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

1999 ◽  
Vol 147 (3) ◽  
pp. 683-693 ◽  
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.

scholarly journals Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

2008 ◽  
Vol 191 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Andreas K. J. Veenendaal ◽  
Charlotta Sundin ◽  
Ariel J. Blocker
Keyword(s):  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Host Cells ◽  
Incomplete Penetrance ◽  
Secretion Systems ◽  
Bacterial Surface ◽  
Type Iii ◽  
Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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

2019 ◽  
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.

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

2004 ◽  
Vol 70 (9) ◽  
pp. 5119-5131 ◽  
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.

scholarly journals Chlamydial Type III Secretion System Is Encoded on Ten Operons Preceded by Sigma 70-Like Promoter Elements

2006 ◽  
Vol 189 (1) ◽  
pp. 198-206 ◽  
Author(s):  
P. Scott Hefty ◽  
Richard S. Stephens
Keyword(s):  
Type Iii Secretion ◽  
Transcriptional Start Site ◽  
Type Iii Secretion System ◽  
Regulatory Elements ◽  
Secretion System ◽  
Secretion Systems ◽  
Promoter Elements ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Genes Encoding

ABSTRACT Many gram-negative bacterial pathogens employ type III secretion systems for infectious processes. Chlamydiae are obligate intracellular bacteria that encode a conserved type III secretion system that is likely requisite for growth. Typically, genes encoding type III secretion systems are located in a single locus; however, for chlamydiae these genes are scattered throughout the genome. Little is known regarding the gene regulatory mechanisms for this essential virulence determinant. To facilitate identification of cis-acting transcriptional regulatory elements, the operon structure was determined. This analysis revealed 10 operons that contained 37 genes associated with the type III secretion system. Linkage within these operons suggests a role in type III secretion for each of these genes, including 13 genes encoding proteins with unknown function. The transcriptional start site for each operon was determined. In conjunction with promoter activity assays, this analysis revealed that the type III secretion system operons encode σ70-like promoter elements. Transcriptional initiation by a sigma factor responsible for constitutive gene expression indicates that undefined activators or repressors regulate developmental stage-specific expression of chlamydial type III secretion system genes.

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

2019 ◽  
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

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.

scholarly journals Mutations in Salmonella Pathogenicity Island 2 (SPI2) Genes Affecting Transcription of SPI1 Genes and Resistance to Antimicrobial Agents

1998 ◽  
Vol 180 (18) ◽  
pp. 4775-4780 ◽  
Author(s):  
Jörg Deiwick ◽  
Thomas Nikolaus ◽  
Jaqueline E. Shea ◽  
Colin Gleeson ◽  
David W. Holden ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Antimicrobial Agents ◽  
Type Iii Secretion System ◽  
Polymyxin B ◽  
Secretion System ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Type Iii ◽  
Genes Encoding

ABSTRACT The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of S. typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC,prgK, and hilA, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

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

1998 ◽  
Vol 62 (2) ◽  
pp. 379-433 ◽  
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.

scholarly journals Identification of Substrates and Chaperone from the Yersinia enterocolitica 1B Ysa Type III Secretion System

2003 ◽  
Vol 71 (1) ◽  
pp. 242-253 ◽  
Author(s):  
Boris Foultier ◽  
Paul Troisfontaines ◽  
Didier Vertommen ◽  
Marie-Noëlle Marenne ◽  
Mark Rider ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Secretion System ◽  
Open Reading Frames ◽  
Target Cells ◽  
Type Iii ◽  
Genes Encoding ◽  
Reading Frames

ABSTRACT All pathogenic Yersinia enterocolitica strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37°C. In addition, biovar 1B Y. enterocolitica strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames, sycB, yspB, yspC, yspD, yspA, and acpY, neighbor the ysa genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of Shigella flexneri and SPI-1 of Salmonella enterica. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both Y. enterocolitica TTS systems. YspC could also be secreted by S. flexneri, but no functional complementation of ipaC was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the yopE promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.

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

2020 ◽  
Vol 113 (6) ◽  
pp. 1240-1254 ◽  
Author(s):  
Sibel Westerhausen ◽  
Melanie Nowak ◽  
Claudia E. Torres‐Vargas ◽  
Ursula Bilitewski ◽  
Erwin Bohn ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Secretion Systems ◽  
Time Analysis ◽  
The Real ◽  
Type Iii ◽  
Real Time Analysis ◽  
Type Iii Secretion Systems ◽  
Bacterial Type

scholarly journals Regulation of the Ysa Type III Secretion System of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR

2004 ◽  
Vol 186 (13) ◽  
pp. 4056-4066 ◽  
Author(s):  
Kimberly A. Walker ◽  
Virginia L. Miller
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii ◽  
Regulatory Cascade ◽  
Two Component

ABSTRACT Yersinia enterocolitica biovar 1B contains two type III secretion systems (TTSSs), the plasmid-encoded Ysc-Yop system and the chromosomally encoded Ysa-Ysp system. Proteins secreted from the Ysa TTSS (Ysps) have only been detected in vitro when cells are cultured at 26°C in a high-NaCl medium. However, the exact role of the Ysa TTSS is unclear. Thus, investigations into the regulation of this system may help elucidate the role of the Ysps during the life cycle of Y. enterocolitica. Here we present evidence that the AraC-like regulator YsaE acts together with the chaperone SycB to regulate transcription of the sycByspBCDA operon, a phenomenon similar to that seen in the closely related Salmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs. Deletion of either sycB or ysaE results in a twofold reduction in the activity of a sycB-lacZ fusion compared to the wild type. In a reconstituted Escherichia coli system, transcription of sycB was activated sixfold only when both YsaE and SycB were present, demonstrating that they are necessary for activation. ysrR and ysrS are located near the ysa genes and encode a putative two-component regulatory system. Mutations in either gene indicated that both YsrR and YsrS were required for secretion of Ysps. In addition, transcription from sycB-lacZ and ysaE-lacZ fusions was decreased 6.5- and 25-fold, respectively, in the ysrS mutant compared to the wild type. Furthermore, in the absence of NaCl, the activity of ysaE-lacZ was reduced 25-fold in the wild-type and ΔysrS strains, indicating that YsrS is probably required for the salt-dependent expression of the ysa locus. These results suggest that the putative two-component system YsrRS may be a key element in the regulatory cascade for the Ysa TTSS.

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