The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD(+). Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD(+) glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a new NAD(+) glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms

Etude du protéome membranaire de souches d'Acinetobacter baumannii multirésistantes aux antibiotiques

Le développement des épidémies d'infections à Acinetobacter baumannii en milieu hospitalier pose le problème de l'émergence des bactéries multirésistantes. Deux causes majeures peuvent expliquer cette résistance : l'existence d'enzymes de dégradation spécifiques ou la modification de la perméabilité membranaire. Nous avons analysé le protéome des membranes interne et externe de la souche type ATCC 19606 et identifié 135 protéines membranaires. La comparaison de ces protéomes entre celle-ci et une souche clinique résistante (CHU de Rouen) a permis de montrer chez cet isolat une modification structurale de CarO, des isoformes d'OmpW, la sous-expression de PonB et l'accumulation de protéines précurseurs des biofilms. L'analyse du sous-protéome membranaire d'une seconde souche résistante a montré la sous-expression des protéines CarO et montré sa capacité à former des canaux ioniques.The recent increase of Acinetobacter baumannii outbreaks in hospital environment highlights problems to antibiotic multi-drug resistance. This feature can be explained by the production of antibiotic degrading enzymes or by alterations in the cell wall permeability. We investigated inner and outer membrane subproteomes of the ATCC 19606 type strain and identified 135 membrane proteins. By comparing the subproteomes of the reference strain with those obtained from a MDR isolate (Rouen Hospital), we showed that this isolate exhibits some structural modifications in CarO, different isoforms of OmpW, under-expresses PonB and accumulates proteins involved in the first steps of biofilm formation. The analysis of the outer membrane proteins profile of a second MDR strain highlighted the under-expression of CarO and an OprD-like proteins. We consequently investigated structural and functional properties of CarO and highlighted its ability to form ionic channels.ROUEN-BU Sciences (764512102) / SudocROUEN-BU Sciences Madrillet (765752101) / SudocSudocFranceF

Weaving of bacterial cellulose by the Bcs secretion systems

International audienceAbstract Cellulose is the most abundant biological compound on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in many diverse bacterial species. While bacterial cellulose was first described in the 19th century, it was not until this last decade that a string of structural works provided insights into how the cellulose synthase BcsA, assisted by its inner-membrane partner BcsB, senses c-di-GMP to simultaneously polymerize its substrate and extrude the nascent polysaccharide across the inner bacterial membrane. It is now established that bacterial cellulose can be produced by several distinct types of cellulose secretion systems and that in addition to BcsAB, they can feature multiple accessory subunits, often indispensable for polysaccharide production. Importantly, the last years mark significant progress in our understanding not only of cellulose polymerization per se, but also of the bigger picture of bacterial signaling, secretion system assembly, biofilm formation and host tissue colonization, as well as of structural and functional parallels of this dominant biosynthetic process between the bacterial and eukaryotic domains of life. Here we review current mechanistic knowledge on bacterial cellulose secretion with focus on the structure, assembly and cooperativity of Bcs secretion system components

Structure-function relationships of CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

International audienceOBJECTIVES: In the context of the increasing worldwide occurrence of imipenem-resistant Acinetobacter baumannii strains, we investigated a possible porin-mediated mechanism relating to the carbapenem resistance-associated outer membrane protein, CarO. The aim of this study was to determine whether this porin may be a diffusion pathway for carbapenems in A. baumannii. METHODS: By analysing and comparing the sequences of CarO with protein databanks, we identified two major groups of sequences that we named CarOa and CarOb. We overproduced in Escherichia coli, extracted, purified by affinity chromatography and refolded in Triton X-100 rCarO from both groups. Their functional properties were investigated and compared by reconstitution in planar lipid bilayers. RESULTS: This functional study showed that rCarOa and rCarOb exhibit identical single-channel conductances (i.e. 20 pS in 1 M KCl) and similar poor cationic selectivity. Both channels were not specific towards meropenem and glutamic acid and poorly specific towards arginine, but they presented a marked specificity towards imipenem. From the calculated binding constants, we highlight that the CarOb channel was twice as specific as the CarOa channel for this antibiotic. Moreover, the CarOa channel could facilitate ornithine diffusion when the CarOb channel would not. CONCLUSIONS: We provide here the first evidence that CarO channels possess an imipenem (but not meropenem) binding site, and that their specificities depend on their primary structure. Any decrease in CarO expression would thus reduce the susceptibility of A. baumannii to this antibiotic

Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M(w) and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels

Global Comparison of the Membrane Subproteomes between a Multidrug-Resistant Acinetobacter baumannii Strain and a Reference Strain

International audienc

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis