Evidence of illegitimate recombination between two pasteurellaceae plasmids resulting in a novel multi-resistance replicon, pM3362MDR, in Actinobacillus pleuropneumoniae

Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries bla ROB−1 , sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required

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

Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis

Resistoma associado ao mobiloma na família Pasteurellaceae e caracterização de RNAs pequenos transportados por vesículas extracelulares produzidas por Actinobacillus pleuropneumoniae

The Pasteurellaceae family comprises several species of Gram-negative, commensal or pathogenic bacteria of human and animal hosts. Some members of the Pasteurellaceae family are responsible for causing diseases of economic impact in food-producing animals, such as swine. In this sector, significant expenses are associated with the use of antimicrobials for the treatment of infections caused by species of the Pasteurellaceae family, which are often persistent and multidrug- resistant. Actinobacillus pleuropneumoniae (App) is a member of the Pasteurellaceae family and causes porcine pleuropneumonia, responsible for important economic losses in pig farming worldwide. A. pleuropneumoniae is a primary pathogen and has several virulence factors such as: capsule, LPS, iron siderophores and Apx exotoxins. In addition, they are capable of producing extracellular vesicles (BEVs – Bacterial Extracellular Vesicles), which represents another associated virulence factor, given the involvement in the dissemination of mobile genetic elements (MGEs) and antimicrobial resistance genes (AMR) and innovative abilities that leverage the virulence of App. Thus, the objectives of this work were: i. to evaluate the representativeness of MGEs associated with AMR in members of the Pasteurellaceae family, including App and ii. characterize the BEVs produced by App and their nucleic acid content, focusing on RNA and virulence. The investigation of the resistome associated with the mobilome in Pasteurellaceae was carried out from in silico analysis using complete genomes available in databases in order to identify and characterize MGEs with emphasis on the presence of resistance genes associated with these elements. Through these analyzes, 20 novel integrative and conjugative elements (ICEs) and 23 novel prophages were found, in addition to expanding the characterization of elements previously described for the family. From the results obtained, it was found that MGEs are responsible for carrying most of the resistance genes identified in bacteria of the Pasteurellaceae family, and can be disseminated by different mechanisms of horizontal gene transfer (HGT), such as vesiduction (via BEVs). Thus, in the second part of the work, the BEVs of App were obtained, characterized and the DNA and RNA content investigated under different conditions. Vesicles were investigated for size, protein and lipid content, including LPS type, and nucleic acid content. The characterization of the BEVs revealed a protein profile similar to that found in the outer membrane of App, highlighting the lipid profile, not investigated so far for this specie. The results revealed that the condition of anaerobic growth affects several aspects of BEV production, such as quantity and relative size. The BEVs carry as nucleic acid content, DNA and RNA, the latter being the most abundant and therefore characterized in this work. Transcriptome analysis of the BEVs produced by App revealed that these vesicles carry several types of RNAs, such as tRNAs and small (sRNAs), the latter of which have already been described for App. In this work, 13 new sRNAs for App were described, 12 of which are present in BEVs. Some sRNAs found in BEVs are enriched in vesicles in relation to cells and are present in the intravesicular portion, protected from the action of nucleases. Among the sRNAs found in the BEVs of App, Rna01, previously identified by our group, was selected for further analysis. Target analysis of this sRNA revealed that it is associated with the regulation of several genes encoding membrane proteins, mainly in response to stress conditions, which was confirmed through quantitative expression analyses. Through analyzes carried out with mutant strains for Rna01, it was observed that the production of BEVs was also affected, revealing the participation of this sRNA in this process in App. This work provides a complete characterization of MGEs and AMR in the Pasteurellaceae family and is also the first work to perform a complete characterization of BEVs produced by App under different conditions, in addition to confirming the presence and functionality of sRNAs in BEVs produced by App. Thus, several unpublished information and guidelines for actions aimed at controlling porcine pleuropneumonia. Keywords: Pasteurellaceae. Actinobacillus pleuropneumoniae. Pleuropneumonia. Mobile genetic elements. Antimicrobial resistance. Bacterial extracellular vesicles. Nucleic acids. small RNAs.A família Pasteurellaceae compreende diversas espécies de bactérias Gram- negativas, comensais ou patogênicas de hospedeiros humanos e animais. Alguns membros da família Pasteurellaceae são responsáveis por causar doenças de impacto econômico em animais de produção, como suínos. Neste setor, gasto expressivos estão associados ao uso de antimicrobianos para o tratamento de infecções causadas por espécies da família Pasteurellaceae, muitas vezes persistentes e multirresistentes a drogas. Actinobacillus pleuropneumoniae (App) é um membro da família Pasteurellaceae e causa a pleuropneumonia suína, responsável por prejuízos econômicos importantes na suinocultura em escala global. A. pleuropneumoniae é um patógeno primário e apresenta diversos fatores de virulência como: cápsula, LPS, sideróforos para ferro e exotoxinas Apx. Além disso, são capazes de produzir vesículas extracelulares (BEVs – Bacterial Extracellular Vesicles), o que representa mais um fator de virulência associado, visto o envolvimento na disseminação de elementos genéticos móveis (EGMs) e genes de resistência a antimicrobianos (AMR) e habilidades inovadoras que potencializam a virulência de App. Assim, os objetivos deste trabalho foram: i. avaliar a representatividade de EGMs associados com AMR em membros da família Pasteurellaceae, incluindo App e ii. caracterizar as BEVs produzidas por App e seu conteúdo de ácidos nucleicos, com foco em RNA e virulência. A investigação do resistoma associado ao mobiloma em Pasteurellaceae foi realizada a partir de análises in silico utilizando genomas completos disponíveis em bancos de dados com o intuito de identificar e caracterizar EGMs com ênfase na presença de genes de resistência associados a esses elementos. Através dessas análises foram encontrados 20 novos elementos integrativos e conjugativos (ICEs) e 23 novos profagos, além de ampliação da caracterização de elementos previamente descritos para a família. A partir dos resultados obtidos foi constatado que EGMs são responsáveis por carrear a maioria dos genes de resistência identificados nas bactérias da família Pasteurellaceae, podendo serem disseminados por diferentes mecanismos de transferência horizontal de genes (THG), como a vesidução (via BEVs). Assim, na segunda parte do trabalho, as BEVs de App foram obtidas, caracterizadas e o conteúdo de DNA e RNA investigado em diferentes de condições. As vesículas foram investigadas quanto ao tamanho, conteúdo proteico e lipídico, incluindo tipo de LPS, e conteúdo de ácidos nucleicos. A caracterização das BEVs revelou um perfil de proteínas semelhante ao encontrado na membrana externa de App, destacando o perfil dos lipídeos, não abordado até então para essa espécie. Os resultados revelaram que a condição de crescimento em anaerobiose afeta diversos aspectos da produção das BEVs, como quantidade e tamanho relativo. As BEVs apresentam como conteúdo de ácidos nucleicos, DNA e RNA, sendo o último mais abundante e por isso caracterizado neste trabalho. Análises de transcriptoma das BEVs produzidas por App revelou que essas vesículas transportam diversos tipos de RNAs, como tRNAs e small RNAs (sRNAs), sendo os últimos já descritos para App. Neste trabalho foram descritos 13 novos sRNAs para App, sendo 12 deles presentes em BEVs. Alguns sRNAs encontrados nas BEVs estão enriquecidos nas vesículas em relação às células e estão presentes na porção intravesicular, protegido da ação de nucleases. Dentre os sRNAs encontrados nas BEVs de App, o Rna01, previamente identificado por nosso grupo, foi selecionado para análises posteriores. Análises de alvos desse sRNA revelaram que ele está associado com a regulação de diversos genes que codificam proteínas de membrana, principalmente em resposta a condições de estresse, que foi confirmado através de análises de expressão quantitativa. Através de análises desenvolvidas com linhagens mutantes para Rna01, foi observado que a produção de BEVs também foi afetada, revelando a participação desse sRNA nesse processo em App. Esse trabalho traz uma completa caracterização de EGMs e AMR na família Pasteurellaceae e é também o primeiro trabalho a realizar uma caracterização completa das BEVs produzidas por App em diferentes condições, além de confirmar a presença e a funcionalidade de sRNAs em BEVs produzidas por App. Assim, várias informações inéditas e norteadoras de ações que visam o controle da pleuropneumonia suína. Palavras-chave: Pasteurellaceae. Actinobacillus pleuropneumoniae. Pleuropneumonia. Elementos genéticos móveis. Resistência antimicrobiana. Vesículas extracelulares bacterianas. Ácidos nucleicos. RNAs pequenos.Conselho Nacional de Desenvolvimento Científico e Tecnológic

p518, a small floR plasmid from a south american isolate of Actinobacillus pleuropneumoniae

A small (3.9 kb) plasmid (p518), conferring resistance to florfenicol (MIC >8 μg/mL) and chloramphenicol (MIC >8 μg/mL) was isolated from an Actinobacillus pleuropneumoniae clinical isolate from Southeastern Brazil. To date, this is the smallest florfenicol resistance plasmid isolated from a member of the Pasteurellaceae. The complete nucleotide of this plasmid revealed a unique gene arrangement compared to previously reported florfenicol resistance plasmids found in other members of the Pasteurellaceae. In addition to the floR gene and a lysR gene, common to various florfenicol resistance plasmids, p518 also encodes strA and a partial strB sequence. An origin of replication (oriV) similar to that in the broad host range plasmid, pLS88, was identified in p518, and transformation into Escherichia coli MFDpir confirmed the ability to replicate in other species. Mobilisation genes appear to have been lost, with only a partial mobC sequence remaining, and attempts to transfer p518 from a conjugal donor strain (E. coli MFDpir) were not successful, suggesting this plasmid is not mobilisable. Similarly, attempts to transfer p518 into a competent A. pleuropneumoniae strain, MIDG2331, by natural transformation were also not successful. These results suggest that p518 may be only transferred by vertical descent

Comparative Genomics of Actinobacillus pleuropneumoniae Serotype 8 Reveals the Importance of Prophages in the Genetic Variability of the Species

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia. Currently, there are 18 different serotypes; the serotype 8 is the most widely distributed in the United States, Canada, United Kingdom, and southeastern Brazil. In this study, genomes of seven A. pleuropneumoniae serotype 8 clinical isolates were compared to the other genomes of twelve serotypes. The analyses of serotype 8 genomes resulted in a set of 2352 protein-coding sequences. Of these sequences, 76.6% are present in all serotypes, 18.5% are shared with some serotypes, and 4.9% were differential. This differential portion was characterized as a series of hypothetical and regulatory protein sequences: mobile element sequence. Synteny analysis demonstrated possible events of gene recombination and acquisition by horizontal gene transfer (HGT) in this species. A total of 30 sequences related to prophages were identified in the genomes. These sequences represented 0.3 to 3.5% of the genome of the strains analyzed, and 16 of them contained complete prophages. Similarity analysis between complete prophage sequences evidenced a possible HGT with species belonging to the family Pasteurellaceae. Thus, mobile genetic elements, such as prophages, are important components of the differential portion of the A. pleuropneumoniae genome and demonstrate a central role in the evolution of the species. This study represents the first study done to understand the genome of A. pleuropneumoniae serotype 8

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