分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-DN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-DN72 that assemble together between 2 membrane proximal domains of the EccB1-DN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-18
摘要: Autotransporters deliver virulence factors to the bacterial surface by translocating an effector passenger domain through a membrane-anchored barrel structure. Although passenger domains are diverse, those found in enteric bacteria autotransporters, including AIDA-I in diffusely adhering Escherichia coli (DAEC) and TibA in enterotoxigenic E. coli, are commonly glycosylated. We show that AIDA-I is heptosylated within the bacterial cytoplasm by autotransporter adhesin heptosyltransferase (AAH) and its paralogue AAH2. AIDA-I heptosylation determines DAEC adhesion to host cells. AAH/AAH2 define a bacterial autotransporter heptosyltransferase (BAHT) family that contains ferric ion and adopts a dodecamer assembly. Structural analyses of the heptosylated TibA passenger domain reveal 35 heptose conjugates forming patterned and solenoid-like arrays on the surface of a beta helix. Additionally, CARC, the AIDA-like autotransporter from Citrobacter rodentium, is essential for colonization in mice and requires heptosylation by its cognate BAHT. Our study establishes a bacterial glycosylation system that regulates virulence and is essential for pathogenesis.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Pneumolysin is one of the major virulence factors elaborated by Streptococcus pneumoniae; this toxin is a member of the cholesterol-dependent cytolysins. Engagement of cholesterol induces the formation of a multi-subunit complex by pneumolysin that lyses host cells by forming pores on the membrane. Because pneumolysin released by bacteria which have been killed by conventional antibiotics is still active, agents capable of directly attacking the toxin are considered advantageous against antimicrobials in the treatment of S. pneumoniae infections. Here we found that the phytosterol, beta-sitosterol, effectively protects against cell lysis caused by pneumolysin. This compound interacts with the toxin at Thr459 and Leu460, two sites important for being recognized by its natural ligand, cholesterol. Similar to cholesterol, beta-sitosterol induces pneumolysin oligomerization. This compound also protects cells from damage by other cholesterol-dependent toxins. Finally, this compound protects mice against S. pneumoniae infection. Thus, beta-sitosterol is a candidate for the development of anti-virulence agents against pathogens that rely on cholesterol-dependent toxins for successful infections.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-15
摘要: Shiga-like toxins (Stxs), produced by pathogenic Escherichia coli, are a major virulence factor involved in severe diseases in human and animals. These toxins are ribosome-inactivating proteins, and treatment for diseases caused by them is not available. Therefore, there is an urgent need for agents capable of effectively targeting this lethal toxin. In this study, we identified baicalin, a flavonoid compound used in Chinese traditional medicine, as a compound against Shiga-like toxin 2 (Stx2). We found that baicalin significantly improves renal function and reduces Stx2-induced lethality in mice. Further experiments revealed that baicalin induces the formation of oligomers by the toxin by direct binding. We also identified the residues important for such interactions and analyzed their roles in binding baicalin by biophysical and biochemical analyses. Our results establish baicalin as a candidate compound for the development of therapeutics against diseases caused by Stxs.