Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections

Cellular responses of Candida albicans to phagocytosis and the extracellular activities of neutrophils are critical to counteract carbohydrate starvation, oxidative and nitrosative stress

Acknowledgments We thank Alexander Johnson (yhb1D/D), Karl Kuchler (sodD/D mutants), Janet Quinn (hog1D/D, hog1/cap1D/D, trx1D/D) and Peter Staib (ssu1D/D) for providing mutant strains. We acknowledge helpful discussions with our colleagues from the Microbial Pathogenicity Mechanisms Department, Fungal Septomics and the Microbial Biochemistry and Physiology Research Group at the Hans Kno¨ll Institute (HKI), specially Ilse D. Jacobsen, Duncan Wilson, Sascha Brunke, Lydia Kasper, Franziska Gerwien, Sea´na Duggan, Katrin Haupt, Kerstin Hu¨nniger, and Matthias Brock, as well as from our partners in the FINSysB Network. Author Contributions Conceived and designed the experiments: PM HW IMB AJPB OK BH. Performed the experiments: PM CD HW. Analyzed the data: PM HW IMB AJPB OK BH. Wrote the paper: PM HW OK AJPB BH.Peer reviewedPublisher PD

Lactate signalling regulates fungal β-glucan masking and immune evasion

AJPB: This work was supported by the European Research Council (STRIFE, ERC- 2009-AdG-249793), The UK Medical Research Council (MR/M026663/1), the UK Biotechnology and Biological Research Council (BB/K017365/1), the Wellcome Trust (080088; 097377). ERB: This work was supported by the UK Biotechnology and Biological Research Council (BB/M014525/1). GMA: Supported by the CNPq-Brazil (Science without Borders fellowship 202976/2014-9). GDB: Wellcome Trust (102705). CAM: This work was supported by the UK Medical Research Council (G0400284). DMM: This work was supported by UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/K000306/1). NARG/JW: Wellcome Trust (086827, 075470,101873) and Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377). ALL: This work was supported by the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPostprin

Improving the oral health of older people in care homes (TOPIC): a protocol for a feasibility study

BACKGROUND: Evidence for interventions promoting oral health amongst care home residents is weak. The National Institute for Health and Care Excellence (NICE) guideline NG48 aims to maintain and improve the oral health of care home residents. A co-design process that worked with residents and care home staff to understand how the NG48 guideline could be best implemented in practice has been undertaken to refine a complex intervention. The aim of this study is to assess the feasibility of the intervention to inform a future larger scale definitive trial. METHODS: This is a protocol for a pragmatic cluster randomised controlled trial with a 12-month follow-up that will be undertaken in 12 care homes across two sites (six in London, six in Northern Ireland). Care homes randomised to the intervention arm (n = 6) will receive the complex intervention based on the NG48 guideline, whilst care homes randomised to the control arm (n = 6) will continue with routine practice. The intervention will include a training package for care home staff to promote knowledge and skills in oral health promotion, the use of the Oral Health Assessment Tool on residents by trained care home staff, and a 'support worker assisted' daily tooth-brushing regime with toothpaste containing 1500 ppm fluoride. An average of ten residents, aged 65 years or over who have at least one natural tooth, will be recruited in each care home resulting in a recruited sample of 120 participants. Assessments will be undertaken at baseline, 6 months and 12 months, and will include a dental examination and questionnaires on general health and oral health administered by a research assistant. A parallel process evaluation involving semi-structured interviews will be undertaken to explore how the intervention could be embedded in standard practice. Rates of recruitment and retention, and intervention fidelity will also be recorded. A cost-consequence model will determine the relevance of different outcome measures in the decision-making context. DISCUSSION: The study will provide valuable information for trialists, policymakers, clinicians and care home staff on the feasibility and associated costs of oral health promotion in UK care homes. TRIAL REGISTRATION: ISRCTN10276613. Registered on 17th April 2020. http://www.isrctn.com/ISRCTN10276613

A Novel Assay to Trace Proliferation History In Vivo Reveals that Enhanced Divisional Kinetics Accompany Loss of Hematopoietic Stem Cell Self-Renewal

BACKGROUND: The maintenance of lifelong blood cell production ultimately rests on rare hematopoietic stem cells (HSCs) that reside in the bone marrow microenvironment. HSCs are traditionally viewed as mitotically quiescent relative to their committed progeny. However, traditional techniques for assessing proliferation activity in vivo, such as measurement of BrdU uptake, are incompatible with preservation of cellular viability. Previous studies of HSC proliferation kinetics in vivo have therefore precluded direct functional evaluation of multi-potency and self-renewal, the hallmark properties of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: We developed a non-invasive labeling technique that allowed us to identify and isolate candidate HSCs and early hematopoietic progenitor cells based on their differential in vivo proliferation kinetics. Such cells were functionally evaluated for their abilities to multi-lineage reconstitute myeloablated hosts. CONCLUSIONS: Although at least a few HSC divisions per se did not influence HSC function, enhanced kinetics of divisional activity in steady state preceded the phenotypic changes that accompanied loss of HSC self-renewal. Therefore, mitotic quiescence of HSCs, relative to their committed progeny, is key to maintain the unique functional and molecular properties of HSCs

Nasopharyngeal Colonization and Invasive Disease Are Enhanced by the Cell Wall Hydrolases LytB and LytC of Streptococcus pneumoniae

Background: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. Methodology/Principal Findings: We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. Conclusions/Significance: This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factor

The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

The Transcriptional Repressor TupA in Aspergillus niger Is Involved in Controlling Gene Expression Related to Cell Wall Biosynthesis, Development, and Nitrogen Source Availability.

The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the DeltatupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37 degrees C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism

Longitudinal associations between television in the bedroom and body fatness in a UK cohort study.

OBJECTIVE: To assess longitudinal associations between screen-based media use (television (TV) and computer hours, having a TV in the bedroom) and body fatness among UK children. METHODS: Participants were 12 556 children from the UK Millennium Cohort Study who were followed from age 7 to age 11 years. Associations were assessed between screen-based media use and the following outcomes: body mass index (BMI), fat mass index (FMI), and overweight. RESULTS: In fully adjusted models, having a bedroom TV at age 7 years was associated with significantly higher BMI and FMI (excess BMI for boys=0.29, 95% confidence interval (CI) 0.06-0.52; excess BMI for girls=0.57, 95% CI 0.31-0.84; excess FMI for boys=0.20, 95% CI 0.04-0.37; excess FMI for girls=0.39, 95% CI 0.21-0.57) and increased risk of being overweight (relative risk (RR) for boys=1.21, 95% CI 1.07-1.36; RR for girls=1.31, 95% CI 1.15-1.48) at age 11 years, compared with having no bedroom TV. Hours spent watching TV or digital versatile disks were associated with increased risk of overweight among girls only. Computer use at age 7 years was not related to later body fatness for either gender. CONCLUSION: Having a TV in the child's bedroom was an independent risk factor for overweight and increased body fatness in this nationally representative sample of UK children. Childhood obesity prevention strategies should consider TVs in children's bedrooms as a risk factor for obesity.International Journal of Obesity advance online publication, 27 June 2017; doi:10.1038/ijo.2017.129

Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes