Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, ß- and γ- TLO gene function.

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, ß- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOß2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloß2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Subgingival microbial diversity and respiratory decline: A cross-sectional study.

AIM: To investigate whether there is an association between subgingival microbial diversity and reduced respiratory function. MATERIALS AND METHODS: A group of dentate 58-72-year-old men in Northern Ireland had a comprehensive periodontal examination including subgingival plaque sampling. DNA was extracted from plaque samples and the V1-V3 regions of the 16S rRNA gene were analysed by high-throughput sequencing and a microbial diversity index (MDI) was derived. Spirometry measurements were made using a wedge bellows spirometer. The primary outcome variable of interest was the percentage of predicted forced expiratory volume in 1 s (% predicted FEV1 ). Analysis included multiple linear regression with adjustment for various confounders. RESULTS: Five-hundred and seven men were included in the analysis. The mean age was 63.6 years (SD = 3.1). Of these, 304 (60.0%) men had no or mild periodontitis, 105 (20.7%) had moderate periodontitis and 98 (19.3%) had severe periodontitis. Multiple linear regression analysis showed that a one unit increase in MDI was associated with a 0.71% loss (95% confidence interval: 0.06%-1.35%; p = .03) in % predicted FEV1 after adjustment for all confounders. CONCLUSIONS: In this group of dentate men from Northern Ireland, subgingival microbial diversity was associated with reduced respiratory function.

A dentifrice containing salivary enzymes and xylitol exhibits superior antimicrobial activity in vitro against adherent Streptococcus mutans compared to a chlorhexidine dentifrice.

Human saliva contains natural antimicrobial enzymes. In this in-vitro study, we evaluate the antimicrobial activity of a dentifrice containing a salivary enzyme complex (SEC) with xylitol versus a standard 0.12% chlorhexidine (CHX) dentifrice. Adherent cells of Streptococcus gordonii, Strep. mutans, Actinomyces naeslundii, Fusobacterium nucleatum subsp polymorphum, and Corynebacterium matruchotii were exposed to SEC-xylitol and CHX dentifrices for 2 min and viable CFUs were enumerated. Exposure to the SEC-xylitol dentifrice resulted in a significant reduction in bacterial viability, which was greater than that shown by the CHX dentifrice, against all organisms tested. The SEC-xylitol dentifrice also exhibited greater antimicrobial activity against all organsims in well diffusion assays compared to CHX. Dentifrice activity was also evaluated against a three species community of Strep. gordonii, Strep. mutans, and Coryne. matruchotii using bacterial live/dead stain. The SEC-xylitol dentifrice was at least as effective as CHX in removal of the multispecies community. The combination of SEC and xylitol generates a highly effective antimicrobial dentifrice with greater antibacterial activity than a standard 0.12% CHX formulations. SEC and xylitol combinations are worthy of further investigation for routine use and in the management of gingivitis and periodontal disease.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.

Role of Mediator in virulence and antifungal drug resistance in pathogenic fungi.

Mediator complex has recently emerged as an important regulator of gene expression in pathogenic fungi. Mediator is a multi-subunit complex of polypeptides involved in transcriptional activation in eukaryotes, with roles including preinitiation complex (PIC) assembly and chromatin remodeling. Within the last decade, Mediator has been shown to play an integral role in regulating virulence gene expression and drug resistance in human fungal pathogens. In some fungi, specific Mediator subunits have been shown to be required for virulence. In Candida species, duplication and expansion of Mediator subunit encoding genes has occurred on at least three occasions (CgMED15 in C. glabrata and MED2/TLO in C. albicans and C. dubliniensis) suggesting important roles for Mediator in the evolution of these pathogens. This review summarises recent developments in our understanding of Mediator in fungal pathogens and the potential for the development of therapeutic drugs to target Mediator functions.

Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of 'free' Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology.

The effect of a decontamination protocol on contaminated titanium dental implant surfaces with different surface topography in edentulous patients.

Objectives: To investigate if it is possible to achieve complete decontamination of dental implant surfaces with different surface characteristics.Materials and methods: Twelve implant pieces with an Osseotite® surface and 12 implant pieces with a Ti-Unite® surface were attached on to the complete lower dentures of six patients and were allowed to accumulate plaque for 30 days. When retrieved, the implant decontamination protocol used, involved both mechanical (PeriBrush™) and chemical (3% H2O2) decontamination. The number of colony forming units per millilitre was determined and the dominant micro-organisms in selected samples was identified by 16s rRNA gene amplicon sequencing. The effect of the titanium brush on the implant surface was examined by SEM.Results: Complete decontamination was achieved in five out of 24 implants (four Osseotite® and one Ti-Unite®). The mean CFU/ml detected after decontamination were 464.48 for Osseotite® and 729.09 for Ti-Unite® implants. On the surface of the implants in which complete decontamination was not achieved, all of the predominant bacteria identified were streptococci except for one which was identified as micrococcus. SEM images revealed that the surface features of the decontaminated implants were not significantly altered.Conclusions: Mechanical decontamination using a titanium brush supplemented with chemical treatment for one minute (3% H2O2) can achieve complete decontamination of implant surfaces in edentulous patients.

Telomeric ORFs (TLOs) in Candida spp. Encode mediator subunits that regulate distinct virulence traits.

The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ null mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity.

Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (valproic acid, trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 µM); while low concentrations (1 µM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14 days) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14 days. 59% of genes were down-regulated at 24 h and 36% at 14 days, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7 d, 14 days) and enzyme activity (48 h, 14 days). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. J. Cell. Physiol. 231: 798-816, 2016. © 2015 Wiley Periodicals, Inc.

An in vivo comparison of internal bacterial colonization in two dental implant systems: identification of a pathogenic reservoir.

OBJECTIVES: The aim of this study was to compare internal bacterial colonization in two implant systems, one screw root form (SRF) with an external hexagon connection and one plateau root form (PRF) with a Morse taper internal connection. MATERIALS AND METHODS: Thirty-two implants; 12 SRF and 20 PRF, were sampled in 15 patients. All implants had been in function for at least 6 months prior to sampling. The implant restoration was removed and 10 µl of sterile saline was introduced into the implant well via a sterile glass syringe. The saline was drawn back up and transferred to the laboratory for microbiological analysis. The number of aerobic and anaerobic colony forming units per millilitre was determined and the dominant micro-organism in each sample was identified by 16s rRNA gene amplicon sequencing. RESULTS: There was a significant difference between bleeding on probing around the SRF implants (3%) and the PRF implants (28%) (p = 0.0496). Bacterial colonization was identified at 11 SRF and 19 PRF implants. The numbers of anaerobic bacteria recovered from PRF implants was significantly higher than that from SRF implants (p = 0.0002). Streptococcus species and Enterococcus faecalis were found to dominate. CONCLUSIONS: This in vivo study demonstrated bacterial colonization in both types of implant systems, irrespective of the type of connection. Significantly greater anaerobic counts were found in the Morse taper internal connection implants.

Response of extraintestinal pathogenic Escherichia coli to human serum reveals a protective role for Rcs-regulated exopolysaccharide colanic acid.

Extraintestinal Escherichia coli (ExPEC) organisms are the leading cause of Gram-negative bacterial bloodstream infections. These bacteria adapt to survival in the bloodstream through expression of factors involved in scavenging of nutrients and resisting the killing activity of serum. In this study, the transcriptional response of a prototypic ExPEC strain (CFT073) to human serum was investigated. Resistance of CFT073 to the bactericidal properties of serum involved increased expression of envelope stress regulators, including CpxR, σE, and RcsB. Many of the upregulated genes induced by active serum were regulated by the Rcs two-component system. This system is triggered by envelope stress such as changes to cell wall integrity. RcsB-mediated serum resistance was conferred through induction of the exopolysaccharide colanic acid. Production of this exopolysaccharide may be protective while cell wall damage caused by serum components is repaired.

Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P < 0.001). However, when the yeast cells were grown at 37°C, no significant difference between the adhesion of C. dubliniensis genotype 1 and C. albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P < 0.001). Using surface plasmon resonance analysis, C. dubliniensis isolates were found to adhere in significantly greater numbers than C. albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

Fusobacterium nucleatum subsp. polymorphum recovered from malignant and potentially malignant oral disease exhibit heterogeneity in adhesion phenotypes and adhesin gene copy number, shaped by inter-subspecies horizontal gene transfer and recombination-derived mosaicism.

Fusobacterium nucleatum is an anaerobic commensal of the oral cavity associated with periodontitis and extra-oral diseases, including colorectal cancer. Previous studies have shown an increased relative abundance of this bacterium associated with oral dysplasia or within oral tumours. Using direct culture, we found that 75 % of Fusobacterium species isolated from malignant or potentially malignant oral mucosa were F. nucleatum subsp. polymorphum. Whole genome sequencing and pangenome analysis with Panaroo was carried out on 76 F. nucleatum subsp. polymorphum genomes. F. nucleatum subsp. polymorphum was shown to possesses a relatively small core genome of 1604 genes in a pangenome of 7363 genes. Phylogenetic analysis based on the core genome shows the isolates can be separated into three main clades with no obvious genotypic associations with disease. Isolates recovered from healthy and diseased sites in the same patient are generally highly related. A large repertoire of adhesins belonging to the type V secretion system (TVSS) could be identified with major variation in repertoire and copy number between strains. Analysis of intergenic recombination using fastGEAR showed that adhesin complement is shaped by horizontal gene transfer and recombination. Recombination events at TVSS adhesin genes were not only common between lineages of subspecies polymorphum, but also between different subspecies of F. nucleatum. Strains of subspecies polymorphum with low copy numbers of TVSS adhesin encoding genes tended to have the weakest adhesion to oral keratinocytes. This study highlights the genetic heterogeneity of F. nucleatum subsp. polymorphum and provides a new framework for defining virulence in this organism.

Influence of site and smoking on malignant transformation in the oral cavity: Is the microbiome the missing link?

The tongue and floor of the mouth are high-risk sites for oral squamous cell carcinoma (OSCC), while smoking is its most significant risk factor. Recently, questions have been raised as to the role of the oral microbiome in OSCC because of a wealth of evidence demonstrating that the microbiome of OSCC differs from that of healthy mucosa. However, oral site and smoking also have a significant impact on oral microbial communities, and to date, the role these factors play in influencing the dysbiotic microbial communities of OSCC and precursor lesions has not been considered. This review aims to examine the influence of site and smoking on the oral microbiome and, in turn, whether these microbiome changes could be involved in oral carcinogenesis.

Does fluoride exposure impact on the human microbiome?

Fluoride is added to drinking water in some countries to prevent tooth decay (caries). There is no conclusive evidence that community water fluoridation (CWF) at WHO recommended concentrations for caries prevention has any harmful effects. However, research is ongoing regarding potential effects of ingested fluoride on human neurodevelopment and endocrine dysfunction. Simultaneously, research has emerged highlighting the significance of the human microbiome in gastrointestinal and immune health. In this review we evaluate the literature examining the effect of fluoride exposure on the human microbiome. Unfortunately, none of the studies retrieved examined the effects of ingested fluoridated water on the human microbiome. Animal studies generally examined acute fluoride toxicity following ingestion of fluoridated food and water and conclude that fluoride exposure can detrimentally perturb the normal microbiome. These data are difficult to extrapolate to physiologically relevant human exposure dose ranges and the significance to humans living in areas with CWF requires further investigation. Conversely, evidence suggests that the use of fluoride containing oral hygiene products may have beneficial effects on the oral microbiome regarding caries prevention. Overall, while fluoride exposure does appear to impact the human and animal microbiome, the long-term consequences of this requires further study.

The role of the Mediator complex in fungal pathogenesis and response to antifungal agents.

Mediator is a complex of polypeptides that plays a central role in the recruitment of RNA polymerase II to promoters and subsequent transcriptional activation in eukaryotic organisms. Studies have now shown that Mediator has a role in regulating expression of genes implicated in virulence and antifungal drug resistance in pathogenic fungi. The roles of specific Mediator subunits have been investigated in several species of pathogenic fungi, particularly in the most pathogenic yeast Candida albicans. Uniquely, pathogenic yeast also present several interesting examples of divergence in Mediator structure and function, most notably in C. glabrata, which possesses two orthologues of Med15, and in C. albicans, which has a massively expanded family of Med2 orthologues known as the TLO gene family. This review highlights specific examples of recent progress in characterizing the role of Mediator in pathogenic fungi.

Smoking, tooth loss and oral hygiene practices have significant and site-specific impacts on the microbiome of oral mucosal surfaces: a cross-sectional study.

We investigated bacterial colonisation patterns of healthy mucosa (buccal, tongue, palate and floor of mouth) in a cohort of adults in order to determine how smoking, tooth loss, plaque levels and oral hygiene practices impacted on mucosal colonisation. A total of 322 swabs were recovered from 256 participants, of whom 46% were current smokers. We analysed colonization by sequencing the V1-V3 regions of the 16S rRNA gene. Palate and tongue microbiomes generally exhibited greater biodiversity than buccal and floor of mouth. Although Neisseria, Lautropia and Haemophilus spp. showed reduced abundance in smokers, buccal mucosa specifically showed a significant increase in Prevotella spp., whereas tongue and floor of mouth tended towards increased abundance of Streptococcus spp. Unexpectedly, tooth brushing frequency had a greater impact on mucosal community structure than plaque levels. Tooth loss was associated with significant reductions in mucosal biodiversity and had site-specific impacts, with buccal communities showing increased abundance of periodontitis-associated species and Rothia mucilaginosa, whereas tongue communities exhibited increased abundance of several streptococcal OTUs and reduced abundance of Haemophilus spp. This study highlights the complex relationship between mucosal colonisation and host factors, highlighting the need for careful consideration of these factors in mucosal microbiome studies.

The Oral Microbiome in Treatment-Naïve Paediatric IBD Patients Exhibits Dysbiosis Related to Disease Severity that Resolves Following Therapy.

BACKGROUND: There is a limited literature describing the oral microbiome and its diagnostic potential in paediatric inflammatory bowel disease [IBD]. METHODS: We examined the dorsum tongue microbiome by V1-V2 sequencing in a cohort of 156 treatment-naïve children diagnosed with IBD compared to 102 healthy control children. Microbiome changes over time following treatment were examined in a subset of patients and associations between IBD diagnosis and dysbiosis were explored. RESULTS: Analysis of community structure of the microbiome in tongue samples revealed that IBD samples diverged significantly from healthy control samples [PERMANOVA p = 0.0009] and exhibited a reduced abundance of Clostridia in addition to several major oral genera [Veillonella, Prevotella and Fusobacterium species] with an increased abundance of streptococci. This dysbiosis was more marked in patients with severe disease. Higher levels of the potential pathobionts Klebsiella and Pseudomonas spp. were also associated with IBD. In terms of predicted functions, the IBD oral microbiome was potentially more acidogenic and exhibited reduced capacity for B vitamin biosynthesis. We used a machine learning approach to develop a predictive model of IBD which exhibited a mean-prediction AUC [area under the ROC curve] of 0.762. Finally, we examined a subset of 53 patients following 12 months of therapy and could show resolution of oral dysbiosis as demonstrated by a shift towards a healthy community structure and a significant reduction in oral dysbiosis. CONCLUSION: Oral dysbiosis found in children with IBD is related to disease severity and resolves over time following successful IBD treatment.

Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans.

Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.