Sophie Goyard

Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote‐harbouring macrophages and for quantitative real‐time monitoring of parasitism features in living mice

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite‐infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracel‐lular amastigote‐harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real‐time bioluminescent assay is not only sensitive but also more rapid than culture‐base techniques allowing to monitor parasite‐load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti‐leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.

Contribution of the glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans

The fungal pathogen Candida albicans forms therapeutically challenging biofilms on biomedical implants. Using a transcript profiling approach genes whose expression is favoured upon biofilm growth compared with planktonic growth have been previously identified. Knock‐out mutants for 38 of these genes were constructed, six of which showed a specific defect in biofilm formation. Among these genes, TYE7 that encodes a transcriptional activator of glycolytic genes in planktonic and biofilm growth conditions was identified as being required for the cohesiveness of biofilms. Biofilms formed by the tye7Δ knock‐out mutant showed a hyperfilamentous morphology, and growth of this mutant on solid medium under hypoxia was also associated with the production of hyphae. Similar to TYE7 inactivation, inhibition of glycolysis or ATP synthesis using oxalate or an uncoupler, respectively, triggered morphogenesis when a wild‐type strain was grown under hypoxia. These treatments also induced the formation of weakly cohesive, hyper‐filamentous biofilms by a wild‐type strain. Our data indicate that a hypoxic environment is generated within C. albicans biofilms and that continued biofilm development requires a Tye7p‐dependent upregulation of glycolytic genes necessary to adapt to hypoxia and prevent uncontrolled hyphal formation. Thus, adaptation to hypoxia is an integral component of biofilm formation in C. albicans.

The SUN41 and SUN42 genes are essential for cell separation in Candida albicans.

Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete‐specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall‐degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter‐balancing cell wall degradation to avoid cell lysis upon cell separation.

Optimization of topical therapy for Leishmania major localized cutaneous leishmaniasis using a reliable C57BL/6 Model.

Background Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized cutaneous leishmaniasis (LCL). Promising ointments are in the final stages of development. One main objective was to help optimize the treatment modalities of human LCL with WR279396, a topical formulation of aminoglycosides that was recently proven to be efficient and safe for use in humans. Methodology/Principal Findings C57BL/6 mice were inoculated in the ear with luciferase transgenic L. major and then treated with WR279396. The treatment period spanned lesion onset, and the evolution of clinical signs and bioluminescent parasite loads could be followed for several months without killing the mice. As judged by clinical healing and a 1.5-3 log parasite load decrease in less than 2 weeks, the 94% efficacy of 10 daily applications of WR279396 in mice was very similar to what had been previously observed in clinical trials. When WR279396 was applied with an occlusive dressing, parasitological and clinical efficacy was significantly increased and no rebound of parasite load was observed. In addition, 5 applications under occlusion were more efficient when done every other day for 10 days than daily for 5 days, showing that length of therapy is a more important determinant of treatment efficacy than the total dose topically applied. Conclusions/Significance Occlusion has a significant adjuvant effect on aminoglycoside ointment therapy of experimental cutaneaous leishmaniasis (CL), a concept that might apply to other antileishmanial or antimicrobial ointments. Generated in a laboratory mouse-based model that closely mimics the course of LCL in humans, our results support a schedule based on discontinuous applications for a few weeks rather than several daily applications for a few days.

A Versatile Overexpression Strategy in the Pathogenic Yeast Candida albicans: Identification of Regulators of Morphogenesis and Fitness

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.

Characterization of BpH3, an H-NS-like protein in Bordetella pertussis

This study describes the characterization of BpH3, a Bordetella pertussis DNA‐binding protein. Sequence analysis reveals significant homology with the H‐NS sequence of Escherichia coli and Haemophilus influenzae, particularly in the C‐terminal part of the proteins. Our results provide evidence that H‐NS and BpH3 display functional homology. First, expression of BpH3 in an hns mutant results in restoration of motility, an H‐NS‐dependent phenotype. This effect is dependent on the level of BpH3 expression and results from transcriptional activation of the flagellar master operon. Second, the high level of β‐glucosidase associated with hns mutations is reversed to the low wild‐type level in the presence of BpH3. Third, BpH3 is able, like H‐NS, to preferentially bind in vitro to curved DNA fragments, such as flhDC and bla promoter regions. Our results are the first demonstration that proteins homologous to H‐NS exist in bacteria phylogenetically distant from H. influenzae and enterobacteria.

Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. I. Parasitological, hematological and pathological parameters.

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.

Protein O-Mannosyltransferase Isoforms Regulate Biofilm Formation in Candida albicans

ABSTRACT Five isoforms of protein mannosyltransferase (Pmt) O-mannosylate secretory proteins in Candida albicans. pmt mutants were differentially defective for biofilm formation on plastic in static and flow-through systems, and a Pmt inhibitor blocked early stages of biofilm formation. Conceptually, Pmt inhibition may prevent surface anchoring and biofilm-dependent resistance of fungal pathogens.

Lipid droplet formation, their localization and dynamics during leishmania major macrophage infection

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

Non-Invasive In Vivo Study of the Trypanosoma vivax Infectious Process Consolidates the Brain Commitment in Late Infections

Trypanosoma vivax, one of the leading parasites responsible for Animal African Trypanosomosis (Nagana), is generally cyclically transmitted by Glossina spp. but in areas devoid of the tsetse flies in Africa or in Latin American countries is mechanically transmitted across vertebrate hosts by other haematophagous insects, including tabanids. We followed on from our recent studies on the maintenance of this parasite in vivo and in vitro, and its genetic manipulation, by constructing a West African IL1392 T. vivax strain that stably expresses firefly luciferase and is fully virulent for immunocompetent mice. We report here on a study where murine infection with this strain was monitored in vivo using a non-invasive method. Study findings fully support the use of this strain in the assessment of parasite dynamics in vivo since a strong correlation was found between whole body light emission measured over the course of the infection and parasitemia determined microscopically. In addition, parasitemia and survival rates were very similar for mice infected by the intraperitoneal and sub-cutaneous routes, except for a longer prepatent period following sub-cutaneous inoculation with the parasite. Our results clearly show that when administered by the subcutaneous route, the parasite is retained few days in the skin close to the inoculation site where it multiplies before passing into the bloodstream. Ex vivo bioluminescence analyses of organs isolated from infected mice corroborated our previous histopathological observations with parasite infiltration into spleen, liver and lungs. Finally, our study reinforces previous observations on the presence of the parasite in the central nervous system and consequently the brain commitment in the very late phases of the experimental infection.