Protease domain and transmembrane domain of the type VII secretion mycosin protease determine system-specific functioning in mycobacteria

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to -5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, the mycosin protease (MycP), is not part of the core complex, but is essential for secretion, as it stabilizes this membrane complex. Here, we investigated which MycP domain is required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane (TM) domain are required for the ESX system-specific function of mycosins. In addition, we observed that the TM domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in the interaction with and the stabilization of their respective ESX complexes

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain

Targeting and translocation of the two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway.

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins

Bacterial secretion chaperones:The mycobacterial type VII case

Chaperones are central players in maintaining the proteostasis in all living cells. Besides highly conserved generic chaperones that assist protein folding and assembly in the cytosol, additional more specific chaperones have evolved to ensure the successful trafficking of proteins with extra-cytoplasmic locations. Associated with the distinctive secretion systems present in bacteria, different dedicated chaperones have been described that not only keep secretory proteins in a translocation competent state, but often are also involved in substrate targeting to the specific translocation channel. Recently, a new class of such chaperones has been identified that are involved in the specific recognition of substrates transported via the type VII secretion pathway in mycobacteria. In this minireview, we provide an overview of the different bacterial chaperones with a focus on their roles in protein secretion and will discuss in detail the roles of mycobacterial type VII secretion chaperones in substrate recognition and targeting

Species‐specific secretion of ESX‐5 type VII substrates is determined by the linker 2 of EccC 5

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC5 , possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC5 . This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC5 , showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis

Protein export into and across the atypical diderm cell envelope of mycobacteria

Mycobacteria are Actinobacteria, which is a phylum of high-GC Gram-positive bacteria. Among the wide range of Mycobacterium spp. are several important pathogens. Most notable of these is Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Although the number of TB cases and deaths show a declining trend in the past decade, the World Health Organization recently warned that “current actions and investments in research are falling far short” (http://www.who.int/mediacentre/news/releases/2016/tuberculosis-investments-short/en/). Over 1.3 million deaths and up to 10.0 million new infections were attributed to M. tuberculosis in 2017, which makes M. tuberculosis the most deadly infectious agent in the world, surpassing HIV and the malaria parasite (1). One issue facing efforts to control TB is that the live attenuated bacillus Calmette-Guérin (BCG) vaccine strain is not able to provide lifelong protection against M. tuberculosis (2). Another problem is the increased incidence of infections caused by multidrug-resistant M. tuberculosis strains. Consequently, major research efforts focus on understanding M. tuberculosis pathogenesis and physiology to advance the development of new anti-TB vaccines and antibiotics

Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.

<p>* The <i>eccC</i>_<i>5</i> NBD mutants appear to have a dominant negative effect on the functioning of endogenous EccC₅.</p><p>^$ Colonies showed a strong growth defect, i.e. colonies were visible only after 17 days, compared to 10 days for the wild-type strain.</p><p>Replacement of pMV-<i>eccBC</i>_<i>5</i> by the input DNA was scored. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. “+” indicates that more than 100 colonies were detected after electroporation with the indicated vector. “–” indicates between 0–20 colonies were found after electroporation. These latter colonies were shown by PCR to still contain the original vector, indicating illegitimate recombination or spontaneous antibiotic resistance. Results are representative data of three independent experiments.</p><p>Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.</p

Role of NBDs domains of EccC5 in ESX-5 dependent secretion and membrane complex assembly.

<p>A) Predicted transmembrane domains (dark grey), and NBD (light grey) of EccC₅ are indicated. The positions of relevant residues are depicted with a black bar. The numbers represent the position in amino acids. B) Secretion of PE_PGRS proteins in the different EccC₅ mutant strains was analyzed by immunoblot of supernatants and cell pellets of wild-type (WT) <i>M</i>. <i>marinum</i> and the <i>eccC5</i> deletion strain <i>(</i>Δ<i>eccC5</i>) complemented with various <i>eccC5</i> mutated genes. GroEL2 staining was used as a control for lysis and equal loading. C) Immunoblot analysis of EccC₅ expression in isolated membranes of indicated strains. D) Blue native PAGE and immunoblot analysis using an anti-EccD₅ antibody of the ESX-5 membrane of <i>M</i>. <i>marinum</i> Δ<i>eccC5</i>::<i>mspA</i>, complemented either with an empty vector (-) or with various <i>eccC5</i> mutated genes. For all samples that contained EccC₅ variants the characteristic pattern of ESX-5 membrane complexes was observed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.ref028" target="_blank">28</a>], consisting of the largest ~1.5 MDa complex and two additional smaller subcomplexes (indicated by the three arrowheads).</p

Secretion analysis of ESX-5 mutant strains.

<p>A) A schematic representation of the ESX-5 region of <i>M</i>. <i>marinum</i> with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain <i>M</i>. <i>marinum</i>::<i>mspA</i> defective in ESX-5 dependent secretion. B) Secretion analysis of <i>M</i>.<i>marinum</i>::<i>mspA (</i>WT::<i>mspA)</i>, a <i>mycP5</i> transposon mutant (<i>mycP5</i>::<i>tn</i>, corresponding to LA9 in (A)) and the complemented version of this strain (<i>mycP5</i>::<i>tn</i>-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB₅ and EspG₅ was analyzed by immunoblotting of total cell lysates of wild-type <i>M</i>. <i>marinum</i> (WT), the <i>Δesx-5</i>::<i>mspA</i> mutant and the complemented <i>Δesx-5</i>::<i>esx-5tub</i> strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).</p