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.

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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.     

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.     

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.     

Predation of Notiophilus (Coleoptera: Carabidae) on Collembola as a predator-prey teaching model

The carabid beetle Notiophilus preys readily on the collembolan Folsomia Candida Willem in very simple experimental conditions. Some features of this predator-prey system are outlined to emphasize its use as a teaching practical. Experiments with another potential collembolan prey, Onychiurus burmeisteri (Lubbock), are described in the context of developing the method for more advanced studies.     

Leishmania tropica: the effect of darkness and light on biological activities in vitro

Leishmania parasites can be exposed to effects of light in their vectors and hosts, at various periods. However, there is no information about the effects of light on Leishmania parasites. The aim of this study is to investigate the effects of light on various cell parameters of Leishmania tropica, in vitro. All experiments were conducted on L. tropica promastigotes and amastigote-macrophage cultures, using flow cytometric analysis, MTT and phenol–sulfuric acid assay, DAPI and Giemsa. The results showed that the morphology of parasites has changed; the cell cycle has been affected and this caused parasites to remain at G0/G1 phase. Furthermore the proliferation, infectivity, glucose consumption and mitochondrial dehydrogenase activities of parasites were decreased. Thus, for the first time, in this study, the effects of light on biological activities of Leishmania parasites were shown. These new information about parasites’ biology, would be very important to investigate the effects of light on the parasites in infected vectors and hosts.     

The monophyletic origin of a remarkable sexual system in akentrogonid rhizocephalan parasites: A molecular and larval structural study

We use sequences from the nuclear ribosomal genes, 18S and 28S to analyze the phylogeny of the Rhizocephala Akentrogonida including two species, Clistosaccus paguri and Chthamalophilus delagei, that are critical for understanding rhizocephalan evolution but have not previously been part of a molecularly based study. In addition we use light and scanning electron microscopy to compare the cypris larvae of C. paguri, Sylon hippolytes and two species of the family Thompsoniidae, since this larval stage offers a suite of characters for analyzing the evolution of these otherwise highly reduced parasites. The Rhizocephala Akentrogonida form a monophyletic group nested within a paraphyletic “Kentrogonida”. C. paguri and S. hippolytes are sistergroups confirming the monophyly of the Clistosaccidae that was originally based on similarities in the cypris larvae. We find numerous LM and SEM level similarities between the two species, many of which appear to be correlated with their specialized sexual system, where male cyprids use an antennule to implant cells into the virgin female parasite. Some of these traits are also found in cyprids of the thompsoniid species. We conclude that the special cypris morphology and the implantation of males by antennular penetration was present in the stem species to the Thompsoniidae and the Clistosaccidae and emphasize the power of larval characters in rhizocephalan systematics. C. delagei is a sister group to Boschmaella balani and the two are nested deep within the Akentrogonida. This confirms the monophyly of the Chthamalophilidae and falsifies the theory that C. delagei should represent the most primitive extant rhizocephalan. Instead, chthamalophilid rhizocephalans represent some of the most highly advanced members of the parasitic barnacles.     

Gorgoderina vitelliloba: interaction with frog leucocytes in vivo and in vitro

Ultrastructural observations were made on the response in vivo of adult Rana temporaria to Gorgoderina vitelliloba and the interaction between host cells and the parasite in vitro. In both cases, the cellular attack on the fluke was most intense 21-23 days postinfection. Host leucocytes (believed to be eosinophils) lay flat against the surface of the parasite. Degranulation of their electron-dense, peroxidase-positive granules occurred and vacuoles formed. The outer plasmalemma of the tegument was breached and eosinophils migrated over the basal lamina, stripping the tegument from the surface of the fluke. The cellular response was so vigorous in vitro that the tegument was sometimes completely lost. In vivo, damage was slight, and the majority of the parasites successfully completed their migration to the bladder, where there was no further cellular response, and tegumental repair occurred. The fact that the migration of the fluke from the kidney to the bladder occurred at the same time as the peak of the cellular response may not be coincidental, and it is suggested that the cellular attack may initiate migration from the kidneys.