Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5.

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 .

EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF.

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.

Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis.

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Šresolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.

Analysis of SecA2-dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector.

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2-dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2-dependent substrates. Immunoblotting procedures confirmed SecA2-dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG-overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2-dependent membrane localization of PknG is an important determinant for M. marinum virulence.

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

Mycobacteria use specialized ESX secretion systems to transport proteins across their cell membranes in order to manipulate their environment. In pathogenic Mycobacterium tuberculosis there are five paralogous ESX secretion systems, named ESX-1 through ESX-5. Each system includes a subtilisin-like protease (mycosin or MycP) as a core component essential for secretion. Here we report crystal structures of MycP1 and MycP3, the mycosins expressed by the ESX-1 and ESX-3 systems, respectively. In both mycosins the putative propeptide wraps around the catalytic domain and does not occlude the active site. The extensive contacts between the putative propeptide and catalytic domain, which include a disulfide bond, suggest that the N-terminal extension is an integral part of the active mycosin. The catalytic residues of MycP1 and MycP3 are located in a deep active site groove in contrast with an exposed active site in majority of subtilisins. We show that MycP1 specifically cleaves ESX-1 secretion-associated protein B (EspB) in vitro at residues Ala358 and Ala386. We also systematically characterize the specificity of MycP1 using peptide libraries, and show that it has evolved a narrow specificity relative to other subtilisins. Finally, comparison of the MycP1 and MycP3 structures suggest that both enzymes have stringent and different specificity profiles that result from the structurally distinct active site pockets, which could explain the system specific functioning of these proteases.

Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

Differential expression of a virulence factor in pathogenic and non-pathogenic mycobacteria.

The pathogenicity of mycobacterial infections depends on virulence factors that mediate survival inside host macrophages. These virulence factors are generally believed to be specific for pathogenic species and absent or mutated in non-pathogenic strains. The serine/threonine protein kinase G (PknG) mediates survival of mycobacteria within macrophages by blocking lysosomal delivery. Here we describe a gene of the non-pathogenic species Mycobacterium smegmatis that is 78% identical with pknG of Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG). When cloned into expression vectors, the M. smegmatis pknG orthologue produced an active kinase and performed the same function as its M. bovis BCG counterpart in intracellular survival. In addition, similar levels of pknG transcripts were found in M. bovis BCG and M. smegmatis. However, virtually no translation product of chromosomal pknG could be detected in M. smegmatis both after in vitro growth and after macrophage infection. This lack of efficient translation was shown to be caused by regulatory elements in the upstream region of the M. smegmatis gene. The data reveal dramatically increased translational efficiency of a virulence gene in a pathogenic mycobacterium compared with a non-pathogenic mycobacterium suggesting that changes in expression levels may underlie evolution of pknG and other pathogenicity genes in mycobacterium.

Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins.

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs.

Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5.

An effective host immune response to mycobacterial infection must control pathogen dissemination without inducing immunopathology. Constitutive overexpression of mycobacterial heat shock protein (myHsp70) is associated with impaired bacterial persistence, but the immune-mediated mechanisms are unknown. We found that myHsp70, in addition to enhancing antigen delivery to human dendritic cells, signaled through the CCR5 chemokine receptor, promoting dendritic cell aggregation, immune synapse formation between dendritic cells and T cells, and the generation of effector immune responses. Thus, CCR5 acts as a pattern-recognition receptor for myHsp70, which may have implications for both the pathophysiology of tuberculosis and the use of myHsps in tumor-directed immunotherapy.

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle.

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.

Interaction of pathogenic mycobacteria with the host immune system.

Pathogenic mycobacteria, in particular Mycobacterium tuberculosis, the causative agent of tuberculosis, have the remarkable capacity to circumvent destruction within one of the most hostile cell types of a vertebrate host: the macrophage. The ability of pathogenic mycobacteria to survive inside macrophages has been known for more than 30 years; yet, only recently have advances in molecular genetics, biochemistry, immunology, as well as global analysis of gene expression, started to unravel the strategies utilized by these pathogens for intracellular persistence. In addition, the definition of key molecules that are important for intracellular survival opens the possibility to develop new drugs to combat mycobacterial diseases.

The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface.

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.

Targeting and translocation of 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.

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.