RESUMO
A 48-year-old man presented with a sustained fever. Abdominal computed tomography revealed multilocular liver abscesses. He underwent percutaneous needle aspiration, yielding straw-colored pus. Gram staining revealed Gram-negative coccobacilli. The organism grew only on chocolate II agar in a 7% carbon dioxide atmosphere. Identification of Aggregatibacter aphrophilus was confirmed using mass spectrometry and 16S rRNA gene sequencing. He was successfully treated with antibiotics. Liver abscess caused by A. aphrophilus is extremely rare. We herein report the first such case in Japan. Even fastidious organisms, such as A. aphrophilus, should be correctly identified using mass spectrometry or 16S rRNA gene sequencing for adequate treatment.
Assuntos
Aggregatibacter aphrophilus/genética , Aggregatibacter aphrophilus/patogenicidade , Antibacterianos/uso terapêutico , Abscesso Hepático/tratamento farmacológico , Abscesso Hepático/etiologia , Infecções por Pasteurellaceae/tratamento farmacológico , Infecções por Pasteurellaceae/etiologia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S , Resultado do TratamentoRESUMO
OBJECTIVE: To test the hypothesis that virulence genes of Aggregatibacter actinomycetemcomitans can be expressed and confer fitness advantages in the closely related Aggregatibacter aphrophilus. DESIGN: Clinical isolates of A. aphrophilus were screened for natural competence with marked genomic DNA from A. actinomycetemcomitans and A. aphrophilus. The gene katA of A. actinomycetemcomitans D7S-1 and its flanking regions were constructed and inserted into a comparable locus in the genome of a naturally competent A. aphrophilus strain by a markerless protocol via natural transformation. Mutants of A. actinomycetemcomitans with or without katA were also constructed by a similar protocol. Discs soaked with either 0.03% hydrogen peroxide or broth culture of Streptococcus gordonii Challis were placed on the agar with cultures of A. actinomycetemcomitans or A. aphrophilus. The size of the growth inhibition zone associated with the disc was measured after 2-day culture. RESULTS: Five of the 13A. aphrophilus strains exhibited a transformation frequency of 10-6 or higher. The intra- and inter-species transformation frequencies were comparable. The inhibition zones for katA-negative strains of A. actinomycetemcomitans or A. aphrophilus were 3- to 7-fold larger than those associated with katA-positive strains (p<0.05). CONCLUSIONS: There was no apparent species barrier for the transfer and expression of A. actinomycetemcomitans katA in A. aphrophilus. The inserted A. actinomycetemcomitans-specific katA gene in A. aphrophilus strain NJ8700 conferred resistance to inhibition by hydrogen peroxide or S. gordonii. The potential to swap genes between these two closely related oral species may be an alternative approach for investigating the virulence determinants of A. actinomycetemcomitans.
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter aphrophilus/genética , Catalase/genética , Aggregatibacter actinomycetemcomitans/enzimologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Aggregatibacter aphrophilus/enzimologia , Aggregatibacter aphrophilus/patogenicidade , Técnicas de Transferência de Genes , Ilhas Genômicas , Genômica , Peróxido de Hidrogênio/farmacologia , Mutagênese Insercional , Reação em Cadeia da Polimerase , VirulênciaAssuntos
Aggregatibacter aphrophilus , Endocardite Bacteriana/diagnóstico , Endocardite Bacteriana/microbiologia , Anuloplastia da Valva Mitral , Valva Mitral/microbiologia , Infecções por Pasteurellaceae/diagnóstico , Infecções por Pasteurellaceae/microbiologia , Aggregatibacter aphrophilus/genética , Aggregatibacter aphrophilus/isolamento & purificação , DNA Bacteriano/genética , Endocardite Bacteriana/patologia , Endocardite Bacteriana/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Valva Mitral/patologia , Valva Mitral/cirurgia , Infecções por Pasteurellaceae/patologia , Infecções por Pasteurellaceae/cirurgia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Taiwan , Resultado do TratamentoRESUMO
Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e' (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e' isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved distinct adaptation strategies to the human oral cavity.
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter aphrophilus/genética , Evolução Molecular , Toxinas Bacterianas/genética , Composição de Bases/genética , DNA Bacteriano/genética , DNA Concatenado/genética , Transferência Genética Horizontal/genética , Genes Bacterianos/genética , Heterogeneidade Genética , Especiação Genética , Variação Genética/genética , Genoma Bacteriano/genética , Ilhas Genômicas/genética , Humanos , Boca/microbiologia , Filogenia , Subunidades Proteicas/genética , Análise de Sequência de DNA , SorogrupoRESUMO
UNLABELLED: Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes. The nontypeable Haemophilus influenzae glycosyltransferase HMW1C mediates unconventional N-linked glycosylation of the adhesive protein HMW1, which is encoded in a two-partner secretion system gene cluster that also encodes HMW1C. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. In the present study, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C that are not encoded near a gene encoding an obvious acceptor protein. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild-type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function. IMPORTANCE: This work emphasizes the importance of glycosylation for proper function of bacterial proteins. Here we show that the Kingella kingae Knh and the Aggregatibacter aphrophilus EmaA trimeric autotransporter proteins are N-glycosylated by novel homologues of the Haemophilus influenzae HMW1C glycosyltransferase, highlighting the first examples of trimeric autotransporters that are modified by HMW1C-like enzymes. In the absence of glycosylation, Knh and EmaA lack adhesive activity. This work has relevance to our understanding of bacterial pathogenicity and expression of potential vaccine antigens.
