Biology subjects

Crouzols, A.

Publications and source records attributed to Crouzols, A..

7 recordsLinked to original sources

bioRxiv2026

Gnotobiotic OMM12 mice support sustained Clostridioides difficile colonization without antibiotic pretreatment

Antibiotic-induced disruption of the intestinal microbiota predispose individuals to Clostridioides difficile infection (CDI). Although several animal models reproduce acute CDI, sustained colonization in the presence of a defined resident microbiota remains challenging to study. Here, we show that gnotobiotic OMM12 mice support sustained colonization for at least 20 days following oral challenge with either the reference CD630{Delta}erm strain or the clinical R20291 (RT027) strain, without prior antibiotic treatment. Early infection induced transient weight loss, rapid intestinal inflammation and transient shifts in the relative abundance of resident bacterial species. By day 20, inflammatory markers had largely resolved, and the resident microbiota had shifted back towards its baseline composition, despite continued detection of C. difficile vegetative cells, spores and toxins. Early bacterial community perturbations were not accompanied by detectable changes in the resident prophage induction. Moreover, whole-genome sequencing of C. difficile clones recovered at day 20 showed no detectable gut-specific genomic adaptation relative to the inoculum. Similar colonization kinetics and clinical outcomes were observed across independent experiments conducted over five years, supporting the consistent reproducibility of this model. Together, OMM12 mice provide a defined, antibiotic-free and experimentally tractable framework to investigate host-microbiota-pathogen interactions during sustained C. difficile colonization and a robust platform for therapeutic development.

microbiology↗

bioRxiv2024

In vitro biofilms of Clostridioides difficile undomesticated strains: Morphology and properties according to strain diversity

Clostridioides difficile is a Gram-positive, spore-forming, obligate anaerobe and an entero-pathogen representing a One Health problem. C. difficile infections are difficult to treat and recurrences frequent. C. difficile biofilms may play a role in persistence. We studied the biofilms formed in vitro by a collection of C. difficile undomesticated strains (n=27, 20 toxigenic and 7 non-toxigenic) of 11 PCR-ribotypes (WRT) and isolated from equids. After growth in rich BHIS medium supplemented with glucose for 48h, the biomass of biofilms stained by crystal violet, i. e. adhesive to the surface, was measured, while intact biofilms were observed in situ, by confocal laser scanning microscopy, to determine their total biovolume and 3D morphology. All biofilms were relatively similar, even though adhesive, stained biofilms of non-toxigenic strains of ribotype 009 significantly displayed highest biomass. These results suggested that strains of ribotype 009 were able to form a highly cohesive and polystyrene-adhesive biofilm than any other strains. 8 common, representative ribotypes in equids, including 6 clinically important ribotypes in humans, were then selected and one strain chosen for each of them. Their biofilms and planktonic cultures grown under the same conditions were compared for sporulation, toxin production and susceptibility to vancomycin at a highly inhibitory concentration (8 MIC). In both the biofilm and planktonic culture of all selected strains, sporulation was low and toxin production undetectable. The addition of vancomycin at T0=24h followed by a 24 hours exposure barely reduced the viability of neither planktonic cultures nor biofilms of any strains. On the contrary, for all selected strains, addition of vancomycin at T0=6h followed by a 24 hours exposure significantly and more efficiently reduced the viability of planktonic cultures than that of biofilms, which was not decreased at all in the case of strains of ribotype WRT 005, 009 and 035. The biofilms of these selected strains of eight ribotypes, compared to their planktonic cultures, therefore showed a higher tolerance to vancomycin, suggesting that they could play a role in persistence.

microbiology↗

bioRxiv2023

Stumpy forms are the predominant transmissible forms of Trypanosoma brucei.

Schuster et al. demonstrated that bloodstream slender forms of African trypanosomes are readily transmissible to young tsetse flies where they can complete their complex life cycle (1). In their experimental conditions, a single slender parasite was sufficient for productive infection. Here, we compared the infectivity of slender and stumpy bloodstream forms in adult flies with a mature immune system, and without using any chemical compounds that would alter the insect immune response and/or promote the infection. After ingestion of slender forms, infected flies were observed only in one out of 24 batches of non-immunocompetent teneral flies and with a high number of parasites. In contrast, infected flies were detected in 75% (18/24) of the batches infected with stumpy parasites, and as few as 10 stumpy parasites produced mature infections in immune adult flies. We discuss that, although Schuster et al. have demonstrated the intrinsic capacity of slender form trypanosomes to infect young and naive tsetse flies, highlighting the remarkable plasticity and adaptability of these protists, this phenomenon is unlikely to significantly contribute to the epidemiology of African trypanosomiases. According to both experimental and field observations, stumpy forms appear to be the most adapted forms for African trypanosome transmission from the mammalian host to the tsetse fly vector in natural conditions.

microbiology↗

bioRxiv2023

FLAgellum Member 8 modulates extravasation and extravascular distribution of African trypanosomes.

In the mammalian host, the biology of tissue-dwelling Trypanosoma brucei parasites is not completely understood, especially the mechanisms involved in their extravascular colonization. The trypanosome flagellum is an essential organelle in multiple aspects of the parasites development. The flagellar protein termed FLAgellar Member 8 (FLAM8) acts as a docking platform for a pool of Cyclic AMP response protein 3 (CARP3) that is involved in signaling. FLAM8 exhibits a stage-specific distribution suggesting specific functions in the mammalian and vector stages of the parasite. Analyses of knockdown and knockout trypanosomes in their mammalian forms demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes can establish and maintain an infection in the blood circulation and differentiate into insect transmissible forms. However, quantitative bioluminescence imaging and gene expression analysis revealed that FLAM8-null parasites exhibit a significantly impaired dissemination in the extravascular compartment, that is restored by the addition of a single rescue copy of FLAM8. In vitro trans-endothelial migration assays revealed significant defects in trypanosomes lacking FLAM8. FLAM8 is the first flagellar component shown to modulate T. brucei distribution in the host tissues, possibly through sensing functions, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin. Author SummaryTrypanosoma brucei parasites cause neglected tropical diseases termed human and animal African trypanosomiases. Transmitted by the bite of an infected tsetse fly, upon deposition in the skin of a mammalian host, these parasites occupy both the vasculature and extravascular tissues. Currently, the biology of tissue-resident parasites is not well understood, and the parasite factors that mediate extravascular colonization are not known. Using quantitative in vivo bioluminescence imaging and ex vivo gene expression quantification in host infected tissues and blood, we reveal that the flagellar parasite protein FLAM8 modulates the extravascular dissemination of trypanosomes in the mammalian host. FLAM8 is known to act as a docking platform for signaling complexes in the flagellum, but we observe that it does not influence parasite differentiation into transmissible stages. However, we show that the absence of FLAM8 results in the loss of a key component of the flagellar adenylate cyclase signaling complexes, and reduces parasite migration through endothelial cell monolayers, suggesting that FLAM8 is important for parasite exchanges between the intravascular and the extravascular compartments. This work identifies a key trypanosome flagellar component involved in host-parasite interactions, including the modulation of parasite tropism and extravascular dissemination.

microbiology↗

bioRxiv2021

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasites mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.

molecular biology↗

bioRxiv2021

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

molecular biology↗

bioRxiv2021

FLAgellum Member 8 modulates extravascular trypanosome distribution in the mammalian host

The African trypanosome flagellum is an essential organelle in multiple aspects of the parasites development. Here, we investigated the role of a flagellar protein termed FLAgellar Member 8 (FLAM8) that is specifically distributed along the entire flagellum in trypanosomes stages of the vertebrate host. Analyses of knockdown and knockout trypanosomes demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and slender to stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes are able to establish and maintain the infection in the blood circulation, and to differentiate into insect transmissible forms. However, quantitative bioluminescence imaging revealed that FLAM8-null parasites exhibit an impaired dissemination in the extravascular compartment, that is partially restored by the addition of a single rescue copy of FLAM8. Interestingly, among all dissected organs scrutinized individually, only the skin of mice infected with FLAM8-deprived parasites showed a significant reduction in extravascular trypanosome population as compared to mice infected with parental controls. To our knowledge, FLAM8 is the first example of a flagellar protein that modulates T. brucei parasite distribution in the host tissues, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin. Take awayO_LIFLAM8 is dispensable in vitro for survival, growth, motility and differentiation of T. brucei. C_LIO_LIFLAM8 depletion does not affect parasitemia and bloodstream form differentiation in vivo. C_LIO_LIFLAM8 modulates the extravascular dissemination of trypanosomes in the mammalian host, especially in the skin. C_LI

microbiology↗