Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted β-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.

As part of the human microbiota, the fungus Candida albicans colonizes the oral cavity and other mucosal surfaces of the human body. Commensalism is tightly controlled by complex interactions of the fungus and the host to preclude fungal elimination but also fungal overgrowth and invasion, which can result in disease. As such, defects in antifungal T cell immunity render individuals susceptible to oral thrush due to interrupted immunosurveillance of the oral mucosa. The factors that promote commensalism and ensure persistence of C. albicans in a fully immunocompetent host remain less clear. Using an experimental model of C. albicans oral colonization in mice we explored fungal determinants of commensalism in the oral cavity. Transcript profiling of the oral isolate 101 in the murine tongue tissue revealed a characteristic metabolic profile tailored to the nutrient poor conditions in the stratum corneum of the epithelium where the fungus resides. Metabolic adaptation of isolate 101 was also reflected in enhanced nutrient acquisition when grown on oral mucosa substrates. Persistent colonization of the oral mucosa by C. albicans also correlated inversely with the capacity of the fungus to induce epithelial cell damage and to elicit an inflammatory response. Here we show that these immune evasive properties of isolate 101 are explained by a strong attenuation of a number of virulence genes, including those linked to filamentation. De-repression of the hyphal program by deletion or conditional repression of NRG1 abolished the commensal behaviour of isolate 101, thereby establishing a central role of this factor in the commensal lifestyle of C. albicans in the oral niche of the host.

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Granuloma formation is a hallmark of several infectious diseases, including those caused by Mycobacterium sp. These structures are composed of accumulations of inflammatory cells, and it has been shown that cytokines such as IFN-g and TNF-a are required for granuloma assembly during M. avium infections in mice. Macrophages (MFs) insensitive to IFN-g (MIIG) mice have MFs, monocytes, and dendritic cells that are unresponsive to IFN-g. We observed that although IFN-g 2/2 mice present an exacerbated infection, the same is not true for MIIG animals, where the same levels of protection as the wild-type animals were observed in the liver and partial protection in the spleen. Unlike IFN-g 2/2 mice, MIIG mice still develop well-defined granulomas, suggesting that IFN-g-mediated MF activation is not required for granuloma assembly. This work also shows that MIIG animals exhibit increased cell recruitment with higher CD4 + T cells numbers as well as increased IFN-g and TNF-a expression, suggesting that TNF-a may have a role in protection and may compensate the lack of MF response to IFN-g in the MIIG model. TNF-a-deficient MIIG mice (MIIG.TNF-a 2/2) exhibited increased bacterial burdens when compared with MIIG mice. These results suggest that in the absence of IFN-g signaling in MFs, TNF-a has a protective role against M. avium.

Neospora caninum is an obligate intracellular protozoan responsible for abortion and stillbirths in cattle. We previously developed a mucosal vaccination approach using N. caninum membrane proteins and CpG adjuvant that conferred long-term protection against neosporosis in mice. Here, we have extended this approach by alternatively using the carbomer-based adjuvant Carbigen™ in the immunizing preparation. Immunized mice presented higher proportions and numbers of memory CD4+ and CD8+ T cells. Stimulation of spleen, lungs and liver leukocytes with parasite antigens induced a marked production of IFN-γ and IL-17A and, less markedly, IL-4. This balanced response was also evident in that both parasite-specific IgG1 and IgG2c were raised by immunization, together with specific intestinal IgA. Upon intraperitoneal infection with N. caninum, immunized mice presented lower parasitic burdens than sham-immunized controls. In the infected immunized mice, memory CD4+ T cells predominantly expressed T-bet and RORγt, and CD8+ T cells expressing T-bet were found increased. While spleen, lungs and liver leukocytes of both immunized and sham-immunized infected animals produced high amounts of IFN-γ, only the cells from immunized mice responded with high IL-17A production. Since in cattle both IFN-γ and IL-17A have been associated with protective mechanisms against N. caninum infection, the elicited cytokine profile obtained using CarbigenTM as adjuvant indicates that it could be worth exploring for bovine neosporosis vaccination.

No abstract

Introduction The FunHoMic project is a Marie Skłodowska-Curie Innovative Training Network comprising 13 PhD students, 8 academic partners and 3 industry partnersaimingto understand the interplay between fungi, hostsand microbiota to improve prevention and treatment of fungal infections. Importance About 2 billion people suffer fungal infections, which have a mortality rate close to that of malaria or breast cancer. Candida albicans has a high clinical and economic burden, making it of particular interest to the FunHoMic project. 70% of women experience at least one episode of vulvovaginal candidiasis (“thrush”) during their lifetime; 8% suffer recurring infections. C. albicans may live as a commensal but can cause symptoms when the fungus-host-microbiota equilibrium is disrupted. Infections by C. albicans have a significant clinical impact, with fatalities in severe cases. Many factors are associated with C. albicans infections; intensive care, neutropenic and diabetic patients are most at risk of systemic infection. Rising antifungal drug resistance has led to certain C. albicans infections having no treatment option. Aim The FunHoMic consortium combines projectson fungal pathogenesis, immunology, microbial ecology and’omics technologies to understand and exploit interactions between fungus, host and microbiota. Identification of novel bio markers on the fungal side such as genetic polymorphisms or on the host side such asmicrobiota profiles, metabolites and/or immune markers can lead to patient classification based on relative risk of infection. This could be the beginning of personalised management for fungal infections using preventive or therapeutic interventions like new antifungals, immune modulators or Live Biotherapeutic Products (LBPs). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the MarieSklodowska-Curie grant agreement No 812969.