Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Item Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection(Microbiology, 2014-11-14) Pereira, Monalessa Fábia; Rossi, Ciro César; Queiroz, Marisa Vieira de; Martins, Gustavo Ferreira; Isaac, Clement; Bossé, Janine T.; Li, Yanwen; Wren, Brendan W.; Terra, Vanessa Sofia; Cuccui, Jon; Langford, Paul R.; Bazzolli, Denise Mara SoaresActinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 6C but had different LD 50 values, ranging from 10 4 to 10 7 c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.Item Draft Genome Sequences of Six Actinobacillus pleuropneumoniae Serotype 8 Brazilian Clinical Isolates: Insight into New Applications(Genome Announcements, 2015-03-05) Pereira, Monalessa Fábia; Rossi, Ciro César; Carvalho, Fabíola Marques de; Almeida, Luiz Gonzaga Paula de; Souza, Rangel Celso; Vasconcelos, Ana Tereza Ribeiro de; Bazzolli, Denise Mara SoaresActinobacillus pleuropneumoniae is the causative agent of swine pleuropneumonia, a highly contagious disease associated with pigs of all ages that results in severe economic losses to the industry. Here, we report for the first time six genome sequences of A. pleuropneumoniae clinical isolates of serotype 8, found worldwide.Item Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: a new insight into an old approach(Genetics and Molecular Biology, 2013-01-29) Rossi, Ciro César; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de; Bazzolli, Denise Mara SoaresThe OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.Item The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation(Plos One, 2014-11-19) Bossé, Janine T.; Soares-Bazzolli, Denise M.; Li, Yanwen; Wren, Brendan W.; Tucker, Alexander W.; Maskell, Duncan J.; Rycroft, Andrew N.; Langford, Paul R.We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.Item Complete genome sequence of MIDG2331, a genetically tractable serovar 8 clinical isolate of Actinobacillus pleuropneumoniae(Genome Announcements, 2016-01-28) Crespo, Roberto Fernandez; Coupland, Paul; Bossé, Janine T.; Chaudhuri, Roy R.; Chaudhuri, Roy R.; Bazzolli, Denise M.; Leanse, Leon G.; Holden, Matthew T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.We report here the complete annotated genome sequence of a clinical serovar 8 isolate Actinobacillus pleuropneumoniae MIDG2331. Unlike the serovar 8 reference strain 405, MIDG2331 is amenable to genetic manipulation via natural transformation as well as conjugation, making it ideal for studies of gene function.