scholarly journals Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252800
Author(s):  
Lucas Kuhlen ◽  
Steven Johnson ◽  
Jerry Cao ◽  
Justin C. Deme ◽  
Susan M. Lea
Keyword(s):  
Cytoplasmic Domain ◽  
Secretion System ◽  
Membrane Domain ◽  
Oligomeric State ◽  
Biologically Relevant ◽  
Bacterial Flagella ◽  
Ring Assembly ◽  
Cryo Electron Microscopy ◽  
Ring Structures ◽  
Type Three Secretion

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer’s cytoplasmic domain, which stabilises the nonameric ring assembly.

scholarly journals Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

2020 ◽  
Author(s):  
Lucas Kuhlen ◽  
Steven Johnson ◽  
Jerry Y. Cao ◽  
Justin C. Deme ◽  
Susan M. Lea
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Cytoplasmic Domain ◽  
Secretion System ◽  
Full Length ◽  
Cytoplasmic Domains ◽  
Bacterial Flagella ◽  
Type Iii ◽  
Closed State ◽  
N Terminus

AbstractType three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, FlhA/SctV is most often studied as a monomer and only a single structure of an SctV homologue assembled into the biologically relevant nonameric ring is available. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in the assembled ring structure SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer’s cytoplasmic domain, which stabilises the nonameric ring assembly. This represents the first structure of SctV in the open state, the first observation of the SctVL interaction with the adjacent protomer and confirms the importance of FlhAL for the stability of the FlhA nonameric ring.ImportanceBacterial flagella are assembled from proteins secreted through a type III secretion system. A related type III secretion system is found in injectisomes, molecular syringes that bridge three membranes to secrete proteins directly from the bacterial cytoplasm into eukaryotic host cells. The major protein of the export apparatus of type III secretion is made up of a membrane and a cytoplasmic domain, which in the flagellar system can adopt an open or a closed state, is known to form a nonameric ring in vivo. We produced the full-length proteins from both injectisome and flagellar systems in the assembled state. The structures of the cytoplasmic domains demonstrate the conserved principle of the N-terminus of one subunit binding the membrane proximal face of the adjacent subunit to stabilise the assembled ring. Our structure of the homologue from the injectisome also demonstrates that the open state of the cytoplasmic domain is not unique to flagella.

An inhibitory mechanism of action of coiled‐coil peptides against type three secretion system from enteropathogenicEscherichia coli

2019 ◽  
Vol 25 (3) ◽  
Author(s):  
Mariano Larzábal ◽  
Hector A. Baldoni ◽  
Fernando D. Suvire ◽  
Lucrecia M. Curto ◽  
Gabriela E. Gomez ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Coiled Coil ◽  
Secretion System ◽  
Inhibitory Mechanism ◽  
Type Three Secretion System ◽  
Type Three Secretion

scholarly journals Application of Cryo-Electron Microscopy on Drug Discovery

2021 ◽  
Vol 27 (S1) ◽  
pp. 3250-3250
Author(s):  
Viswanath Vittaladevaram ◽  
Kranthi Kuruti
Keyword(s):  
Electron Microscopy ◽  
Protein Complexes ◽  
Lead Discovery ◽  
Biologically Relevant ◽  
Biological Targets ◽  
3 Dimensional ◽  
Drug Molecules ◽  
Cryo Electron Microscopy ◽  
Therapeutic Molecules ◽  
Novel Drug

AbstractThe key aspect for development of novel drug molecules is to perform structural determination of target molecule associated with its ligand. One such tool that provides insights towards structure of molecule is Cryo-electron microscopy which covers biological targets that are intractable. Examination of proteins can be carried out in native state, as the samples are frozen at -175 degree Celsius i.e. cryogenic temperatures. In addition to this, there were no limits for molecular and functional structures of proteins that can be imagined in 3-dimensional form. This includes ligands which unravel mechanisms that are biologically relevant. This will enable to better understand the mechanisms that are used for development of new therapeutics. Application of Cryo-electron microscopy is not limited to protein complexes and is considered as non-specific. Intervention of Cryo-EM would allow to analyse the structures and also able to dissect the interaction with therapeutic molecules. The study determines the usage of cryo-EM for providing resolutions that are acceptable for lead discovery. It also provides support for lead optimization and also for discovery of vaccines and therapeutics.

scholarly journals Structure of the type VI secretion system TssK–TssF–TssG baseplate subcomplex revealed by cryo-electron microscopy

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Young-Jun Park ◽  
Kaitlyn D. Lacourse ◽  
Christian Cambillau ◽  
Frank DiMaio ◽  
Joseph D. Mougous ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Cryo Electron Microscopy

scholarly journals Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns

BMC Genomics ◽  
2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Katherine H. Tanaka ◽  
Antony T. Vincent ◽  
Jean-Guillaume Emond-Rheault ◽  
Marcin Adamczuk ◽  
Michel Frenette ◽  
...  
Keyword(s):  
Secretion System ◽  
Aeromonas Salmonicida ◽  
Type Three Secretion System ◽  
Type Three Secretion

scholarly journals Type Three Secretion System-Dependent Microvascular Thrombosis and Ischemic Enteritis in Human Gut Xenografts Infected with Enteropathogenic Escherichia coli

2017 ◽  
Vol 85 (11) ◽  
Author(s):  
Einat Nissim-Eliraz ◽  
Eilam Nir ◽  
Irit Shoval ◽  
Noga Marsiano ◽  
Israel Nissan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blood Vessels ◽  
Secretion System ◽  
Infection Model ◽  
Apical Surface ◽  
Muscularis Mucosa ◽  
Human Gut ◽  
Type Three Secretion System ◽  
Type Three Secretion ◽  
Ischemic Enteritis

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS), causing the hallmark attaching and effacing (AE) lesions and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in ∼50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels, which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria, which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA, and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.

scholarly journals Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

2021 ◽  
Vol 118 (34) ◽  
pp. e2026719118
Author(s):  
Mar Pérez-Ruiz ◽  
Mar Pulido-Cid ◽  
Juan Román Luque-Ortega ◽  
José María Valpuesta ◽  
Ana Cuervo ◽  
...  
Keyword(s):  
Bacteriophage T7 ◽  
Dna Translocation ◽  
Oligomeric State ◽  
Viral Dna ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Core Proteins ◽  
Core Components ◽  
Bacterial Cytoplasm ◽  
Bacterial Peptidoglycan

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

scholarly journals Expression of the Bradyrhizobium japonicum Type III Secretion System in Legume Nodules and Analysis of the Associated tts box Promoter

2008 ◽  
Vol 21 (8) ◽  
pp. 1087-1093 ◽  
Author(s):  
Susanne Zehner ◽  
Grit Schober ◽  
Mandy Wenzel ◽  
Kathrin Lang ◽  
Michael Göttfert
Keyword(s):  
Type Iii Secretion ◽  
Bradyrhizobium Japonicum ◽  
Response Regulator ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Symbiotic Efficiency ◽  
Type Iii ◽  
Genes Encoding ◽  
Transcriptional Start Sites ◽  
Type Three Secretion

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.

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