pH‐dependent pore‐forming activity of OmpATb from <i>Mycobacterium tuberculosis</i> and characterization of the channel by peptidic dissection

Molle, Virginie; Saint, Nathalie; Campagna, Sylvie; Kremer, Laurent; Lea, E.J.A.; Draper, P.; Molle, Gérard

doi:10.1111/j.1365-2958.2006.05277.x

Cited by 39 publications

(69 citation statements)

References 40 publications

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“…The average singlechannel conductance of 50 channels of the CpnT NTD O2 oligomer was 1.2 ± 0.5 nS (Fig. 3D), which is consistent with the conductance of the unfractionated CpnT NTD sample but distinct from other mycobacterial pore proteins (27)(28)(29)(30). The fast flickering events in the current traces of oligomeric CpnT NTD involve opening and closing of complete channels and of subconductance states (Fig.…”

Section: Significancesupporting

confidence: 60%

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Danilchanka¹,

Sun²,

Pavlenok³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

113

136

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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.transport | pore | secretion

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Section: Significancesupporting

confidence: 60%

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Danilchanka¹,

Sun²,

Pavlenok³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

113

136

View full text Add to dashboard Cite

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“…Despite the well-documented importance of OMPs for the import of nutrients, secretion processes and host-pathogen interactions in gram-negative bacteria 10 , surprisingly few OMPs of mycobacteria are known. The only two well characterized examples of integral OMPs are the porin MspA of M. smegmatis and the channelforming protein OmpA of M. tuberculosis [11][12][13][14] . By contrast, E. coli uses more than 60 proteins to functionalize its outer membrane 15 , none of which has significant sequence similarity to any M. tuberculosis protein.…”

Section: Introductionmentioning

confidence: 99%

Identification of outer membrane proteins of Mycobacterium tuberculosis

Sandie²,

et al. 2008

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SUMMARYThe cell wall of mycobacteria contains an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the β-barrel structure, the absence of hydrophobic α-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the β-strand content and the amphiphilicity of the β-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs of unknown functions by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with β-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis.

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“…1A (fourth panel) shows that all STPKs failed to phosphorylate AcpM. We next addressed the question whether OmpATb, the major porin found in the cell wall of M. tuberculosis (31,38) and which is not related to FAS-II, could be a substrate of the kinases. Our results indicate that none of the kinases were able to phosphorylate OmpATb in vitro (Fig.…”

Section: Stpk-mediated Phosphorylation Of Mycobacterial Fas-ii Enzymes-mentioning

confidence: 99%

“…Purification of recombinant mtFabD, KasA, KasB was performed as described earlier (30). Expression and purification of holo-AcpM and OmpATb was done as reported previously (30,31).…”

Section: Cloning Expression and Purification Of The Eleven Recombinmentioning

confidence: 99%

The Condensing Activities of the Mycobacterium tuberculosis Type II Fatty Acid Synthase Are Differentially Regulated by Phosphorylation

Molle

Brown

Besra

et al. 2006

Journal of Biological Chemistry

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105

119

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Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the ␤-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.Mycobacterium tuberculosis has a unique cell wall structure that accounts for the ability of the bacterium to grow in several contrasting environments and which is responsible for its low membrane permeability, contributing to its resistance to common chemotherapeutic agents (1). The cell wall has been implicated as a direct modulator of interactions between mycobacteria and the environment (2). This envelope, characterized by its high lipid content, comprises an inner membrane barrier composed of mycolic acids anchored to arabinogalactan, linked to peptidoglycan. Mycolic acids are a hallmark of the mycobacterial waxy coat: they represent key virulence factors required for intracellular survival (3, 4) and contribute to the physiopathology of tuberculosis. They consist of very long chains of ␣-branched ␤-hydroxy fatty acids (C 60 -C 90 ), whose biosynthesis is controlled by two elongation systems, the eukaryotic-type fatty acid synthase (FAS-I) 3 and the prokaryotic-like FAS-II (5, 6). FAS-I consists of a single multifunctional polypeptide, catalyzing de novo synthesis of medium length acyl-CoA chains (C 16 -C 26 ), whereas FAS-II comprises several distinct enzymes. It catalyzes similar types of reactions to FAS-I, but functions on acyl carrier protein (AcpM)-bound chains and is incapable of de novo synthesis. The initial substrates of FAS-II are ␤-ketoacylAcpM resulting from the condensation by mtFabH of the acylCoA products of FAS-I with malonyl-AcpM (7, 8). Following reduction by MabA, elimination of water by a yet unidentified dehydratase, and reduction by the enoyl-AcpM reductase InhA, the ␤-ketoacyl-AcpM synthases KasA and KasB catalyze further...

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pH‐dependent pore‐forming activity of OmpATb from Mycobacterium tuberculosis and characterization of the channel by peptidic dissection

Cited by 39 publications

References 40 publications

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Identification of outer membrane proteins of Mycobacterium tuberculosis

The Condensing Activities of the Mycobacterium tuberculosis Type II Fatty Acid Synthase Are Differentially Regulated by Phosphorylation

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