TransfOrming Transnational intErcultural sensitivity for Midwifery students through an inclusive mobility model: A mixed-method evaluation of the TOTEMM project.

BACKGROUND: Contemporary midwifery curricula require that student midwives have insight and understanding of global health practice and intercultural sensitivity. The current mobility model excludes large numbers of students from engaging in transnational learning. OBJECTIVES: 1) to evaluate midwifery students' experiences of blended mobility; 2) to investigate if the combination of virtual and physical mobility activities supported development of intercultural sensitivity and soft skills. DESIGN: Multi-centre mixed-methods study. SETTINGS: Four European Higher Education Institutions located in England, Italy, Estonia and The Netherlands. PARTICIPANTS: Sixty-four midwifery students studying in one of the four partner institutions selected as study sites and who participated in the TOTEMM blended mobility scheme took part in the evaluation. METHODS: Data were collected through two online surveys, face-to-face focus groups and learning analytics. Descriptive summary statistical analysis of survey data was undertaken. Focus group discussions were subjected to thematic analysis. Findings from the quantitative survey and qualitative focus groups were merged using a convergent mixed methods approach. Learning Analytics were interpreted as complementary to the above components, to further triangulate the findings. RESULTS: Both virtual and physical components were evaluated positively by students, with high engagement confirmed by learning analytics. A statistically significant increase in the mean of the Total Intercultural Sensitivity Scale score was seen between the pre- and post-mobility surveys, indicating participation in the TOTEMM mobility model was associated with enhanced intercultural sensitivity. Positive effects on confidence, open-mindedness, empathy, interaction and non-judgment were shared by participants. CONCLUSIONS: TOTEMM is an innovative inclusive approach to enable a diverse student group to benefit from transnational learning, including the development of intercultural sensitivity. The TOTEMM blended mobility model has potential for integration into future midwifery curricula and programmes in the four partner settings involved in TOTEMM and utility for the wider European context.

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

Exploring the experiences of healthcare professionals in South Africa and Uganda around communicating with children about life-threatening conditions: a workshop-based qualitative study to inform the adaptation of communication frameworks for use in these settings.

OBJECTIVES: This study aimed to explore how published communication frameworks could be amended to ensure applicability and cultural appropriateness for professionals to support family-centred conversations by investigating' healthcare professionals' (1) experiences of providing support to families when a caregiver or a dependent child (<18 years old) has a life-threatening condition, (2) perceived challenges for caregivers and healthcare professionals in communicating with children about illness, (3) perceptions of how clinicians could be equipped to facilitate conversations between caregivers and children about an adult or the child's own life-threatening condition and (4) suggestions for amendments to previously published guidelines to ensure cultural relevance in South Africa and Uganda. DESIGN: A qualitative study involving two 2-day workshops with embedded focus group discussions, break out rooms and consensus discussions. SETTING: Health and social care and third sector organisations in South Africa and Uganda. PARTICIPANTS: Thirty-two professionals providing care to families affected by life-threatening conditions in South Africa or Uganda who were aged 18 years or older and able to converse in English. RESULTS: Participants identified obstacles to having conversations with caregivers about children and to telling children about serious illness during consultations. These included patients' beliefs about illness, medicine and death, language barriers between families and the healthcare team, and emotional and practical challenges for professionals in having these conversations. Culturally appropriate adaptations were made to previously published communication frameworks for professionals to support family-centred conversations. CONCLUSIONS: Culturally sensitive communication frameworks could help healthcare professionals to talk with families about what children need to know when they or a caregiver have a serious illness. More broadly, effective communication could be facilitated by promoting healthcare professionals' and communities' understanding of the benefits of telling children about illness within the family. Together these strategies may mitigate the psychological impact of global disease on children and their families.

High-Pressure Freezing and Transmission Electron Microscopy to Visualize the Ultrastructure of the C. auris Cell Wall.

Transmission electron microscopy (TEM) is the main technique used to study the ultrastructure of biological samples. Chemical fixation was considered the main method for preserving samples for TEM; however, it is a relatively slow method of fixation and can result in morphological alterations. Cryofixation using high-pressure freezing (HPF) overcomes the limitations of chemical fixation by preserving samples instantly. Here, we describe our HPF methods optimized for visualizing Candida auris at the ultrastructural level.

Monoclonal Antibodies Targeting Surface-Exposed Epitopes of Candida albicans Cell Wall Proteins Confer In Vivo Protection in an Infection Model.

Monoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.

Breaking bad news in a cross-language context: A qualitative study to develop a set of culturally and linguistically appropriate phrases and techniques with Zulu speaking cancer patients.

OBJECTIVE: Breaking bad news (BBN) in healthcare is common. Guidelines abound but little is documented in an African context. We wanted to describe Zulu speaking patients' BBN experience and assess their opinions of internationally recommended techniques. METHODS: BBN techniques were highlighted from the literature using systematic review methods. Semi-structured focus group interviews with Zulu speaking cancer patients were conducted. Data were analysed using Framework Analysis. RESULTS: Language concordance was central - regardless of whether this necessitated a nurse acting as translator. While non-abandonment, empathy and maintenance of hope was valued by participants, an oft-expressed belief in positive outcomes accounted for mixed responses to phrases implying ambiguity. In contrast, "I wish" phrases were appreciated. Silence received mixed responses with a strong dislike for silence as a front for non-disclosure. CONCLUSION: Language-related concerns dictated the bulk of participants BBN perspectives. While cultural and linguistic differences exist, good communication skills, empathy and the maintenance of hope remain central. PRACTICE IMPLICATIONS: BBN in a language in which the patient is fluent, whether mediated or not, should be the standard of care. Cultural and linguistic variance must be born in mind and clinicians should become familiar with the preferences of the communities they serve.

Crosstalk between the calcineurin and cell wall integrity pathways prevents chitin overexpression in Candida albicans.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise ß-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.

Weight loss, hypertension and mental well-being improvements during COVID-19 with a multicomponent health promotion programme on Zoom: a service evaluation in primary care.

BACKGROUND: Obesity is a risk factor for complications from SARS-CoV-2 infection, increasing the need for effective weight management measures in primary care. However, in the UK, COVID-19 restrictions have hampered primary care weight management referral and delivery, and COVID-19 related weight gain has been reported. The present study evaluated outcomes from a multicomponent weight loss and health promotion programme in UK primary care, delivered remotely due to COVID-19 restrictions. METHOD: Patients with obesity, type 2 diabetes or pre-diabetes attended six 90 min sessions over 10 weeks on Zoom. The dietary component comprised a low-carbohydrate 'real food' approach, augmented with education on physical activity, intermittent fasting, gut health, stress management, sleep and behaviour change. Anthropometric and cardiometabolic data were self-reported. Mental well-being was assessed with the Warwick Edinburgh Mental Wellbeing Scale. Subjective outcomes and participant feedback about the programme were collected with an anonymous online survey. RESULTS: Twenty participants completed the programme. Weight loss and improvements in body mass index, waist circumference, systolic and diastolic blood pressure and mental well-being achieved statistical and clinical significance. Mean weight loss (5.8 kg) represented a 6.5% weight loss. Participants' subjective outcomes included weight loss without hunger (67%) and increased confidence in their ability to improve health (83%). All participants reported the usage of Zoom to access the programme as acceptable with 83% reporting it worked well. CONCLUSION: A multicomponent weight loss and health promotion programme with a low-carbohydrate dietary component, clinically and statistically significantly improved health outcomes including weight status, blood pressure and mental well-being in a group of primary care patients when delivered remotely. Further research is warranted.

Dependence on Mincle and Dectin-2 Varies With Multiple Candida Species During Systemic Infection.

More than 95% of invasive Candida infections are caused by four Candida spp. (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis). C-type lectin-like receptors (CLRs), such as Dectin-1, Dectin-2, and Mincle mediate immune responses to C. albicans. Dectin-1 promotes clearance of C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis, however, dependence on Dectin-1 for specific immune responses varies with the different Candida spp. Dectin-2 is important for host immunity to C. albicans and C. glabrata, and Mincle is important for the immune response to C. albicans. However, whether Dectin-2 drives host immunity to C. tropicalis or C. parapsilosis, and whether Mincle mediates host immunity to C. glabrata, C. tropicalis or C. parapsilosis is unknown. Therefore, we compared the roles of Dectin-2 and Mincle in response to these four Candida spp. We demonstrate that these four Candida spp. cell walls have differential mannan contents. Mincle and Dectin-2 play a key role in regulating cytokine production in response to these four Candida spp. and Dectin-2 is also important for clearance of all four Candida spp. during systemic infection. However, Mincle was only important for clearance of C. tropicalis during systemic infection. Our data indicate that multiple Candida spp. have different mannan contents, and dependence on the mannan-detecting CLRs, Mincle, and Dectin-2 varies between different Candida spp. during systemic infection.

Midwifery students' views and experiences of birth on mainstream factual television.

OBJECTIVES: To investigate midwifery students' experiences of viewing childbirth on mainstream factual television and to explore implications for student career intentions and potential pedagogical uses of television excerpts in midwifery education. DESIGN: Twenty-two undergraduate midwifery students at one of two universities took place in focus groups between February and June 2019. Ethical approval was obtained at both sites. Thematic analysis was employed to generate key themes from the data. SETTING: Two UK universities based in the East Midlands and East Yorkshire regions of England. PARTICIPANTS: Twenty-two midwifery students at any stage of their studies. FINDINGS: Researchers generated four key themes from the data a) Changed Perspectives on Televised Childbirth, b) Representations of Midwives and Social Implications, c) Representation of Childbirth and Social Implications and d) The Role of Televising Childbirth in Midwifery Education. KEY CONCLUSIONS: Midwifery students often experience a change of perspective on birth on television as they acquire new knowledge and skills. They recognise the potential social implications of how childbirth and midwifery are represented on television. Pedagogical use of televised birth has potential benefits but needs further investigation in the context of midwifery education. IMPLICATIONS FOR PRACTICE: Midwifery students are likely to begin their studies with pre-existing views and experiences around how birth is represented on mainstream factual television. They may need support to reflect on these to consider their expectations of the profession, to effectively support childbearing women and to potentially influence future production of media images of childbirth.

Erratum to "Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation" [Cell Surf. 1 (2018) 43-56].

[This corrects the article DOI: 10.1016/j.tcsw.2018.03.002.].

Caspofungin Induced Cell Wall Changes of Candida Species Influences Macrophage Interactions.

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of ß-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii, which had naturally more ß-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and ß-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells.

Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans' tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.IMPORTANCECandida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

Dependence on Dectin-1 Varies With Multiple Candida Species.

Four Candida spp. (albicans, glabrata, tropicalis, parapsilosis) cause >95% of invasive Candida infections. C. albicans elicits immune responses via pathogen recognition receptors including C-type lectin-like receptors (CLRs). The CLR, Dectin-1 is important for host immunity to C. albicans and C. glabrata, however, whether Dectin-1 is important for host defense against C. tropicalis or C. parapsilosis is unknown. Therefore, we compared the involvement of Dectin-1 in response to these four diverse Candida spp. We found that Dectin-1 mediates innate cytokine responses to these Candida spp. in a species- and cell-dependent manner. Dectin-1 KO mice succumbed to infection with highly virulent C. albicans while they mostly survived infection with less virulent Candida spp. However, Dectin-1 KO mice displayed increased fungal burden following infection with each Candida spp. Additionally, T cells from Dectin-1 KO mice displayed enhanced effector functions likely due to the inability of Dectin-1 KO mice to clear the infections. Together, these data indicate that Dectin-1 is important for host defense to multiple Candida spp., although the specific roles for Dectin-1 varies with different Candida spp.

Inhibition of Classical and Alternative Modes of Respiration in Candida albicans Leads to Cell Wall Remodeling and Increased Macrophage Recognition.

The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis, and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we made use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans We found that use of the nitric oxide donor sodium nitroprusside (SNP) and of the alternative oxidase inhibitor salicylhydroxamic acid (SHAM) prevents respiration and leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo We propose that treatment with SNP plus SHAM (SNP+SHAM) leads to transcriptional changes that drive cell wall rearrangement but which also prime cells to activate the transition to hyphal growth. In line with this, we found that pretreatment of C. albicans with SNP+SHAM led to an increase in virulence. Our data reveal strong links between respiration, cell wall remodeling, and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals that are not damaging to the mammalian host but that we need to develop a deeper understanding of the roles of mitochondria in cellular signaling if they are to be developed successfully as a target for new antifungals.IMPORTANCE Current approaches to tackling fungal infections are limited, and new targets must be identified to protect against the emergence of resistant strains. We investigated the potential of targeting mitochondria, which are organelles required for energy production, growth, and virulence, in the human fungal pathogen Candida albicans Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However, release of C. albicans cells from respiratory inhibition appears to activate a stress response that increases the levels of traits associated with virulence. Our results make it clear that mitochondria represent a valid target for the development of antifungal strategies but that we must determine the mechanisms by which they regulate stress signaling and virulence ahead of successful therapeutic advance.

The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

The type VI secretion system deploys antifungal effectors against microbial competitors.

Interactions between bacterial and fungal cells shape many polymicrobial communities. Bacteria elaborate diverse strategies to interact and compete with other organisms, including the deployment of protein secretion systems. The type VI secretion system (T6SS) delivers toxic effector proteins into host eukaryotic cells and competitor bacterial cells, but, surprisingly, T6SS-delivered effectors targeting fungal cells have not been reported. Here we show that the 'antibacterial' T6SS of Serratia marcescens can act against fungal cells, including pathogenic Candida species, and identify the previously undescribed effector proteins responsible. These antifungal effectors, Tfe1 and Tfe2, have distinct impacts on the target cell, but both can ultimately cause fungal cell death. 'In competition' proteomics analysis revealed that T6SS-mediated delivery of Tfe2 disrupts nutrient uptake and amino acid metabolism in fungal cells, and leads to the induction of autophagy. Intoxication by Tfe1, in contrast, causes a loss of plasma membrane potential. Our findings extend the repertoire of the T6SS and suggest that antifungal T6SSs represent widespread and important determinants of the outcome of bacterial-fungal interactions.

Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.