Grégory Jouvion

Deep Dermatophytosis and Inherited CARD9 Deficiency

BACKGROUND Deep dermatophytosis is a severe and sometimes life-threatening fungal infection caused by dermatophytes. It is characterized by extensive dermal and subcutaneous tissue invasion and by frequent dissemination to the lymph nodes and, occasionally, the central nervous system. The condition is different from common superficial dermatophyte infection and has been reported in patients with no known immunodeficiency. Patients are mostly from North African, consanguineous, multiplex families, which strongly suggests a mendelian genetic cause. METHODS We studied the clinical features of deep dermatophytosis in 17 patients with no known immunodeficiency from eight unrelated Tunisian, Algerian, and Moroccan families. Because CARD9 (caspase recruitment domain-containing protein 9) deficiency has been reported in an Iranian family with invasive fungal infections, we also sequenced CARD9 in the patients. RESULTS Four patients died, at 28, 29, 37, and 39 years of age, with clinically active deep dermatophytosis. No other severe infections, fungal or otherwise, were reported in the surviving patients, who ranged in age from 37 to 75 years. The 15 Algerian and Tunisian patients, from seven unrelated families, had a homozygous Q289X CARD9 allele, due to a founder effect. The 2 Moroccan siblings were homozygous for the R101C CARD9 allele. Both alleles are rare deleterious variants. The familial segregation of these alleles was consistent with autosomal recessive inheritance and complete clinical penetrance. CONCLUSIONS All the patients with deep dermatophytosis had autosomal recessive CARD9 deficiency. Deep dermatophytosis appears to be an important clinical manifestation of CARD9 deficiency. (Funded by Agence Nationale pour la Recherche and others.).

Liver-Resident Macrophage Necroptosis Orchestrates Type 1 Microbicidal Inflammation and Type-2-Mediated Tissue Repair during Bacterial Infection

Kupffer cells, the phagocytes of fetal origin that line the liver sinusoids, are key contributors of host defense against enteroinvasive bacteria. Here, we found that infection by Listeria monocytogenes induced the early necroptotic death of Kupffer cells, which was followed by monocyte recruitment and an anti-bacterial type 1 inflammatory response. Kupffer cell death also triggered a type 2 response that involved the hepatocyte-derived alarmin interleukin-33 (IL-33) and basophil-derived interleukin-4 (IL-4). This led to the alternative activation of the monocyte-derived macrophages recruited to the liver, which thereby replaced ablated Kupffer cells and restored liver homeostasis. Kupffer cell death is therefore a key signal orchestrating type 1 microbicidal inflammation and type-2-mediated liver repair upon infection. This indicates that beyond the classical dichotomy of type 1 and type 2 responses, these responses can develop sequentially in the context of a bacterial infection and act interdependently, orchestrating liver immune responses and return to homeostasis, respectively.

Characterization of a Serine/Threonine Kinase Involved in Virulence of Staphylococcus aureus

Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Deltastk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis

ABSTRACT Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.

Genetic variability and integration of Merkel cell polyomavirus in Merkel cell carcinoma.

Merkel cell polyomavirus (MCPyV) is associated to Merkel cell carcinoma (MCC). We studied 113 MCC tumoral skin lesions originating from 97 patients. MCPyV detection was higher in fresh-frozen (FF) biopsies (94%) than in formalin-fixed paraffin-embedded biopsies (39-47%). Mean viral load in FF tumor was of 7.5 copies per cell with a very wide range (0.01-95.4). Nineteen complete sequences of LTAg were obtained, mainly from FF biopsies when the viral load was high. Seventeen showed stop codons, all localized downstream of the pRb protein binding domain. Sequence comparison and phylogenetic analysis showed that all sequences clustered in the large C clade of MCPyV strains. MCPyV integration was demonstrated in 19 out of 27 FF MCC DNA biopsies without evidence of specific host cellular genome integration site. In 13/19 cases, the viral junction was located within the second exon of the LTAg, after the pRB binding domain.

In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis

BackgroundInvasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain.ResultsIn this study, we demonstrate that bioluminescence imaging can track the progression of IA at different anatomic locations in a murine model of disease that recapitulates the natural route of infection. To define the temporal and functional requirements of distinct innate immune cellular subsets in host defense against respiratory A. fumigatus infection, we examined the development and progression of IA using bioluminescence imaging and histopathologic analysis in mice with four different types of pharmacologic or numeric defects in innate immune function that target resident and recruited phagocyte subsets. While bioluminescence imaging can track the progression and location of invasive disease in vivo, signals can be attenuated by severe inflammation and associated tissue hypoxia. However, especially under non-inflammatory conditions, such as cyclophosphamide treatment, an increasing bioluminescence signal reflects the increasing biomass of alive fungal cells.ConclusionsImaging studies allowed an in vivo correlation between the onset, peak, and kinetics of hyphal tissue invasion from the lung under conditions of functional or numeric inactivation of phagocytes and sheds light on the germination speed of conidia under the different immunosuppression regimens. Conditions of high inflammation -either mediated by neutrophil influx under corticosteroid treatment or by monocytes recruited during antibody-mediated depletion of neutrophils- were associated with rapid conidial germination and caused an early rise in bioluminescence post-infection. In contrast, 80% alveolar macrophage depletion failed to trigger a bioluminescent signal, consistent with the notion that neutrophil recruitment is essential for early host defense, while alveolar macrophage depletion can be functionally compensated.

Systemic Delivery of Allogenic Muscle Stem Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dogs clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Molecular Characterization of a Streptococcus gallolyticus Genomic Island Encoding a Pilus Involved in Endocarditis

BACKGROUND Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. METHODS S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. RESULTS We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. CONCLUSIONS This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Comparative Study of Injury Models for Studying Muscle Regeneration in Mice

Background A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. Methods We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®. Results We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a “dead zone” devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models. Conclusions Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.

Noninvasive Imaging Technologies Reveal Edema Toxin as a Key Virulence Factor in Anthrax

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection.