Anne Mathy

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.

Secreted subtilisin Sub3 from Microsporum canis is required for adherence to but not for invasion of the epidermis.

Background  Microsporum canis is a pathogenic dermatophyte that causes a superficial cutaneous mycosis, mainly in cats and humans. Proteolytic enzymes, including subtilisins, have been postulated to be key factors involved in adherence and invasion of the stratum corneum and keratinized epidermal structures.

Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats and humans. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. Here, a model was developed to study the adherence of M. canis to feline corneocytes through the use of a reconstructed interfollicular feline epidermis (RFE). In this model, adherence of arthroconidia to RFE was found to be time-dependent, starting at 2 h post-inoculation and still increasing at 6 h. Chymostatin, a serine protease inhibitor, inhibited M. canis adherence to RFE by 53%. Moreover, two mAbs against the keratinolytic protease subtilisin 3 (Sub3) inhibited M. canis adherence to RFE by 23%, suggesting that subtilisins, and Sub3 in particular, are involved in the adherence process.

Immunization and dermatophytes.

Purpose of reviewDespite the availability of effective vaccines for certain animal species, vaccination against dermatophytosis requires improvement and further development in both animals and humans. This review provides an update on the current situation and focuses on recent advances in host–dermatophyte relationships that could have implications for future vaccination against the most prevalent of the fungal diseases. Recent findingsNumerous dermatophytic virulence factors have recently been isolated and characterized at the molecular level, notably secreted proteases involved in the invasion of the keratin network. Their precise roles in the different steps of the infectious process and in immunopathogenesis are being studied, while all aspects of the host immune response against dermatophytes, including the innate response, are becoming increasingly documented. In addition, new molecular tools are now available for studying dermatophytes, which will accelerate research on this topic. SummaryThe growth of knowledge concerning all aspects of the host–dermatophyte relationship should contribute towards sound strategies for the development of effective and safe vaccines against dermatophytosis.

THE CARRIAGE OF LARVAL ECHINOCOCCUS MULTILOCULARIS AND OTHER CESTODES BY THE MUSK RAT (ONDATRA ZIBETHICUS) ALONG THE OURTHE RIVER AND ITS TRIBUTARIES (BELGIUM)

In Belgium, the carriage of Echinococcus multilocularis by the red fox (Vulpes vulpes) can be very high in some areas. This study was designed to evaluate the carriage of the larval form of E. multilocularis and other cestodes in a musk rat (Ondatra zibethicus) population trapped along the Ourthe River (southeastern Belgium). Six hundred fifty-seven musk rats were necropsied, and the larval cestodes of the abdominal and pleural cavities were identified. For E. multilocularis, the fertility of the cysts was verified in 58 liver samples. The following species were found: Taenia taeniaeformis (65.8%), Taenia martis (22.2%), E. multilocularis (22.1%), Taenia polyacantha (2.6%), and Taenia crassiceps (0.9%). Results were analyzed according to the site of capture (upper, middle, and lower Ourthe). There was a highly significant relationship between the carriage of E. multilocularis and the site of capture (the prevalence being higher in the upper part of the river). This difference could be due to different geoclimatic conditions. All but one hepatic lesion were found to contain protoscoleces of E. multilocularis (98.8%). The musk rat is probably infected through the consumption of plant material contaminated by the foxs feces. The red fox can occasionally prey on musk rats, but the musk rat cadavers that are left on the river banks by the trappers are probably also consumed. This could favor the maintenance of E. multilocularis life cycle. In conclusion, the musk rat seems to be highly susceptible to E. multilocularis and in Belgium could play the role of reservoir; when present this species could represent an inexpensive and sensitive bioindicator for the study and monitoring of the zoonosis.

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.

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.