Contribution of the Gut Microbiota in P28GST-Mediated Anti-Inflammatory Effects: Experimental and Clinical Insights.

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn's disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn's disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules.

Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii.

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.

Antifungal and cytotoxic activity of withanolides from Acnistus arborescens.

Three compounds were isolated from Acnistus arborescens, a tree commonly used in South and Central America in traditional medicine against several infectious diseases, some of which are caused by fungi. Bioassay-guided fractionation of a MeOH extract of leaves, based on its anti-Pneumocystis carinii activity, led to the isolation of compounds 1-3. Mono- and bidimensional NMR analyses enabled identification of two new withanolides, (20R,22R)-5beta,6beta-epoxy-4beta,12beta,20-trihydroxy-1-oxowith-2-en-24-enolide (1) and (20R,22R)-16beta-acetoxy-3beta,4beta;5beta,6beta-diepoxy-12beta,20-dihydroxy-1-oxowith-24-enolide (2), and withanolide D (3). Antifungal activity on 13 fungi responsible for human infections (five dermatophytes, one nondermatophyte mold, six yeasts, and Pneumocystis carinii) was examined. Cytotoxicity of these compounds was also evaluated in vitro.

High-speed cell sorting of infectious trophic and cystic forms of Pneumocystis carinii.

The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6+/-0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.

The Pneumocystis life cycle

First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.