High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

AbstractType III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multi-ring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

Download Full-text

High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916331116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24786-24795 ◽

Cited By ~ 11

Author(s):

Carmen Butan ◽

Maria Lara-Tejero ◽

Wenwei Li ◽

Jun Liu ◽

Jorge E. Galán

Keyword(s):

High Resolution ◽

Protein Secretion ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Protein Translocation ◽

Membrane Remodeling ◽

Secretion Systems ◽

Type Iii ◽

The Core

Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

Download Full-text

YplA Is Exported by the Ysc, Ysa, and Flagellar Type III Secretion Systems of Yersinia enterocolitica

Journal of Bacteriology ◽

10.1128/jb.184.5.1324-1334.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1324-1334 ◽

Cited By ~ 77

Author(s):

Briana M. Young ◽

Glenn M. Young

Keyword(s):

Yersinia Enterocolitica ◽

Protein Secretion ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Chloride Concentration ◽

Effector Proteins ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Type Iii Protein Secretion

ABSTRACT Yersinia enterocolitica maintains three different pathways for type III protein secretion. Each pathway requires the activity of a specific multicomponent apparatus or type III secretion system (TTSS). Two of the TTSSs are categorized as contact-dependent systems which have been shown in a number of different symbiotic and pathogenic bacteria to influence interactions with host organisms by targeting effector proteins into the cytosol of eukaryotic cells. The third TTSS is required for the assembly of flagella and the secretion of the phospholipase YplA, which has been implicated in Y. enterocolitica virulence. In this study, YplA was expressed from a constitutive promoter in strains that contained only a single TTSS. It was determined that each of the three TTSSs is individually sufficient for YplA secretion. Environmental factors such as temperature, calcium availability, and sodium chloride concentration affected the contribution of each system to extracellular protein secretion and, under some conditions, more than one TTSS appeared to operate simultaneously. This suggests that some proteins might normally be exported by more than one TTSS in Y. enterocolitca.

Download Full-text

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

10.1101/745471 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sibel Westerhausen ◽

Melanie Nowak ◽

Claudia Torres-Vargas ◽

Ursula Bilitewski ◽

Erwin Bohn ◽

...

Keyword(s):

Protein Secretion ◽

High Throughput ◽

Type Iii Secretion ◽

Molecular Mechanisms ◽

Host Cells ◽

Target Cells ◽

Secretion Systems ◽

Type Iii ◽

Nanoluc Luciferase ◽

Type Iii Secretion Systems

AbstractThe elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.ImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

Download Full-text

The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar and Virulence Type III Secretion Systems

mBio ◽

10.1128/mbio.00818-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 12

Author(s):

Steven Johnson ◽

Lucas Kuhlen ◽

Justin C. Deme ◽

Patrizia Abrusci ◽

Susan M. Lea

Keyword(s):

Type Iii Secretion ◽

Complex Analysis ◽

Secretion Systems ◽

Core Complex ◽

Type Iii ◽

The Core ◽

Cryo Electron Microscopy ◽

Type Iii Secretion Systems ◽

Equivalent Complex ◽

Resolution Structure

ABSTRACT Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex. IMPORTANCE Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.

Download Full-text

Comparison of ATPase-Encoding Type III Secretion System hrcN Genes in Biocontrol Fluorescent Pseudomonads and in Phytopathogenic Proteobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5119-5131.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5119-5131 ◽

Cited By ~ 45

Author(s):

Fabio Rezzonico ◽

Geneviève Défago ◽

Yvan Moënne-Loccoz

Keyword(s):

Pseudomonas Syringae ◽

Protein Secretion ◽

Type Iii Secretion ◽

Fluorescent Pseudomonads ◽

Secretion Systems ◽

Associated Bacteria ◽

Type Iii ◽

Encoding Gene ◽

Type Iii Protein Secretion ◽

Protein Secretion Systems

ABSTRACT Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.

Download Full-text

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.05017-11 ◽

2012 ◽

Vol 76 (2) ◽

pp. 262-310 ◽

Cited By ~ 253

Author(s):

D. Buttner

Keyword(s):

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Protein Export ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems

Download Full-text

The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

The Journal of Cell Biology ◽

10.1083/jcb.147.3.683 ◽

1999 ◽

Vol 147 (3) ◽

pp. 683-693 ◽

Cited By ~ 363

Author(s):

Ariel Blocker ◽

Pierre Gounon ◽

Eric Larquet ◽

Kirsten Niebuhr ◽

Véronique Cabiaux ◽

...

Keyword(s):

Protein Secretion ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Coupled Processes ◽

Critical Threshold ◽

Secretion Systems ◽

Type Iii ◽

Rbc Membrane ◽

Genes Encoding ◽

Electron Microscopy Analysis

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

Download Full-text

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.

Download Full-text

Negative Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-5-0682 ◽

2010 ◽

Vol 23 (5) ◽

pp. 682-701 ◽

Cited By ~ 26

Author(s):

Inmaculada Ortiz-Martín ◽

Richard Thwaites ◽

John W. Mansfield ◽

Carmen R. Beuzón

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

In Planta ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Bacterial Fitness

Many plant-pathogenic bacteria require type III secretion systems (T3SS) to cause disease in compatible hosts and to induce the hypersensitive response in resistant plants. T3SS gene expression is induced within the plant and responds to host and environmental factors. In Pseudomonas syringae, expression is downregulated by the Lon protease in rich medium and by HrpV under inducing conditions. HrpV acts as an anti-activator by binding HrpS. HrpG, which can also bind HrpV, has been reported to act as an anti-anti-activator. Previous studies have used mostly in vitro inducing conditions, different pathovars, and methodology. We have used single and double lon and hrpV mutants of P. syringae pv. phaseolicola 1448a, as well as strains ectopically expressing the regulators, to examine their role in coordinating expression of the T3SS. We applied real-time polymerase chain reaction to analyze gene expression both in vitro and in planta, and assessed bacterial fitness using competitive indices. Our results indicate that i) Lon downregulates expression of the hrp/hrc genes in all conditions, probably by constitutively degrading naturally unstable HrpR; ii) HrpV and HrpT downregulate expression of the hrp/hrc genes in all conditions; and iii) HrpG has an additional, HrpV-independent role, regulating expression of the hrpC operon.

Download Full-text