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

2019 ◽  
Author(s):  
C. M. Bunduc ◽  
R. Ummels ◽  
W. Bitter ◽  
E.N.G. Houben
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Effector Proteins ◽  
Specific Substrate ◽  
Secretion Systems ◽  
Binding Domains ◽  
Type Vii Secretion ◽  
Wide Range ◽  
Species Specific ◽  
System 1

AbstractType VII secretion systems (T7SSs) are used by mycobacteria to translocate a wide range of effector proteins across their diderm cell envelope. These systems, also known as ESX systems, have crucial roles for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We previously observed species-specificity in the secretion of the PE_PGRS proteins by the ESX-5 system [1], in that the M. tuberculosis ESX-5 system was unable to fully complement an M. marinum esx-5 mutant. In this study, we established that the responsible factor for this is the central membrane ATPase EccC5, which has three nucleotide binding domains (NBDs). By creating chimeric M. marinum/M. tuberculosis EccC5 constructs, we observed that PE_PGRS secretion is mediated only in the presence of an EccC5 containing the cognate linker 2, irrespective of the origin of the EccC5 backbone. This region is responsible for linking the first two NBDs and for keeping the first NBD in an inhibited state. Notably, this region is disordered in a EccC crystal structure and is particularly extended in EccC proteins of the different ESX-5 systems. These results indicate that this region is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5.

scholarly journals Structure of the extracellular region of the bacterial type VIIb secretion system subunit EsaA

2020 ◽  
Author(s):  
Timothy A. Klein ◽  
Dirk W. Grebenc ◽  
Shil Y. Gandhi ◽  
Vraj S. Shah ◽  
Youngchang Kim ◽  
...  
Keyword(s):  
Structural Information ◽  
Cell Envelope ◽  
Critical Role ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Type Vii Secretion ◽  
Streptococcus Gallolyticus ◽  
Extracellular Region

SummaryGram-positive bacteria use type VII secretion systems (T7SSs) to export effector proteins that manipulate the physiology of nearby prokaryotic and eukaryotic cells. Several mycobacterial T7SSs have established roles in virulence. By contrast, recent work has demonstrated that the genetically distinct T7SSb pathway found in Firmicutes bacteria more often functions to mediate interbacterial competition. A lack of structural information on the T7SSb has limited the understanding of effector export by this protein secretion apparatus. In this work, we present the 2.4Å crystal structure of the extracellular region of the elusive T7SSb subunit EsaA from Streptococcus gallolyticus. Our structure reveals that homodimeric EsaA is an elongated, arrow-shaped protein with a surface-accessible ‘tip’, which serves as a receptor for lytic bacteriophages in some species of bacteria. Because it is the only T7SSb subunit large enough to traverse the thick peptidoglycan layer of Firmicutes bacteria, we propose that EsaA plays a critical role in transporting effectors across the entirety of the Gram-positive cell envelope.

scholarly journals Structural insights into substrate recognition by the type VII secretion system

2019 ◽  
Vol 11 (2) ◽  
pp. 124-137 ◽  
Author(s):  
Shuhui Wang ◽  
Kaixuan Zhou ◽  
Xiaolin Yang ◽  
Bing Zhang ◽  
Yao Zhao ◽  
...  
Keyword(s):  
Substrate Recognition ◽  
Secretion Systems ◽  
Atp Binding Site ◽  
Type Vii Secretion ◽  
The Family ◽  
Type Vii Secretion System ◽  
Pass Through ◽  
System 1 ◽  
Substrate Proteins ◽  
Structural Insights

AbstractType VII secretion systems (T7SSs) are found in many disease related bacteria including Mycobacterium tuberculosis (Mtb). ESX-1 [early secreted antigen 6 kilodaltons (ESAT-6) system 1] is one of the five subtypes (ESX-1~5) of T7SSs in Mtb, where it delivers virulence factors into host macrophages during infection. However, little is known about the molecular details as to how this occurs. Here, we provide high-resolution crystal structures of the C-terminal ATPase3 domains of EccC subunits from four different Mtb T7SS subtypes. These structures adopt a classic RecA-like ɑ/β fold with a conserved Mg-ATP binding site. The structure of EccCb1 in complex with the C-terminal peptide of EsxB identifies the location of substrate recognition site and shows how the specific signaling module “LxxxMxF” for Mtb ESX-1 binds to this site resulting in a translation of the bulge loop. A comparison of all the ATPase3 structures shows there are significant differences in the shape and composition of the signal recognition pockets across the family, suggesting that distinct signaling sequences of substrates are required to be specifically recognized by different T7SSs. A hexameric model of the EccC-ATPase3 is proposed and shows the recognition pocket is located near the central substrate translocation channel. The diameter of the channel is ~25-Å, with a size that would allow helix-bundle shaped substrate proteins to bind and pass through. Thus, our work provides new molecular insights into substrate recognition for Mtb T7SS subtypes and also a possible transportation mechanism for substrate and/or virulence factor secretion.

scholarly journals Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
M. Sloan Siegrist ◽  
Magnus Steigedal ◽  
Rushdy Ahmad ◽  
Alka Mehra ◽  
Marte S. Dragset ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Iron Acquisition ◽  
Multiple Reaction Monitoring ◽  
Protein Export ◽  
Secretion Systems ◽  
Experimental Conditions ◽  
Content Type ◽  
Type Vii Secretion ◽  
Wide Range

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP 3ms mutant, failed to export the native Esx-3 substrates EsxH ms and EsxG ms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxH ms and EsxG ms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxG ms and EsxH ms secretion in the ΔmycP 3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

scholarly journals PE5-PPE4-EspG3 trimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

2020 ◽  
Author(s):  
Zachary A. Williamson ◽  
Catherine T. Chaton ◽  
William A. Ciocca ◽  
Natalia Korotkova ◽  
Konstantin V. Korotkov
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Secretion System ◽  
Multiple Factors ◽  
Type Vii Secretion ◽  
Shape Complementarity ◽  
Numerous Type ◽  
Unique Shape ◽  
Insight Into

ABSTRACTMycobacterium tuberculosis (Mtb) has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. Three such systems; ESX-1, ESX-3, and ESX-5; have been shown to each secrete a unique set of substrates. A large class of these substrates secreted by these three systems are the PE and PPE families of proteins. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems and how each EspG is recognizing its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG trimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm trimer, from the ESX-3 system. Our trimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between an ‘open’ and ‘closed’ form, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 trimers, the PE-PPE heterodimer of our ESX-3 trimer is interacting with it’s chaperone at a drastically different angle, and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate amongst non-cognate PE-PPE pairs.

scholarly journals A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

2021 ◽  
Vol 17 (12) ◽  
pp. e1010121
Author(s):  
Brady L. Spencer ◽  
Uday Tak ◽  
Jéssica C. Mendonça ◽  
Prescilla E. Nagao ◽  
Michael Niederweis ◽  
...  
Keyword(s):  
Group B Streptococcus ◽  
Bioinformatic Analysis ◽  
Effector Proteins ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Gram Positive Bacteria ◽  
Type Vii Secretion ◽  
Type Vii Secretion System ◽  
Group B

Type VII secretion systems (T7SS) have been identified in Actinobacteria and Firmicutes and have been shown to secrete effector proteins with functions in virulence, host toxicity, and/or interbacterial killing in a few genera. Bioinformatic analysis indicates that isolates of Group B Streptococcus (GBS) encode at least four distinct subtypes of T7SS machinery, three of which encode adjacent putative T7SS effectors with WXG and LXG motifs. However, the function of T7SS in GBS pathogenesis is unknown. Here we assessed the role of the most abundant GBS T7SS subtype during GBS pathogenesis. In a murine model of hematogenous meningitis, mice infected with GBS lacking a functional T7SS or lacking the secreted WXG100 effector EsxA exhibited less mortality, lower bacterial burdens in tissues, and decreased inflammation in the brain compared to mice infected with the parental GBS strain. We further showed that this T7SS induces cytotoxicity in brain endothelium and that EsxA contributes to these cytotoxicity phenotypes in a WXG motif-dependent manner. Finally, we determined that EsxA is a pore-forming protein, thus demonstrating the first role for a non-mycobacterial EsxA homolog in pore formation. This work reveals the importance of a T7SS in host–GBS interactions and has implications for T7SS effector function in other Gram-positive bacteria.

scholarly journals Structure and dynamics of the ESX-5 type VII secretion system of Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Catalin M. Bunduc ◽  
Dirk Fahrenkamp ◽  
Jiri Wald ◽  
Roy Ummels ◽  
Wilbert Bitter ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Transport ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Transport Systems ◽  
Secretion Systems ◽  
Type Vii Secretion ◽  
Membrane Complex ◽  
Type Vii Secretion System ◽  
Inner Membrane Complex

AbstractMycobacterium tuberculosis causes one of the most important infectious diseases in humans, leading to 1.5 million deaths every year. Specialized protein transport systems, called type VII secretion systems (T7SSs), are central for its virulence, but also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the first structure of an intact T7SS inner membrane complex of M. tuberculosis. We show how the 2.32 MDa, 165 transmembrane helices-containing ESX-5 assembly is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber formed by three EccB5 dimers, with the proteolytic sites facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, highlighting the importance of this component for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three four-transmembrane helix bundles, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations, likely reflecting different secretion states. Our work suggests a novel mechanism of protein transport and provides a structural scaffold to aid drug development against the major human pathogen.

scholarly journals PE5–PPE4–EspG3 heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

2020 ◽  
Vol 295 (36) ◽  
pp. 12706-12715
Author(s):  
Zachary A. Williamson ◽  
Catherine T. Chaton ◽  
William A. Ciocca ◽  
Natalia Korotkova ◽  
Konstantin V. Korotkov
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Secretion System ◽  
Multiple Factors ◽  
Type Vii Secretion ◽  
Shape Complementarity ◽  
Numerous Type ◽  
Unique Shape ◽  
Insight Into

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE–PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE–PPE proteins is currently unknown. The only current structural characterization of PE–PPE–EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt–PPE4mt–EspG3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between “open” and “closed” forms, and this movement is likely functionally relevant in the unloading of PE–PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE–PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE–EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE–PPE pairs.

scholarly journals Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

2016 ◽  
Vol 84 (8) ◽  
pp. 2255-2263 ◽  
Author(s):  
Emir Tinaztepe ◽  
Jun-Rong Wei ◽  
Jenelle Raynowska ◽  
Cynthia Portal-Celhay ◽  
Victor Thompson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Bacterial Pathogen ◽  
Transcriptional Response ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vii Secretion ◽  
Significant Difference ◽  
Iron And Zinc

More people die every year fromMycobacterium tuberculosisinfection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogenMycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. InM. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. WithM. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of theesx-3locus to these metals. While iron regulated theesx-3expression in bothM. tuberculosisandM. smegmatis, there is a significant difference in the dynamics of this regulation. InM. smegmatis, theesx-3locus behaved like other iron-regulated genes such asmbtB. InM. tuberculosis, both iron and zinc modestly repressedesx-3expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction ofM. tuberculosiswith macrophages, leading to impaired intracellularM. tuberculosissurvival. Our findings detail the regulatory differences ofesx-3inM. tuberculosisandM. smegmatisand demonstrate the importance of metal-dependent regulation of ESX-3 for virulence inM. tuberculosis.

scholarly journals Assembly and mechanisms of bacterial type IV secretion machines

2012 ◽  
Vol 367 (1592) ◽  
pp. 1073-1087 ◽  
Author(s):  
Ellen L. Zechner ◽  
Silvia Lang ◽  
Joel F. Schildbach
Keyword(s):  
Cell Surface ◽  
Cell Envelope ◽  
Recipient Cell ◽  
Antibiotic Resistance Genes ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv ◽  
Wide Range ◽  
Cell Envelopes

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.

scholarly journals Phosphatidylinositol-3,4,5-Triphosphate and Cellular Signaling: Implications for Obesity and Diabetes

2015 ◽  
Vol 35 (4) ◽  
pp. 1253-1275 ◽  
Author(s):  
Prasenjit Manna ◽  
Sushil K. Jain
Keyword(s):  
Metabolic Diseases ◽  
Specific Binding ◽  
Cell Metabolism ◽  
Effector Proteins ◽  
Current Status ◽  
Cellular Functions ◽  
Binding Domains ◽  
Obesity And Diabetes ◽  
Impaired Metabolism ◽  
Wide Range

Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is one of the most important phosphoinositides and is capable of activating a wide range of proteins through its interaction with their specific binding domains. Localization and activation of these effector proteins regulate a number of cellular functions, including cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking, and cell metabolism. Phosphoinositides have been investigated as an important agonist-dependent second messenger in the regulation of diverse physiological events depending upon the phosphorylation status of their inositol group. Dysregulation in formation as well as metabolism of phosphoinositides is associated with various pathophysiological disorders such as inflammation, allergy, cardiovascular diseases, cancer, and metabolic diseases. Recent studies have demonstrated that the impaired metabolism of PtdIns(3,4,5)P3 is a prime mediator of insulin resistance associated with various metabolic diseases including obesity and diabetes. This review examines the current status of the role of PtdIns(3,4,5)P3 signaling in the regulation of various cellular functions and the implications of dysregulated PtdIns(3,4,5)P3 signaling in obesity, diabetes, and their associated complications.

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