental strains under at least one condition, revealing that App sRNAs are involved in virulence regulation. Notably, simple deletion mutants showed a gain of function phenotype, which has not yet been described for any other sRNA gene deletion mutant in the Pasteurellaceae family. In addition, a mutant showed a strong attenuation of virulence, being a promising live-attenuated vaccine candidate against App. In silico analysis revealed the presence of App sRNAs in six genera of the Pasteurellaceae family. Several putative targets were found and categorized in different functions, evidencing a complex gene regulation network by these sRNAs. From these results, we propose a model of action via Arrc14 in which it acts as a negative regulator in amino and nucleotide sugars metabolism. Our results allow us to conclude that the sRNAs studied here interfere differently in virulence regulation of Actinobacillus pleuropneumoniae serotype 8. We also conclude, based on the data obtained for RNA01, that the WT_Δrna01 line has potential to be used in the development of a live attenuated vaccine to prevent and control Actinobacillus pleuropneumoniae infections

Formigas (Hymenoptera: Formicidae) como vetores de bactérias em dois hospitais do município de Divinópolis, Estado de Minas Gerais

A presença de formigas (Hymenoptera: Formicidae) em ambientes hospitalares pode constituir um problema de saúde pública, especialmente por serem vetores mecânicos de organismos patogênicos. O trabalho teve como objetivo realizar o levantamento de formigas e analisar a presença de bactérias a elas associadas em dois hospitais regionais de médio porte da cidade de Divinópolis, MG. As coletas foram realizadas mensalmente, durante um período de seis meses. Foram coletadas formigas Pheidole sp1 e sp2, Linepithema humile, Wasmannia auropunctata, Camponotus sp1 e sp2, Odontomachus sp, Solenopsis sp, Acromyrmex sp e Tapinoma melenocephalum. Observou-se que estas transportavam mecanicamente Pseudomonas aeruginosa, Enterococcus, Streptococcus, Staphylococcus patogênico e não patogênico e Escherichia coli. Tais resultados evidenciam a propensão à ocorrência de infecções hospitalares nesses locais pela transmissão mecânica de agentes patogênicos por formigas

Levantamento de espécies de Odonata associadas à tanques de piscicultura e efeito de Bacillus thuringiensis var. israelensis sobre ninfas de Pantala flavescens (Fabricius, 1798) - DOI: 10.4025/actascibiolsci.v26i1.1655

Several psiculture stations that deal with fingerlings or ornamental fishes rearing have presented some problems with larvae preying, post-larvae and fingerlings by Odonata Order insect nymphs. Thus, the aim of this work was to survey the Odonata species present in fish-raising tanks in two towns of the Midwest region of Minas Gerais, and also to evaluate the effect of Bacillus thuringiensis var. Barjac israelensis on Pantala flavescens nymphs (Fabricius, 1798) (Odonata: Libellulidae). Fortnightly collections were performed over a three month period. The adult insects were captured with entomological nets and the nymphs with fine mesh sieves, coupled to wooden handles. The captured nymphs were taken to the laboratory where they were individualized in 2L plastic foam boxes and sealed in its upper extremity with tulle. Soon after the adults emergence, they were killed, packed into envelopes and sent to be identified. The laboratory experiments were conducted in an acclimatized room at 25 ± 2°C, RH of 70 ± 10% and 12-hour photophase. Second instar P. flavescens nymphs were packed individually into plastic foam boxes containing 500mL of chlorine free water each one. When they were in the third, fifth and seventh instars, they were treated with B. thuringiensis var. israelensis through the microbial product Vectobac® in granulate formulation. The product was directly applied to the rearing container water at the concentration denoted for the control of culicidae larvae, two superior concentrations, two inferior and also another one, where the product was not applied. The evaluations were done at 24, 48 and 72 hours after the product application, recording the dead insects number in each treatment. In both experiments, the nymphs were fed with Culicidae larvae and new-born fishes of the species Poecilia cf. vivipara Schneider, 1801. The following species were identified: Ischnura fluvialis Selys, 1876; Aphylla theodorina (Navas, 1933); Brachymesia furcata (Hagen, 1861); Erythrodiplax fusca (Rambur, 1842); Miathyria marcella (Selys, 1857); Micrathyria almeidai Santos, 1945; Micrathyria hesperis Ris, 1911; Orthemis discolor (Burmeister, 1839); Perithemis mooma Kirby, 1889 and P. flavescens. There were no significant microbial product effects on the studied speciesVárias estações de piscicultura que trabalham com a produção de alevinos ou peixes ornamentais têm apresentado problemas relacionados à predação de larvas, pós-larvas e alevinos por ninfas de insetos da Ordem Odonata. Dessa forma, objetivou-se fazer um levantamento de espécies de odonatos presentes em tanques de criação de peixes em dois municípios da região centro-oeste de Minas Gerais, assim como avaliar o efeito de Bacillus thuringiensis var. israelensis de Barjac sobre ninfas de Pantala flavescens (Fabricius, 1798) (Odonata: Libellulidae). Durante um período de três meses, foram realizadas coletas quinzenais, sendo os insetos adultos capturados com o auxílio de redes entomológicas e as ninfas, através de peneiras de malha fina, acopladas a cabos de madeira. As ninfas capturadas foram levadas para o laboratório onde foram individualizadas em caixas de isopor com capacidade para 2L, vedadas em sua extremidade superior com filó. Logo após a emergência dos adultos, esses foram mortos, acondicionados em envelopes e enviados para serem identificados. Os experimentos laboratoriais foram conduzidos em sala climatizada à 25 ± 2°C, UR de 70 ± 10% e fotofase de 12 horas. Ninfas de segundo ínstar de P. flavescens foram individualmente acondicionadas em caixas de isopor contendo cada uma 500mL de água livre de cloro e, estando essas no terceiro, quinto e sétimo ínstares, foram tratadas com B. thuringiensis var. israelensis, através do produto microbiano Vectobac®, em formulação granulada. O produto foi aplicado diretamente na água do recipiente de criação, em concentração indicada para o controle de larvas de culicídeos, duas concentrações superiores e duas inferiores a essa, além da testemunha, na qual não se aplicou o produto. As avaliações foram realizadas a 24, 48 e 72 horas após a aplicação do produto, registrando-se o número de insetos mortos em cada tratamento. Em ambos os experimentos, as ninfas foram alimentadas com larvas de mosquito da Família Culicidae e peixes recém-nascidos da espécie Poecilia cf. vivípara (Schneider, 1801). Foram identificadas as seguintes espécies: Ischnura fluvialis Selys, 1876; Aphylla theodorina (Navas, 1933); Brachymesia furcata (Hagen, 1861); Erythrodiplax fusca (Rambur, 1842); Miathyria marcella (Selys, 1857); Micrathyria almeidai Santos, 1945; Micrathyria hesperis Ris, 1911; Orthemis discolor (Burmeister, 1839); Perithemis mooma Kirby, 1889 e P. flavescens. Não houve efeito significativo do produto microbiano sobre a espécie estudad

Data_Sheet_1_Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.docx

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.</p

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data