Differential Detergent Extraction of Mycobacterium marinum Cell Envelope Proteins Identifies an Extensively Modified Threonine-Rich Outer Membrane Protein with Channel Activity

Abstract

e A striking characteristic of mycobacteria is the presence of an unusual outer membrane which forms a thick permeability barrier and provides resistance to many antibiotics. Although specialized proteins must reside in this layer, only few mycolate outer membrane (MOM) proteins have been identified to date. Their discovery is complicated by difficulties in obtaining good separation of mycobacterial inner and outer membranes. During our efforts to identify novel mycobacterial outer membrane proteins (MOMPs), we discovered that we can enrich for MOMPs using differential solubilization of mycobacterial cell envelopes. Subsequently, these different fractions were analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This proteomic analysis confirmed that our marker proteins for inner membrane and MOM were found in their expected fractions and revealed a few interesting candidate MOMPs. A number of these putative MOMPs were further analyzed for their expression and localization in the cell envelope. One identified MOMP, MMAR_0617 of Mycobacterium marinum, was purified and demonstrated to form a large oligomeric complex. Importantly, this protein showed a clear single-channel conductance of 0.8 ؎ 0.1 ns upon reconstitution into artificial planar lipid bilayers. The most surprising feature of MMAR_0617 is a long C-terminal threonine-rich domain with extensive modifications. In summary, we have identified a novel mycobacterial outer membrane porin with unusual properties.

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

“…Despite our using a variety of proteases (trypsin, Glu-C, and pepsin) to digest natively purified Cnm, no peptides corresponding to the B domain were identified (data not shown). Similar observations have been made with other bacterial glycoproteins, for which enzymatic deglycosylation and standard MS analyses were not successful (41,42). Nevertheless, the glycosylated nature of Cnm and the likely presence of N-acetylglucosamine residues were confirmed using lectin-binding assays.…”

Understanding specificity of the mycosin proteases in ESX/type VII secretion by structural and functional analysis

“…The various M. marinum strains were grown at 30°C in Middlebrook 7H9 - broth (Difco) supplemented with 0.2% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC) (BBC) and 0.05% Tween 80. Expression and subcellular localization of the HA-tagged proteins in M. marinum were analyzed by subcellular fractionation followed by SDS-PAGE and immunoblotting as previously described (Houben et al, 2012b; van der Woude et al, 2013). Briefly, bacteria were grown until an OD 600 of ~1, harvested by centrifugation and resuspended in PBS, 250 mM sucrose.…”

Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

“…The molecular machinery described for this diderm-mycolate bacterial “Type VII secretion system” is an export pathway (protein transport across the cytoplasmic membrane) just as the Sec and Tat systems are (Economou et al, 2006; Desvaux et al, 2009a; Houben et al, 2012). In fact, no translocon in the mycolate outer membrane (MOM), which would truly enable protein secretion and thus form a complete secretion pathway, has been uncovered as yet in diderm-mycolate bacteria [the secretion of proteins exported in the first instance by the Wss, Sec, and Tat could then be completed by the very same MOM translocon, or different MOM translocons specific to each of these export systems (Desvaux et al, 2009a)] (Niederweis, 2003; Converse and Cox, 2005; Ize and Palmer, 2006; Digiuseppe Champion and Cox, 2007; Song et al, 2008; Desvaux et al, 2009a; Niederweis et al, 2010; Stoop et al, 2012; Freudl, 2013; Van Der Woude et al, 2013). This “Type VII secretion system” nomenclature in diderm-mycolate bacteria is clearly not compatible with the numerical classification basically designed to describe OM translocation systems in diderm-LPS bacteria (Salmond and Reeves, 1993; Economou et al, 2006; Desvaux et al, 2009a).…”

“…As in monodermata, some proteins with no N-terminal SP can be translocated via Sec in a SecA2-dependent manner (Feltcher and Braunstein, 2012). While it is clear some exported proteins (i.e., first translocated across the CM by these export systems) are further secreted into the extracellular milieu (i.e., translocated across the MOM), no mycomembrane machinery allowing the translocation across the MOM have been identified to date (Niederweis et al, 2010; Houben et al, 2012; Ligon et al, 2012; Freudl, 2013; Van Der Woude et al, 2013). In other no words (Table 1), no protein secretion system has been sensu stricto identified as yet in diderm-mycolate bacteria.…”