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1.
Int J Mol Sci ; 25(18)2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39337285

RESUMO

Infection with Campylobacter jejuni is the major cause of human gastroenteritis in the United States and Europe, leading to debilitating autoimmune sequelae in many cases. While considerable progress has been made in detailing the infectious cycle of C. jejuni, a full understanding of the molecular mechanisms responsible for virulence remains to be elucidated. Here, we apply a novel approach by modulating protein expression on the pathogen's ribosomes by inactivating a highly conserved rRNA methyltransferase. Loss of the RsmA methyltransferase results in a more motile strain with greater adhesive and cell-invasive properties. These phenotypical effects correlate with enhanced expression of specific proteins related to flagellar formation and function, together with enzymes involved in cell wall/membrane and amino acid synthesis. Despite the enhancement of certain virulent traits, the null strain grows poorly on minimal media and is rapidly out-competed by the wild-type strain. Complementation with an active copy of the rsmA gene rescues most of the traits changed in the mutant. However, the complemented strain overexpresses rsmA and displays new flaws, including loss of the spiral cell shape, which is distinctive for C. jejuni. Proteins linked with altered virulence and morphology are identified here by mass spectrometry proteomic analyses of the strains.


Assuntos
Proteínas de Bactérias , Campylobacter jejuni , Metiltransferases , Ribossomos , Campylobacter jejuni/patogenicidade , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Metilação , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Campylobacter/microbiologia , Proteômica/métodos
2.
Antibiotics (Basel) ; 11(7)2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35884218

RESUMO

Campylobacteriosis seems to be a growing problem worldwide. Apart from the most common sources of numerous Campylobacter species, such as poultry and other farm animals, dogs may be an underrated reservoir of this pathogen. Our goal was to establish the frequency of occurrence, antimicrobial resistance, and detection of chosen virulence factor genes in genomes of canine Campylobacter isolates. Campylobacter isolates frequency in dogs from shelters, and private origin was 13%. All of the tested virulence factor genes were found in 28 of 31 isolates. We determined high resistance levels to the ciprofloxacin and ampicillin and moderate tetracycline resistance. For C. jejuni shelter isolates, genetic diversity was also determined using PFGE. Our results indicate that dogs may be the reservoir of potentially diverse, potentially virulent, and antimicrobial-resistant Campylobacter strains.

3.
Pathogens ; 11(5)2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35631083

RESUMO

In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter's and Gatsons' index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen.

4.
Front Cell Infect Microbiol ; 11: 803730, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35096652

RESUMO

Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribosomes to disrupt the composition of its proteome, and how this in turn provides a means of identifying factors that are essential for infection and pathogenesis. Specifically, inactivation of the C. jejuni Cj0588/TlyA methyltransferase prevents methylation of nucleotide C1920 in the 23S rRNA of its ribosomes and reduces the pathogen's ability to form biofilms, to attach, invade and survive in host cells, and to provoke the innate immune response. Mass spectrometric analyses of C. jejuni TlyA-minus strains revealed an array of subtle changes in the proteome composition. These included reduced amounts of the cytolethal distending toxin (CdtC) and the MlaEFD proteins connected with outer membrane vesicle (OMV) production. Inactivation of the cdtC and mlaEFD genes confirmed the importance of their encoded proteins in establishing infection. Collectively, the data identify a subset of genes required for the onset of human campylobacteriosis, and serve as a proof of principle for use of this approach in detecting proteins involved in bacterial pathogenesis.


Assuntos
Infecções por Campylobacter , Campylobacter jejuni , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/metabolismo , Humanos , Metilação , Ribossomos/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
5.
Int J Mol Sci ; 21(12)2020 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545831

RESUMO

Trueperella pyogenes is an important opportunistic animal pathogen. Different antimicrobials, including aminoglycosides, are used to treat T. pyogenes infections. The aim of the present study was to evaluate aminoglycoside susceptibility and to detect aminoglycoside resistance determinants in 86 T. pyogenes isolates of different origin. Minimum inhibitory concentration of gentamicin, streptomycin, and kanamycin was determined using a standard broth microdilution method. Genetic elements associated with aminoglycoside resistance were investigated by PCR and DNA sequencing. All studied isolates were susceptible to gentamicin, but 32.6% and 11.6% of them were classified as resistant to streptomycin and kanamycin, respectively. A total of 30 (34.9%) isolates contained class 1 integrons. Class 1 integron gene cassettes carrying aminoglycoside resistance genes, aadA11 and aadA9, were found in seven and two isolates, respectively. Additionally, the aadA9 gene found in six isolates was not associated with mobile genetic elements. Moreover, other, not carried by gene cassettes, aminoglycoside resistance genes, strA-strB and aph(3')-IIIa, were also detected. Most importantly, this is the first description of all reported genes in T. pyogenes. Nevertheless, the relevance of the resistance phenotype to genotype was not perfectly matched in 14 isolates. Therefore, further investigations are needed to fully explain aminoglycoside resistance mechanisms in T. pyogenes.


Assuntos
Actinomycetaceae/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Integrons , Actinomycetaceae/genética , Actinomycetaceae/isolamento & purificação , Animais , Animais Selvagens/microbiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Gentamicinas/farmacologia , Canamicina/farmacologia , Gado/microbiologia , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , Estreptomicina/farmacologia
6.
Cell Microbiol ; 22(7): e13199, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32134554

RESUMO

Campylobacter jejuni is a bacterial pathogen that is generally acquired as a zoonotic infection from poultry and animals. Adhesion of C. jejuni to human colorectal epithelial cells is weakened after loss of its cj0588 gene. The Cj0588 protein belongs to the type I group of TlyA (TlyAI ) enzymes, which 2'-O-methylate nucleotide C1920 in 23S rRNA. Slightly longer TlyAII versions of the methyltransferase are found in actinobacterial species including Mycobacterium tuberculosis, and methylate not only C1920 but also nucleotide C1409 in 16S rRNA. Loss of TlyA function attenuates virulence of both M. tuberculosis and C. jejuni. We show here that the traits impaired in C. jejuni null strains can be rescued by complementation not only with the original cj0588 (tlyA I ) but also with a mycobacterial tlyA II gene. There are, however, significant differences in the recombinant phenotypes. While cj0588 restores motility, biofilm formation, adhesion to and invasion of human epithelial cells and stimulation of IL-8 production in a C. jejuni null strain, several of these properties are further enhanced by the mycobacterial tlyA II gene, in some cases to twice the original wild-type level. These findings strongly suggest that subtle changes in rRNA modification patterns can affect protein synthesis in a manner that has serious consequences for bacterial pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Biofilmes , Células CACO-2 , Campylobacter jejuni/genética , Capreomicina , Células Epiteliais , Regulação Bacteriana da Expressão Gênica , Genes de RNAr/genética , Humanos , Macrófagos , Metilação , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Células RAW 264.7 , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Virulência , Fatores de Virulência/genética
7.
Vet Microbiol ; 216: 25-30, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29519521

RESUMO

A total of 43 Campylobacter isolates from poultry, cattle and pigs were investigated for their ability to form biofilm. The studied strains were also screened for motility, adhesion and invasion of Caco-2 cells as well as extracellular DNase activity. The relation between biofilm formation and selected phenotypes was examined. Biofilm formation by the tested strains was found as irrespective from their motility and not associated with colonization abilities of human Caco-2 cells. Results of our study show that Campylobacter isolates from various animal sources are able to form biofilm and invade human Caco-2 cells in vitro.


Assuntos
Infecções por Campylobacter/veterinária , Campylobacter/genética , Campylobacter/isolamento & purificação , Fenótipo , Animais , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Células CACO-2 , Campylobacter/classificação , Campylobacter/patogenicidade , Infecções por Campylobacter/microbiologia , Bovinos , Galinhas , Humanos , Reação em Cadeia da Polimerase , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Suínos , Fatores de Virulência
8.
Artigo em Inglês | MEDLINE | ID: mdl-29404277

RESUMO

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2'-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2'-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2'-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2'-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.


Assuntos
Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/enzimologia , Campylobacter jejuni/fisiologia , Locomoção , RNA Ribossômico 23S/metabolismo , tRNA Metiltransferases/metabolismo , Substituição de Aminoácidos , Campylobacter jejuni/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Metilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Virulência/metabolismo , tRNA Metiltransferases/genética
9.
Biochem Biophys Res Commun ; 448(3): 298-302, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24796671

RESUMO

TlyA proteins belong to 2'-O-methyltransferases. Methylation is a common posttranscriptional RNA modification. The Campylobacter jejuni Cj0588 protein belongs to the TlyA(I) protein family and is a rRNA methyltransferase. Methylation of ribosomal RNA catalyzed by Cj0588 appears to have an impact on the biology of the cell. Presence of the cj0588 gene in bacteria appears to be important for ribosome stability and virulence properties. Absence of the Cj0588 protein causes accumulation of the 50S ribosomal subunits, reduction in the amount of functional 70S ribosomes and confers increase resistance to capreomycin.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Metiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Capreomicina/farmacologia , Farmacorresistência Bacteriana , Genes Bacterianos , Metilação , Metiltransferases/química , Metiltransferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Polirribossomos/metabolismo , Conformação Proteica , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Homologia de Sequência de Aminoácidos , Virulência
10.
Curr Microbiol ; 56(6): 592-6, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18389311

RESUMO

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with amino acid sequences predicted to be homologous to other bacterial hemolysins. The Cj0183 protein exhibits homology to Brachyspira hyodysenteriae TlyC protein, whereas the cj0588 gene product is homologous to TlyA proteins Brachyspira hyodysenteriae, Helicobacter pylori, and Mycobacterium tuberculosis, which play a crucial role in bacterial virulence. The aim of our work was to examine the hemolytic activity and determine the role of cj0183- and cj0588-encoded proteins on the adherence of chosen C. jejuni strains to the Caco-2 cell line by constructing deletion mutants in the mentioned genes. We found out there is no difference in hemolytic activity between both mutants in gene cj0183 and cj0588 and the wild strains. However, Cj0588 protein but not Cj0183 is involved in adherence to the Caco-2 cells.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/fisiologia , Proteínas Hemolisinas/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Células CACO-2 , Campylobacter jejuni/genética , Proteínas Hemolisinas/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência
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