Coiled-Coil Domain of Enteropathogenic Escherichia coli Type III Secreted Protein EspD Is Involved in EspA Filament-Mediated Cell Attachment and Hemolysis

ABSTRACT Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagicEscherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenicE. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

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Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.62.2.379-433.1998 ◽

1998 ◽

Vol 62 (2) ◽

pp. 379-433 ◽

Cited By ~ 1674

Author(s):

Christoph J. Hueck

Keyword(s):

Host Cell ◽

Protein Secretion ◽

Type Iii Secretion ◽

Plant Pathogens ◽

Host Cells ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Animal Pathogens ◽

Type Iii Protein Secretion

SUMMARY Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

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Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.673122 ◽

2021 ◽

Vol 11 ◽

Author(s):

Mariana L. Ferrari ◽

Spyridoula N. Charova ◽

Philippe J. Sansonetti ◽

Efstratios Mylonas ◽

Anastasia D. Gazi

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Secretion Systems ◽

Host Cell Membrane ◽

Type Iii ◽

X Ray Scattering ◽

Type Iii Secretion Systems ◽

Key Factor ◽

Bacterial Cytoplasm ◽

Structural Insights

Bacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with full-length IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states.

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The Extreme C Terminus of Shigella flexneri IpaB Is Required for Regulation of Type III Secretion, Needle Tip Composition, and Binding

Infection and Immunity ◽

10.1128/iai.00645-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1682-1691 ◽

Cited By ~ 33

Author(s):

A. Dorothea Roehrich ◽

Isabel Martinez-Argudo ◽

Steven Johnson ◽

Ariel J. Blocker ◽

Andreas K. J. Veenendaal

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Physical Contact ◽

Host Cells ◽

Cell Contact ◽

Virulence Determinants ◽

Secretion Systems ◽

Host Cell Membrane ◽

Type Iii ◽

C Terminus

ABSTRACT Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a ΔipaB strain. Like the ΔipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the ΔipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an “off” conformation that turns “on” upon host cell contact. Our mutants may adopt a partially “on” conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion.

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Domains of the Shigella flexneri Type III Secretion System IpaB Protein Involved in Secretion Regulation

Infection and Immunity ◽

10.1128/iai.00470-10 ◽

2010 ◽

Vol 78 (12) ◽

pp. 4999-5010 ◽

Cited By ~ 30

Author(s):

Da-Kang Shen ◽

Saroj Saurya ◽

Carolin Wagner ◽

Hiroaki Nishioka ◽

Ariel J. Blocker

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Effector Proteins ◽

Physical Contact ◽

Host Cells ◽

Cell Contact ◽

Secretion Systems ◽

Host Cell Membrane ◽

Type Iii ◽

Secretion Regulation

ABSTRACT Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacterial pathogens. Upon cell contact, they inject effector proteins directly into eukaryotic cells through a needle protruding from the bacterial surface. Host cell sensing occurs through a distal needle “tip complex,” but how this occurs is not understood. The tip complex of quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. It binds needles via its extreme C-terminal coiled coil, thereby likely positioning a large domain containing its hydrophobic regions at the distal tips of needles. In this study, we used short deletion mutants within this domain to search for regions of IpaB involved in secretion regulation. This identified two regions, amino acids 227 to 236 and 297 to 306, the presence of which are required for maintenance of IpaB at the needle tip, secretion regulation, and normal pore formation but not invasion. We therefore propose that removal of either of these regions leads to an inability to block secretion prior to reception of the activation signal and/or a defect in host cell sensing.

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The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli. Vol. 274 (1999) 35969–35974

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)67595-0 ◽

2005 ◽

Vol 280 (19) ◽

pp. 19436

Author(s):

Robin M. Delahay ◽

Stuart Knutton ◽

Robert K. Shaw ◽

Elizabeth L. Hartland ◽

Mark J. Pallen ◽

...

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Coiled Coil ◽

Enteropathogenic Escherichia Coli ◽

Type Iii ◽

Coiled Coil Domain

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Injectisome T3SS Subunits As Potential Chaperones In The Extracellular Export of Pectobacterium Carotovorum Subsp. Carotovorum Bacteriocins Carocin S1 And Carocin S3 Secreted Via Flagellar T3SS

10.21203/rs.3.rs-590298/v1 ◽

2021 ◽

Author(s):

Hang-Cheng Chen ◽

Reymund C. Derilo ◽

Han-Ling Chen ◽

Tzu-Rung Li ◽

Ruchi Briam James S. Lagitnay ◽

...

Keyword(s):

Type Iii Secretion ◽

Insertional Mutagenesis ◽

Plant Pathogens ◽

Soft Rot ◽

Economic Losses ◽

Pectobacterium Carotovorum ◽

Secretion Systems ◽

Unique Type ◽

Bacterial Flagellum ◽

Type Iii

Abstract Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22-6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems.

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Bridging the Gap: Type III Secretion Systems in Plant-Beneficial Bacteria

Microorganisms ◽

10.3390/microorganisms10010187 ◽

2022 ◽

Vol 10 (1) ◽

pp. 187

Author(s):

Antoine Zboralski ◽

Adrien Biessy ◽

Martin Filion

Keyword(s):

Type Iii Secretion ◽

Plant Pathogens ◽

Plant Immunity ◽

Effector Proteins ◽

Secretion Systems ◽

Beneficial Bacteria ◽

Living Plant ◽

Pseudomonas Spp ◽

Type Iii ◽

Type Iii Secretion Systems

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

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Escherichia coli type III secretion system 2 (ETT2) is widely distributed in avian pathogenic Escherichia coli isolates from Eastern China

Epidemiology and Infection ◽

10.1017/s0950268816000820 ◽

2016 ◽

Vol 144 (13) ◽

pp. 2824-2830 ◽

Cited By ~ 5

Author(s):

S. WANG ◽

X. LIU ◽

X. XU ◽

Y. ZHAO ◽

D. YANG ◽

...

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Bacterial Infections ◽

Eastern China ◽

Type Iii Secretion System ◽

Secretion System ◽

Secretion Systems ◽

E Coli ◽

Type Iii ◽

System 2

SUMMARYPathogens utilize type III secretion systems to deliver effector proteins, which facilitate bacterial infections. The Escherichia coli type III secretion system 2 (ETT2) which plays a crucial role in bacterial virulence, is present in the majority of E. coli strains, although ETT2 has undergone widespread mutational attrition. We investigated the distribution and characteristics of ETT2 in avian pathogenic E. coli (APEC) isolates and identified five different ETT2 isoforms, including intact ETT2, in 57·6% (141/245) of the isolates. The ETT2 locus was present in the predominant APEC serotypes O78, O2 and O1. All of the ETT2 loci in the serotype O78 isolates were degenerate, whereas an intact ETT2 locus was mostly present in O1 and O2 serotype strains, which belong to phylogenetic groups B2 and D, respectively. Interestingly, a putative second type III secretion-associated locus (eip locus) was present only in the isolates with an intact ETT2. Moreover, ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human extraintestinal pathogenic E. coli, suggesting that APEC might be a potential risk to human health. However, there was no distinct correlation between ETT2 and other virulence factors in APEC.

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