Mycologie medicale dans l'ouest de la France: Contribution du service de Parasitologie de l'Universite de NANTES (1963–1973)

Résumé Le service de Parasitologie de l'Université de NANTES s'est intéressé aux problèmes de Mycologie hospitalière, d'épidémiologie des mycoses ainsi que d'expérimentation sur les affinités fongiques, à l'échelon régional, et de 1963 à 1973.La contribution clinique a porté essentiellement sur les levuroses étudiées soit au niveau des cavités buccales irradiées, soit chez les malades des services de Réanimation où l'interprétation du rôle pathogène de Candida parapsilosis nécessite des épreuves immunologiques.Nous avons décrit une forme de Blastomycose chéloïdienne à Aureobasidium pullulans.Nos études morphologiques ont surtout porté sur l'observation au microscope électronique à balayage des ultrasculptures présentées par les champignons kératinophiles, ce qui nous a permis de distinguer les différentes espèces du complexe Microsporum gypseum. Il ne semble pourtant pas que cette technique d'observation permette de résoudre tous les problèmes. La conservation des Dermatophytes en eaudistillée par la méthode de Castellani s'est révélée particulièrement remarquable: une souche de M. gypseum a été récupérée après 6 ans de conservation par ce procédé.Notons, en matière d'épidémiologie la prédominance de Trichophyton rubrum sur T. mentagrophytes, survenue récemment: la recrudescence passagère d'Epidermophyton floccosum; la présence d'Arthroderma simii sur une plage. Une étude des champignons kératinophiles telluriques du massif armoricain a porté sur près de 3000 échantillons de terre, ce qui a permis de retrouver, assez rarement d'ailleurs, Arthroderma benhamiae.Nos expérimentations ont porté sur les réponses sérologiques et histologiques de lapins à une imprégnation aspergillaire par instillation intra-trachéale: les résultats obtenus ont été comparés aux examens sérologiques obtenus chez l'homme atteint d'aspergillose. Enfin, nous avons remarqué que le contact levures-cellules sarcomateuses opéré in vitro permet aux levures d'acquérir un pouvoir immunisant antitumoral par emprunt antigénique. La création d'un service de Parasitologie, Mycologie et Immunologie Parasitaire à l'Unité d'Enseignement et de Recherche des Techniques Médicales de NANTES nous a permis non seulement d'aborder les problèmes du diagnostic biologique des mycoses humaines, mais aussi d'entreprendre des enquêtes épidémiologiques dans la région nantaise ainsi que des expériences sur les affinités fongiques.

---

     

Dematin, a component of the erythrocyte membrane skeleton, is internalized by the malaria parasite and associates with Plasmodium 14-3-3

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.     

Toxoplasma gondii Virulence Factor ROP18 Inhibits the Host NF-κB Pathway by Promoting p65 Degradation

The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.     

Optimization of the electrophile of chloronitrobenzamide leads active against Trypanosoma brucei

We previously reported the phenylchloronitrobenzamides (PCNBs), a novel class of compounds active against the species of trypanosomes that cause Human African Trypanosomiasis (HAT). Herein, we explored the potential to adjust the reactivity of the electrophilic chloronitrobenzamide core. These studies identified compound 7d that potently inhibited the growth of trypanosomes (EC₅₀ = 120 nM for Trypanosoma b. brucei, 18 nM for Trypanosoma b. rhodesiense, and 38 nM for Trypanosoma b. gambiense) without significant cytotoxicity against mammalian cell lines (EC₅₀ > 25 μM for HepG2, HEK293, Raji, and BJ cell lines) and also had good stability in microsomal models (t_1/2 > 4 h in both human and mouse). Overall these properties indicate the compound 7d and its analogs are worth further exploration as potential leads for HAT.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Prolyl 4-Hydroxlase Activity Is Essential for Development and Cuticle Formation in the Human Infective Parasitic Nematode Brugia malayi

Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.     

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.     

Positive phototropism is accelerated in Biomphalaria glabrata snails by infection with Schistosoma mansoni

Parasite-induced behavioral changes in their hosts favor to complete the lifecycle of parasites. Schistosome infection is also known to cause physiological changes in infected freshwater snail intermediate hosts. Here, we report, a novel phenomenon in which Schistosoma mansoni, a highly debilitating worm affecting millions of people worldwide, alters the phototropic behavior of Biomphalaria glabrata, the vector snail. S. mansoni-infection enhanced positive phototropism of vector snails and infected snails spent significantly more time in light. Possibly, these behavioral changes help the parasite to be released efficiently from the infected intermediate hosts, and to infect mammalian hosts.