Ludivine Cambier

Mechanisms of skin adherence and invasion by dermatophytes

Dermatophytes are keratinophilic fungi that can be pathogenic for humans and animals by infecting the stratum corneum, nails, claws or hair. The first infection step consists of adherence of arthroconidia to the stratum corneum. The mechanisms and the kinetics of adherence have been investigated using different in vitro and ex vivo experimental models, most notably showing the role of a secreted serine protease from Microsporum canis in fungal adherence to feline corneocytes. After germination of the arthroconidia, dermatophytes invade keratinised structures that have to be digested into short peptides and amino acids to be assimilated. Although many proteases, including keratinolytic ones, have been characterised, the understanding of dermatophyte invasion mechanisms remains speculative. To date, research on mechanisms of dermatophyte infection focused mainly on both secreted endoproteases and exoproteases, but their precise role in both fungal adherence and skin invasion should be further explored.

Assessment of the cutaneous immune response during Arthroderma benhamiae and A. vanbreuseghemii infection using an experimental mouse model

Dermatophytoses are common but poorly understood skin infections. Most in vivo studies have been performed using the guinea pig as the experimental animal model, which has several limitations.

Subtilisin Sub3 is involved in adherence of Microsporum canis to human and animal epidermis

The aim of this study was to assess the role of the secreted keratinolytic subtilisin-like protease Sub3 in adherence of Microsporum canis to epidermis from various susceptible species, in addition to cat for which this role was recently demonstrated. Firstly, we showed by immunostaining that Sub3 is not expressed in arthroconidia from an M. canis SUB3 RNA-silenced strain but is present on the surface of arthroconidia from a SUB3 non-silenced parental strain. Secondly, comparative adherence assays using arthroconidia from both M. canis strains and skin explants from humans, dogs, horses, rabbits, guinea pigs, mice and cats revealed that only 8-16% of arthroconidia from the SUB3 silenced strain adhered to different types of epidermis when compared to the control strain. Attempts to restore fungal adherence by the addition of recombinant Sub3 failed in the tested conditions. Overall results show for the first time that Sub3 is necessary for the adherence of M. canis arthroconidia to epidermis from humans and other animal species than cat, supporting the idea that Sub3 plays a central role in colonization of keratinized host structures by M. canis, whatever the host.

Feline polymorphonuclear neutrophils produce pro-inflammatory cytokines following exposure to Microsporum canis

The mechanisms involved in the establishment of the specific immune response against dermatophytes remain unknown. Polymorphonuclear neutrophils (PMNs) are recruited early during the infection process and participate in the elimination of dermatophytes. They could therefore be involved in the induction of the immune response during dermatophytoses by producing specific cytokines. The aim of this work was to assess the in vitro cytokine production by feline PMNs exposed to living arthroconidia from the dermatophyte species Microsporum canis or stimulated with either a secreted or a structural component of M. canis, the latter consisting of heat-killed arthroconidia. The levels of specific cytokines produced by PMNs were determined by capture ELISA and/or quantitative RT-PCR. Results showed that PMNs secrete TNFα, IL-1β and IL-8 following exposure to M. canis living arthroconidia and stimulation with both a secreted component and heat-killed arthroconidia. The level of IL-8 mRNA was also increased in PMNs stimulated with M. canis living arthroconidia. In conclusion, infective M. canis arthroconidia induce the production of pro-inflammatory cytokines by feline PMNs that can be activated either by secreted or structural fungal components. Our results suggest that these granulocytes are involved in the initiation of the immune response against M. canis.

Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats

Microsporum canis is the main pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis in domestic carnivores. In cats, M. canis causes symptomatic or asymptomatic infection. Recent conflicting data raise the question of whether the clinical status of the infected cat (symptomatic or asymptomatic) is directly correlated to the proteolytic activity of M. canis strains. Here, the transcription of fungalysin and dipeptidyl-peptidase genes (DPP) of M. canis was compared between four strains isolated from symptomatic and asymptomatic cats during the first steps of the infection process, namely in arthroconidia, during adherence of arthroconidia to corneocytes and during early invasion of the epidermis, using a new ex vivo model made of feline epidermis. There was no detectable transcription of the fungalysin genes in arthroconidia or during the first steps of the infection process for any of the tested strains, suggesting that these proteases play a role later in the infection process. Among DPP, the DPP IV gene was the most frequently transcribed both in arthroconidia and later during infection (adherence and invasion), but no significant differences were observed between M. canis strains isolated from symptomatic and asymptomatic cats. This study shows that the clinical aspect of M. canis feline dermatophytosis depends upon factors relating to the host rather than to the proteolytic activity of the infective fungal strain.

Are Th17 Cells Playing a Role in Immunity to Dermatophytosis

Despite their superficial localization in the skin, pathogenic dermatophytes can induce a complex but still misunderstood immune response in their hosts. The cell-mediated immunity (CMI) is correlated with both clinical recovery and protection against reinfection, and CD4+ T lymphocytes have been recognized as a crucial component of the immune defense against dermatophytes. Before the discovery of the Th17 pathway, CMI was considered to be only dependent of Th1 cells, and thus most studies on the immunology of dermatophytosis have focused on the Th1 pathway. Nevertheless, the fine comparative analysis of available scientific data on immunology of dermatophytosis in one hand and on the Th17 pathway mechanisms involved in opportunistic mucosal fungal infections in the other hand reveals that some key elements of the Th17 pathway can be activated by dermatophytes. Stimulation of the Th17 pathway could occur through the activation of some C-type lectin-like receptors and inflammasome in antigen-presenting cells. The Th17 cells could go back to the affected skin and by the production of signature cytokines could induce the effector mechanisms like the recruitment of polymorphonuclear neutrophils and the synthesis of antimicrobial peptides. In conclusion, besides the Th1 pathway, which is important to the immune response against dermatophytes, there are also growing evidences for the involvement of the Th17 pathway.

Relevant Animal Models in Dermatophyte Research

Dermatophytoses are common superficial fungal infections affecting both humans and animals. They are provoked by filamentous fungi called dermatophytes specialized in the degradation of keratinized structures, which allows them to induce skin, hair and nail infections. Despite their high incidence, little investigation has been performed for the understanding of these infections compared to fungal opportunistic infections and most of the studies were based on in vitro experiments. The development of animal models for dermatophyte research is required to evaluate new treatments against dermatophytoses or to increase knowledge about fungal pathogenicity factors or host immune response mechanisms. The guinea pig has been the most often used animal model to evaluate efficacy of antifungal compounds against dermatophytes, while mouse models were preferred to study the immune response generated during the disease. Here, we review the relevant animal models that were developed for dermatophyte research and we discuss the advantages and disadvantages of the selected species, especially guinea pig and mouse.

Inhibition of the keratinolytic subtilisin protease Sub3 from Microsporum canis by its propeptide (proSub3) and evaluation of the capacity of proSub3 to inhibit fungal adherence to feline epidermis

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats, dogs and humans. Proteolytic enzymes have been postulated to be key factors involved in the invasion of the stratum corneum and keratinized epidermal structures. Among these proteases, the secreted subtilisin protease Sub3 was found to be required for adherence of M. canis arthroconidia to feline epidermis. This protease is synthetized as a preproenzyme consisting of a signal peptide followed by the propeptide and the protease domain. In order to assess whether the enzymatic activity of Sub3 could be responsible for the role of the protease in the adherence process, we expressed and characterized the propeptide of Sub3 and demonstrated that this propeptide is a strong inhibitor of its mature enzyme. This propeptide acts as a noncompetitive inhibitor with dissociation constants, K(I) and [Formula: see text] of 170 and 130 nM respectively. When tested for its capacity to inhibit adherence of M. canis to feline epidermis using an ex vivo adherence model made of feline epidermis, the propeptide does not prevent adherence of M. canis arthroconidia because it loses its capacity to inhibit rSub3 following a direct contact with living arthroconidia, presumably through inactivation by fungal membrane-bound proteases.

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.

Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis.

BACKGROUND Polymorphonuclear neutrophils (PMNs), along with macrophages, are the first leukocytes recruited to the site of infection in dermatophytoses and are responsible for the in fine elimination of the fungus. It has been demonstrated that feline PMNs produce pro-inflammatory cytokines after stimulation with Microsporum canis. The activation of these cells results from the recognition of specific PAMPs (pathogen associated molecular patterns) from M. canis by PRRs (pattern recognition receptors) of PMNs. The C-type lectin receptors (CLRs) and toll-like receptors (TLRs) are the two main PRRs in phagocytic cells that recognize fungal components. HYPOTHESIS/OBJECTIVE The aim of this study was to evaluate the expression of TLR-2, TLR-4 and dectin-1 mRNA in feline PMNs exposed to different components from M. canis. METHODS Feline PMNs were stimulated for 2 h or 4 h with either live arthroconidia, heat-killed arthroconidia or secreted components from M. canis. The levels of TLR-2, TLR-4 and dectin-1 mRNA were assessed by RT-qPCR. RESULTS Results showed an increase of TLR-2 and TLR-4 mRNA levels in feline PMNs stimulated with live and heat-killed arthroconidia, but not in those stimulated with the secreted components from M. canis. No significant variation in dectin-1 mRNA expression was observed in PMNs stimulated with the different fungal components. CONCLUSIONS AND CLINICAL IMPORTANCE The overexpression of TLR-2 and TLR-4 mRNAs in stimulated feline PMNs suggests that these receptors are involved in the host immune response through the recognition of M. canis PAMPs.