Marie-Christine Prévost

A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy.

Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21–23. This region contains the αB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. αB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and αB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chro‐matin condensation and large‐scale (—50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c‐Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio‐nuclear redistribution of AIF is prevented by a Bcl‐2 protein specifically targeted to mitochondrial membranes. The pan‐caspase inhibitor Z‐VAD.fmk does not prevent the staurosporin‐induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a ‘necrotic’ phenotype. Both in the presence of Z‐VAD.fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large‐scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase‐independent mitochondrial death effector responsible for partial chromatinolysis.—Daugas, E., Susin, S. A., Zamzami, N., Ferri, K., Irinopoulou, T., Larochette, N., Prévost, M.‐C, Leber, B., Andrews, D., Penninger, J., Kroemer, G. Mitochondrio‐nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14, 729–739 (2000)

Characterization of Reemerging Chikungunya Virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-β response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole.

By creating mutations within the Shigella flexneri ipaB gene, we have demonstrated that the invasion of epithelial cells is a three‐step process encompassing adhesion on the cell surface, entry and lysis of the phagocytic vacuole allowing subsequent access to the cytoplasm. SC403, an insertion mutant which lacks expression of IpaB but still expresses downstream genes, has been particularly studied. It is non‐invasive, does not elicit actin polymerization, but binds to HeLa cells indicating that an adhesion step occurs immediately prior to the entry process. The consequence of the inactivation of ipaB on the intracellular behaviour of S.flexneri was investigated using the macrophage cell line J774. SC403 was unable to lyse the phagocytic vacuole; moreover, this strain did not display the contact mediated haemolytic activity characteristics of Shigella. In addition to being a major component of the invasion complex, IpaB acts as a membrane‐lysing toxin enabling escape to the cytoplasmic compartment.

Are the PE‐PGRS proteins of Mycobacterium tuberculosis variable surface antigens?

Mycobacterium tuberculosis H37Rv contains 67 PE‐PGRS genes, with multiple tandem repetitive sequences, encoding closely related proteins that are exceptionally rich in glycine and alanine. As no functional information was available, 10 of these genes were selected and shown to be expressed in vitro by reverse transcription–polymerase chain reaction (RT–PCR). Antibodies against five PE‐PGRS proteins, raised in mice by DNA vaccination, detected single proteins when the same plasmid constructs used for immunization were expressed in epithelial cells or in reticulocyte extracts, confirming that the PE‐PGRS proteins are antigenic. As expected from the conserved repetitive structure, the antibodies cross‐reacted with more than one PE‐PGRS protein, suggesting that different proteins share common epitopes. PE‐PGRS proteins were detected by West‐ern blotting in five different mycobacterial species (M. tuberculosis, M. bovis BCG, M. smegmatis, M. marinum and M. gordonae) and 11 clinical isolates of M. tuberculosis. Whole‐genome comparisons of M. tuberculosis predicted allelic diversity in the PE‐PGRS family, and this was confirmed by immunoblot studies as size variants were detected in clinical strains. Subcellular fractionation studies and immunoelectron microscopy localized many PE‐PGRS proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE‐PGRS proteins are variable surface antigens.

Human Immunodeficiency Virus Type 1 Entry into Macrophages Mediated by Macropinocytosis

ABSTRACT Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.

Is adipose tissue a place for Mycobacterium tuberculosis persistence

Background Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has the ability to persist in its human host for exceptionally long periods of time. However, little is known about the location of the bacilli in latently infected individuals. Long-term mycobacterial persistence in the lungs has been reported, but this may not sufficiently account for strictly extra-pulmonary TB, which represents 10–15% of the reactivation cases. Methodology/Principal Findings We applied in situ and conventional PCR to sections of adipose tissue samples of various anatomical origins from 19 individuals from Mexico and 20 from France who had died from causes other than TB. M. tuberculosis DNA could be detected by either or both techniques in fat tissue surrounding the kidneys, the stomach, the lymph nodes, the heart and the skin in 9/57 Mexican samples (6/19 individuals), and in 8/26 French samples (6/20 individuals). In addition, mycobacteria could be immuno-detected in perinodal adipose tissue of 1 out of 3 biopsy samples from individuals with active TB. In vitro, using a combination of adipose cell models, including the widely used murine adipose cell line 3T3-L1, as well as primary human adipocytes, we show that after binding to scavenger receptors, M. tuberculosis can enter within adipocytes, where it accumulates intracytoplasmic lipid inclusions and survives in a non-replicating state that is insensitive to the major anti-mycobacterial drug isoniazid. Conclusions/Significance Given the abundance and the wide distribution of the adipose tissue throughout the body, our results suggest that this tissue, among others, might constitute a vast reservoir where the tubercle bacillus could persist for long periods of time, and avoid both killing by antimicrobials and recognition by the host immune system. In addition, M. tuberculosis-infected adipocytes might provide a new model to investigate dormancy and to evaluate new drugs for the treatment of persistent infection.

IpaB mediates macrophage apoptosis induced by Shigella flexneri

Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigellas virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hlylow). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hlylow. Both δipaC hlylow and δipaD hlylow were cytotoxic whilst δipaB hlylow and a hlylow strain cured of shigellas pathogenicity plasmid were not. Furthermore, both δipC ahlylow and δipaD hlylow killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host ceil DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.

Assembly and role of pili in group B streptococci

Streptococcus agalactiae[group B streptococcus (GBS)] is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1‐C2 and srtC3‐C4, with a similar genetic organization and are thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG protein encoding genes, two upstream and one downstream, and a divergently transcribed regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3‐C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3‐C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus‐associated adhesin. Surprisingly, pilus‐like structures can be formed in the absence of the two minor components, i.e. the putative anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Gram‐positive pathogens.