Host Chromatin Regulators Required for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activity in Saccharomyces cerevisiae Model.

Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, AaCdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

The impact of fixed orthodontic appliances on oral microbiome dynamics in Japanese patients.

Fixed orthodontic appliances are common and effective tools to treat malocclusion. Adverse effects of these appliances, such as dental caries and periodontitis, may be associated with alteration of the microbiome. This study investigated the impact of these appliances on the dynamics of the oral microbiome. Seventy-one patients were selected. Supragingival plaque samples were collected before placement (T0) and six months after placement (T1). Saliva samples were collected at T0 and T1, and then when appliance removal (T2). Microbial DNA was analyzed by 16S rRNA meta-sequencing. The diversity analysis indicated dynamic changes in the structure of the oral microbiome. Taxonomic analysis at phylum level showed a significant increase in Bacteroidetes and Saccharibacteria (formally TM7) and decrease in Proteobacteria and Actinobacteria over time, in both plaque and saliva. Genus level analysis of relative abundance indicated a significant increase in anaerobic and facultative anaerobes in both plaque and saliva. Fixed orthodontic appliances induced measurable changes in the oral microbiome. This was characterized by an increase in relative abundance of obligate anaerobes, including periodontal pathogens. It can be concluded that this dysbiosis induced by fixed orthodontic appliances is likely to represent a transitional stage in the shift in microbiome from healthy to periodontitis.

Oral colonisation by antimicrobial-resistant Gram-negative bacteria among long-term care facility residents: prevalence, risk factors, and molecular epidemiology.

BACKGROUND: For residents of long-term care facilities (LTCFs), antimicrobial-resistant bacteria (ARB) are a risk factor, yet their oral colonisation, potentially leading to aspiration pneumonia, remains unclear. This study was undertaken to survey the prevalence, phenotypic characteristics, and molecular epidemiology of antimicrobial-resistant Gram-negative bacteria in the oral cavity of LTCF residents, and to analyse the risk factors for such carriers. METHODS: This study involved 98 residents of a LTCF in Hiroshima City, Japan, aged between 55 and 101 years. Oropharyngeal swabs were collected and plated on screening media for ESBL-producing and carbapenem-resistant bacteria; isolates were identified and tested for antibiotic susceptibility; biofilm formation was tested in vitro; identification of epidemic clones were pre-determined by PCR; resistance genes, sequence types, and whole-genome comparison of strains were conducted using draft genome sequences. Demographic data and clinical characterisations were collected and risk factors analysed. RESULTS: Fifty-four strains from 38% of the residents grew on screening media and comprised predominantly of Acinetobacter spp. (35%), Enterobacteriaceae spp. (22%), and Pseudomonas spp. (19%). All Escherichia coli isolates carried CTX-M-9 group and belonged to the phylogroup B2, O25:H4 ST131 fimH30 lineage. Six Acinetobacter baumannii isolates presented identical molecular characteristics and revealed more biofilm production than the others, strongly suggesting their clonal lineage. One Acinetobacter ursingii isolate displayed extensive resistance to various ß-lactams due to multiple acquired resistance genes. One Pseudomonas aeruginosa isolate showed exceptional resistance to all ß-lactams including carbapenems, aminoglycosides, and a new quinolone, showing a multidrug-resistant Pseudomonas aeruginosa (MDRP) phenotype and remarkable biofilm formation. Genome sequence analysis revealed this isolate was the blaIMP-1-positive clone ST235 in Japan. Strokes (cerebral infarction or cerebral haemorrhage) and percutaneous endoscopic gastrostomy tubes were recognised as risk factors for oral colonisation by ARB in the LTCF residents. CONCLUSIONS: ARB, as defined by growth on screening agar plates, which carried mobile resistance genes or elements or conferred high biofilm formation, were already prevalent in the oral cavity of LTCF residents. Health-care workers involved in oral care should be aware of antimicrobial resistance and pay special attention to transmission prevention and infection control measures to diminish ARB or mobile resistance elements dissemination in LTCFs.

Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model.

BACKGROUND: Aggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity. OBJECTIVE: To investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model. METHODS: We transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type. RESULTS: Out of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity. CONCLUSION: The genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.

CdtC-Induced Processing of Membrane-Bound CdtA Is a Crucial Step in Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Holotoxin Formation.

Aggregatibacter actinomycetemcomitans is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an in vitro processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA'), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA'. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA' and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA' showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation in vivo is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA' forms a complex with CdtC. Subsequent binding of CdtB to the CdtA'/CdtC complex results in CDT holotoxin formation.

Significant increase of oral bacteria in the early pregnancy period in Japanese women.

AIM: Oral microflora during pregnancy is critical to oral health care in the mother and her child. We examined the changes in the oral microbiota between pregnancy and nonpregnancy periods. METHODS: The study was performed using 132 healthy pregnant women enrolled from Hiroshima City Asa Citizens Hospital and 51 healthy nonpregnant women as control. During pregnancy, 132 subjects were assessed for seven microbial species by the cultured method and polymerase chain reaction at the early (7-16 weeks gestation), the middle (17-28 weeks), and the late (29-39 weeks) pregnancy periods. Pregnant women completed a series of questionnaires regarding oral and systemic health and lifestyle habits. RESULTS: The total cultivable microbial counts in the early pregnancy were significantly higher than that of the nonpregnant women (P < 0.05). The incidences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in gingival sulcus during the early and middle pregnancy were significantly higher than the nonpregnant group (P < 0.05), while Prevotella intermedia and Fusobacterium nucleatum did not change. Candida species were more frequently detected during the middle and late pregnancy. CONCLUSION: The data suggest that pregnancy, especially in the early periods, promotes the proliferation of microorganisms in the oral cavity and facilitates a colonization of periodontal pathogens.

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms.

A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 µM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.

Cytolethal distending toxin from Aggregatibacter actinomycetemcomitans induces DNA damage, S/G2 cell cycle arrest, and caspase- independent death in a Saccharomyces cerevisiae model.

Cytolethal distending toxin (CDT) is a bacterial toxin that induces G(2)/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A. actinomycetemcomitans CdtB (AaCdtB) expression induces S/G(2) arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.