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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Aplicación de la técnica manual craneosacral en mujeres que laboran en la Educación Superior / Application of the craniosacral manual technique in women working in Higher Education / Aplicação da técnica manual craniossacral em mulheres que trabalham no Ensino Superior

En la actualidad la mayoría de las personas por lo menos una vez en el año presenta una molestia en la estructura del cuello, considerado como cervicalgia o dolor cervical, el mismo que está ocasionando un impacto considerable en la vida de las personas dentro del ámbito familiar, laboral y en la comunidad, provocando efectos negativos en la calidad de vida y en casos severos puede llegar a ocasionar discapacidad y limitación funcional de los movimientos. Por lo que este estudio presentó como objetivo determinar la efectividad de la aplicación de la técnica manual craneosacral en mujeres que laboran en la Universidad Católica de Santiago de Guayaquil. La metodología que se utilizó fue un enfoque cuantitativo- descriptivo con un diseño experimental del tipo pre experimental, con un corte longitudinal, se utilizó como muestra a 31 mujeres que laboran en diversas áreas de la Universidad Católica Santiago de Guayaquil, a su vez, se utilizó como técnica la encuesta, y el instrumento aplicado para la valoración del dolor cervical fue la escala visual analógica del dolor (EVA). Los resultados de esta técnica respecto a la escala visual analógica (EVA) mediante una valoración inicial y final, reflejaron mejoría en el dolor cervical donde el 68% de las pacientes no presentaron dolor, y el 32% reflejan un dolor leve. Se concluyó, que por medio de la técnica manual craneosacral el dolor disminuyo en un gran porcentaje restableciendo los procesos naturales del equilibrio del cuerpo a través de la terapia manual.

Currently, most people at least once a year have a discomfort in the structure of the neck, considered cervicalgia or cervical pain, which is causing a considerable impact on the lives of people within the family, work and in the community, causing negative effects on the quality of life and in severe cases can lead to disability and functional limitation of movements. Therefore, this study presented the objective of determining the effectiveness of the application of the craniosacral manual technique in women who work at the Catholic University of Santiago de Guayaquil. The methodology used was a quantitative-descriptive approach with an experimental design of the pre-experimental type, with a longitudinal cut, 31 women who work in various areas of the Santiago de Guayaquil Catholic University were used as a sample, in turn, used the survey as a technique, and the instrument applied for the assessment of neck pain was the visual analogue pain scale (VAS). The results of this technique with respect to the visual analog scale (VAS) through an initial and final evaluation, reflected improvement in cervical pain where 68% of the patients did not present pain, and 32% reflected mild pain. It was concluded that through the craniosacral manual technique, the pain decreased by a large percentage, restoring the natural processes of the body's balance through manual therapy.

Atualmente, a maioria das pessoas pelo menos uma vez ao ano apresenta um desconforto na estrutura do pescoço, considerado cervicalgia ou dor cervical, que está causando um impacto considerável na vida das pessoas dentro da família, no trabalho e na comunidade, causando efeitos negativos na qualidade de vida e em casos graves pode levar à incapacidade e limitação funcional dos movimentos. Portanto, este estudo apresentou o objetivo de determinar a eficácia da aplicação da técnica manual craniossacral em mulheres que trabalham na Universidade Católica de Santiago de Guayaquil. A metodologia utilizada foi uma abordagem quantitativo-descritiva com um desenho experimental do tipo pré-experimental, com corte longitudinal, 31 mulheres que trabalham em várias áreas da Universidade Católica de Santiago de Guayaquil foram usadas como amostra, por sua vez, utilizaram a pesquisa como técnica, e o instrumento aplicado para avaliação da cervicalgia foi a escala visual analógica de dor (EVA). Os resultados desta técnica com relação à escala visual analógica (EVA) através de uma avaliação inicial e final, refletiram melhora da dor cervical onde 68% dos pacientes não apresentavam dor, e 32% refletiam dor leve. Concluiu-se que através da técnica manual craniossacral, a dor diminuiu em grande porcentagem, restabelecendo os processos naturais de equilíbrio do corpo através da terapia manual.

A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans.

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species.

Acetaldehyde production by Rothia mucilaginosa isolates from patients with oral leukoplakia.

Rothia mucilaginosa has been found at high abundance on oral leukoplakia (OLK). The ability of clinical isolates to produce acetaldehyde (ACH) from ethanol has not been investigated. The objective of the current study was to determine the capacity of R. mucilaginosa isolates recovered from OLK to generate ACH. Analysis of R. mucilaginosa genomes (n = 70) shows that this species does not normally encode acetaldehyde dehydrogenase (ALDH) required for detoxification of ACH. The predicted OLK metagenome also exhibited reduced ALDH coding capacity. We analysed ACH production in 8 isolates of R. mucilaginosa and showed that this species is capable of generating ACH in the presence of ethanol. The levels of ACH produced (mean = 53 µM) were comparable to those produced by Neisseria mucosa and Candida albicans in parallel assays. These levels were demonstrated to induce oxidative stress in cultured oral keratinocytes. This study shows that R. mucilaginosa can generate ACH from ethanol in vitro at levels which can induce oxidative stress. This organism likely contributes to oral ACH levels following alcohol consumption and the significance of the increased abundance of R. mucilaginosa in patients with potentially malignant disorders requires further investigation.

The Oral Microbiome in Pediatric IBD: A Source of Pathobionts or Biomarkers?

The oral cavity is continuous with the gastrointestinal tract and in children, oral health may be closely linked with the overall health of the GI tract. In the case of pediatric Crohn's disease (CD), oral manifestations are an important clinical indicator of intestinal disease. Recent studies of the microbiome in IBD suggest that translocation of oral microbes to the gut may be a common feature of the microbial dysbiosis which is a signature of both CD and ulcerative colitis (UC). Murine studies suggest that translocation of oral bacteria and yeasts to the lower GI tract may trigger inflammation in susceptible hosts, providing a mechanistic link to the development of IBD. Conversely, some studies have shown that dysbiosis of the oral microbiome may occur, possibly as a result of inflammatory responses and could represent a useful source of biomarkers of GI health. This review summarizes our current knowledge of the oral microbiome in IBD and presents current hypotheses on the potential role of this community in the pathogenesis of these diseases.

The microbiome and oral cancer: More questions than answers.

Recent advances in DNA sequencing technology have facilitated rapid advances in the analysis of the human microbiome and its role in human disease. Several studies have now shown that OSCC and some oral premalignant conditions are associated with alterations in the oral microbiome. These studies raise questions regarding the role of the oral microbiome in the progression of oral malignancies and whether microbiome change is a significant risk factor in the development of oral cancer. This short review summarises current knowledge in the field and highlights questions that require further investigation.

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.

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.

Candida albicans Hyphal Expansion Causes Phagosomal Membrane Damage and Luminal Alkalinization.

Macrophages rely on phagosomal acidity to destroy engulfed microorganisms. To survive this hostile response, opportunistic fungi such as Candida albicans developed strategies to evade the acidic environment. C. albicans is polymorphic and able to convert from yeast to hyphae, and this transition is required to subvert the microbicidal activity of the phagosome. However, the phagosomal lumen, which is acidic and nutrient deprived, is believed to inhibit the yeast-to-hypha transition. To account for this apparent paradox, it was recently proposed that C. albicans produces ammonia that alkalinizes the phagosome, thus facilitating yeast-to-hypha transition. We reexamined the mechanism underlying phagosomal alkalinization by applying dual-wavelength ratiometric pH measurements. The phagosomal membrane was found to be highly permeable to ammonia, which is therefore unlikely to account for the pH elevation. Instead, we find that yeast-to-hypha transition begins within acidic phagosomes and that alkalinization is a consequence of proton leakage induced by excessive membrane distension caused by the expanding hypha.IMPORTANCEC. albicans is the most common cause of nosocomial fungal infection, and over 3 million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that yeast-to-hypha transition occurs within the nutrient-deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause, of hyphal growth.

Expansion of the TLO gene family enhances the virulence of Candida species.

The TLO genes are a family of subtelomeric ORFs in the fungal pathogens Candida albicans and C. dubliniensis encoding a subunit of the Mediator complex homologous to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two. To investigate if expansion of the TLO repertoire in C. dubliniensis has an effect on phenotype and virulence we expressed three representative C. albicans TLO genes (TLOß2, TLOγ11 and TLOα12) in a wild type C. dubliniensis background, under the control of either their native or the ACT1 promoter. Expression of TLOß2 resulted in a hyperfilamentous phenotype, while overexpression of TLOγ11 and TLOα12 resulted in enhanced resistance to oxidative stress. Expression of all three TLO genes from the ACT1 promoter resulted in increased virulence in the Galleria infection model. In order to further investigate if individual TLO genes exhibit differences in function we expressed six representative C. albicans TLO genes in a C. dubliniensis Δtlo1/Δtlo2 double mutant. Differences were observed in the ability of the expressed CaTLOs to complement the various phenotypes of the mutant. All TLO genes with the exception of TLOγ7 could restore filamentation, however only TLOα9, γ11 and α12 could restore chlamydospore formation. Differences in the ability of CaTLO genes to restore growth in the presence of H2O2, calcofluor white, Congo red and at 42°C were observed. Only TLOα3 restored wild-type levels of virulence in the Galleria infection model. These data show that expansion of the TLO gene family in C. dubliniensis results in gain of function and that there is functional diversity amongst members of the gene family. We propose that this expansion of the TLO family contributes to the success of C. albicans as a commensal and opportunistic pathogen.