Laboratory of Pathogens and Host Immunity

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Within-species genetic diversity is thought to reflect population size, history, ecology, and ability to adapt. Using a comprehensive collection of polymorphism data sets covering approximately 3000 animal species, we show that the widely used mitochondrial DNA (mtDNA) marker does not reflect species abundance or ecology: mtDNA diversity is not higher in invertebrates than in vertebrates, in marine than in terrestrial species, or in small than in large organisms. Nuclear loci, in contrast, fit these intuitive expectations. The unexpected mitochondrial diversity distribution is explained by recurrent adaptive evolution, challenging the neutral theory of molecular evolution and questioning the relevance of mtDNA in biodiversity and conservation studies.

Abstract genclone 1.0 is designed for studying clonality and its spatial components using genotype data with molecular markers from haploid or diploid organisms. genclone 1.0 performs the following tasks. (i) discriminates distinct multilocus genotypes (MLGs), and uses permutation and resampling approaches to test for the reliability of sets of loci and sampling units for estimating genotypic and genetic diversity (a procedure also useful for nonclonal organisms); (ii) computes statistics to test for clonal propagation or clonal identity of replicates; (iii) computes various indices describing genotypic diversity; and (iv) summarizes the spatial organization of MLGs with adapted spatial autocorrelation methods and clonal subrange estimates.

Successful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms--organized communities of surface-attached cells--but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

Mycobacterium abscessus is a rapidly growing Mycobacterium causing a wide spectrum of clinical syndromes. It now is recognized as a pulmonary pathogen to which cystic fibrosis patients have a particular susceptibility. The M. abscessus rough (R) variant, devoid of cell-surface glycopeptidolipids (GPLs), causes more severe clinical disease than the smooth (S) variant, but the underlying mechanisms of R-variant virulence remain obscure. Exploiting the optical transparency of zebrafish embryos, we observed that the increased virulence of the M. abscessus R variant compared with the S variant correlated with the loss of GPL production. The virulence of the R variant involved the massive production of serpentine cords, absent during S-variant infection, and the cords initiated abscess formation leading to rapid larval death. Cording occurred within the vasculature and was highly pronounced in the central nervous system (CNS). It appears that M. abscessus is transported to the CNS within macrophages. The release of M. abscessus from apoptotic macrophages initiated the formation of cords that grew too large to be phagocytized by macrophages or neutrophils. This study is a description of the crucial role of cording in the in vivo physiopathology of M. abscessus infection and emphasizes cording as a mechanism of immune evasion.

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.

While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa(+) and tnfa(-) macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa(+) macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic.

Most Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Beside chemical properties and topographical features, mechanical properties of gels have been recently demonstrated to play an important role in various cellular processes, including cell attachment, proliferation, and differentiation. In this work, we used multilayer films made of poly(L-lysine)/Hyaluronan (PLL/HA) of controlled stiffness to investigate the effects of mechanical properties of thin films on skeletal muscle cells (C2C12 cells) differentiation. Prior to differentiation, cells need to adhere and proliferate in growth medium. Stiff films (E(0) > 320 kPa) promoted formation of focal adhesions and organization of the cytoskeleton as well as an enhanced proliferation, whereas soft films were not favorable for cell anchoring, spreading or proliferation. Then C2C12 cells were switched to a low serum containing medium to induce cell differentiation, which was also greatly dependent on film stiffness. Although myogenin and troponin T expressions were only moderately affected by film stiffness, the morphology of the myotubes exhibited striking stiffness-dependent differences. Soft films allowed differentiation only for few days and the myotubes were very short and thick. Cell clumping followed by aggregates detachment could be observed after ~2 to 4 days. On stiffer films, significantly more elongated and thinner myotubes were observed for up to ~ 2 weeks. Myotube striation was also observed but only for the stiffer films. These results demonstrate that film stiffness modulates deeply adhesion, proliferation and differentiation, each of these processes having its own stiffness requirement.

Because the availability of fish genomic data, the number of reported sequences for fish type II helical cytokines is rapidly growing, featuring different IFNs including virus-induced IFNs (IFNphi) and IFN-gamma, and IL-10 with its related cytokines (IL-20, IL-22, and IL-26). Many candidate receptors exist for these cytokines and various authors have postulated which receptor chain would be involved in which functional receptor in fish. To date, only the receptor for zebrafish IFNphi1 has been identified functionally. Three genes encoding virus-induced IFNphis have been reported in zebrafish. In addition to these genes clustered on chromosome 3, we have identified a fourth IFNphi gene on chromosome 12. All these genes possess the intron-exon organization of mammalian lambda IFNs. In the zebrafish larva, all induce the expression of reporter antiviral genes; protection in a viral challenge assay was observed for IFNphi1 and IFNphi2. Using a combination of gain- and loss-of-function experiments, we also show that all zebrafish IFNphis do not bind to the same receptor. Two subgroups of fish virus-induced IFNs have been defined based on conserved cysteines, and we find that this subdivision correlates with receptor usage. Both receptor complexes include a common short chain receptor (CRFB5) and a specific long chain receptor (CRFB1 or CRFB2).

Abstract Efficient delivery of growth or survival factors to cells is one of the most important long‐term challenges of current cell‐based tissue engineering strategies. The extracellular matrix acts as a reservoir for a number of growth factors through interactions with its components. In the matrix, growth factors are protected against circulating proteases and locally concentrated. Thus, the localized and long‐lasting delivery of a matrix‐bound recombinant human bone morphogenetic protein 2 (rhBMP‐2) from a biomaterial surface would mimic in vivo conditions and increase BMP‐2 efficiency by limiting its degradation. Herein, it is shown that crosslinked poly( L ‐lysine)/hyaluronan (HA) layer‐by‐layer films can serve as a reservoir for rhBMP‐2 delivery to myoblasts and induce their differentiation into osteoblasts in a dose‐dependent manner. The amount of rhBMP‐2 loaded in the films is controlled by varying the deposition conditions and the film thickness. Its local concentration in the film is increased up to ≈500‐fold when compared to its initial solution concentration. Its adsorption on the films, as well as its diffusion within the films, is evidenced by microfluorimetry and confocal microscopy observations. A direct interaction of rhBMP‐2 with HA is demonstrated by size‐exclusion chromatography, which could be at the origin of the rhBMP‐2 “trapping” in the film and of its low release from the films. The bioactivity of rhBMP‐2‐loaded films is due neither to film degradation nor to rhBMP‐2 release. The rhBMP‐2‐containing films are extremely resistant and could sustain three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP‐2 immobilization. These films may find applications in the local delivery of immobilized growth factors for tissue‐engineered constructs and for metallic biomaterial surfaces, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP(2))-containing liposomes via its NH(2)-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP(2) binding were located to amino acids 12-115 and 233-310. Both regions contain a KK(X)(n)K/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP(2)-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP(2) with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP(2) dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP(2) binding site in ezrin whose mutagenesis correlates with an altered cellular localization.

Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.

International audience

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Purified human recombinant ezrin cosediments with large liposomes containing phosphatidylserine (PS). This interaction is optimal at low ionic strength. At physiological ionic strength (130 mM KCl) ezrin interacts strongly with liposomes containing > or = 5% phosphatidylinositol-4,5-bisphosphate (PIP2), the residual being phosphatidylcholine (PC). When PIP2 is replaced by phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol (PI) or PS, the interaction is markedly reduced. Furthermore we show, that a purified N-terminal glutathione S-transferase (GST) fusion protein of ezrin (1-309) still has retained the capacity to interact with PIP2-containing liposomes, whereas a C-terminal fusion protein (310-586) has lost this ability.

Polyelectrolyte multilayer films have been well characterized for almost two decades and there is now a growing interest for the development of biomimetic films that could be used in vitro or in vivo to control cellular behaviors. In this review, the important properties of multilayer films designed for cell/surface interactions will be highlighted. The first part will deal with the physico-chemical properties of polypeptide and polysaccharide multilayer films, including their growth, swellability, stability and mechanical properties. In the second part, we will focus on important properties influencing cellular behaviors: i) film biodegradability, ii) film mechanical properties, iii) film bioactivity achieved by either the intrinsic properties of the film components or the insertion of small peptides, proteins, or DNA. In particular, films thicker than one micron are particularly well suited for loading bioactive molecules due to their reservoir capacities.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.