scholarly journals A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

2019 ◽  
Author(s):  
Januka S Athukoralage ◽  
Stephen McMahon ◽  
Changyi Zhang ◽  
Sabine Grüschow ◽  
Shirley Graham ◽  
...  
Keyword(s):  
Active Site ◽  
Cyclic Nucleotides ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Type Iii ◽  
Signalling Molecules ◽  
New Family ◽  
Signalling Molecule ◽  
Defence Enzymes

ABSTRACTThe CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1–3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

scholarly journals Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Extracellular Proteins Involved in Soybean Cultivar-Specific Nodulation Are Associated with Pilus-Like Surface Appendages and Exported by a Type III Protein Secretion System in Sinorhizobium fredii USDA257

2003 ◽  
Vol 16 (7) ◽  
pp. 617-625 ◽  
Author(s):  
Hari B. Krishnan ◽  
Julio Lorio ◽  
Won Seok Kim ◽  
Guoqiao Jiang ◽  
Kil Yong Kim ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Symbiotic Bacterium ◽  
Secretion System ◽  
Extracellular Proteins ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Sinorhizobium Fredii ◽  
Type Iii ◽  
Conserved Genes ◽  
Type Iii Protein Secretion

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

scholarly journals Functional Analysis of Plant Defense Suppression and Activation by the Xanthomonas Core Type III Effector XopX

2015 ◽  
Vol 28 (2) ◽  
pp. 180-194 ◽  
Author(s):  
William Stork ◽  
Jung-Gun Kim ◽  
Mary Beth Mudgett
Keyword(s):  
Plant Defense ◽  
Defense Responses ◽  
Effector Proteins ◽  
Immune Signaling ◽  
Type Iii ◽  
Effector Triggered Immunity ◽  
Gene Transcripts ◽  
Type Iii Secretion Effector ◽  
Susceptible Tomato

Many phytopathogenic type III secretion effector proteins (T3Es) have been shown to target and suppress plant immune signaling but perturbation of the plant immune system by T3Es can also elicit a plant response. XopX is a “core” Xanthomonas T3E that contributes to growth and symptom development during Xanthomonas euvesicatoria infection of tomato but its functional role is undefined. We tested the effect of XopX on several aspects of plant immune signaling. XopX promoted ethylene production and plant cell death (PCD) during X. euvesicatoria infection of susceptible tomato and in transient expression assays in Nicotiana benthamiana, which is consistent with its requirement for the development of X. euvesicatoria-induced disease symptoms. Additionally, although XopX suppressed flagellin-induced reactive oxygen species, it promoted the accumulation of pattern-triggered immunity (PTI) gene transcripts. Surprisingly, XopX coexpression with other PCD elicitors resulted in delayed PCD, suggesting antagonism between XopX-dependent PCD and other PCD pathways. However, we found no evidence that XopX contributed to the suppression of effector-triggered immunity during X. euvesicatoria–tomato interactions, suggesting that XopX's primary virulence role is to modulate PTI. These results highlight the dual role of a core Xanthomonas T3E in simultaneously suppressing and activating plant defense responses.

The role of Engrailed in establishing the dorsoventral axis of the chick limb

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2317-2324 ◽  
Author(s):  
C. Logan ◽  
A. Hornbruch ◽  
I. Campbell ◽  
A. Lumsden
Keyword(s):  
Expression Patterns ◽  
Limb Bud ◽  
Dorsoventral Patterning ◽  
Signalling Molecules ◽  
Endogenous Expression ◽  
Signalling Molecule ◽  
Normal Expression ◽  
Dorsal Ectoderm ◽  
Mutation Analyses

Expression and mutation analyses in mice suggest that the homeobox-containing gene Engrailed (En) plays a role in dorsoventral patterning of the limb. During the initial stages of limb bud outgrowth, En-1 mRNA and protein are uniformly distributed throughout the ventral limb bud ectoderm. Limbs of En-1(−/−) mice display a double dorsal phenotype suggesting that normal expression of En-1 in the ventral ectoderm is required to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectodermal ridge (AER), suggesting that En-1 is also required for proper formation of the AER. To further investigate the role En plays in dorsoventral patterning and AER formation, we have used the replication competent retroviral vector, RCAS, to mis-express mouse En-1 in the early chick limb bud. We show that ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectodermal marker Wnt7a, with a resultant decrease in Lmx1 expression in underlying dorsal mesenchyme. Furthermore, the AER is disrupted morphologically and the expression patterns of the AER signalling molecules Fgf-8 and Fgf-4 are altered. Consistent with recent evidence that there is a reciprocal interaction between signalling molecules in the dorsal ectoderm, AER, and zone of polarising activity (ZPA), loss of Wnt7a, Fgf-8 and Fgf-4 expression leads to a decrease in expression of the signalling molecule Shh in the ZPA. These results strongly support the idea that, in its normal domain of expression, En-1 represses Wnt7a-mediated dorsal differentiation by limiting the expression of Wnt7a to the dorsal ectoderm. Furthermore, our results provide additional evidence that En-1 is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity.

scholarly journals Role of the Salmonella Pathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, Two Salmonella Effector Proteins Encoded Outside of SPI-1

2003 ◽  
Vol 185 (23) ◽  
pp. 6950-6967 ◽  
Author(s):  
Kristin Ehrbar ◽  
Andrea Friebel ◽  
Samuel I. Miller ◽  
Wolf-Dietrich Hardt
Keyword(s):  
Type Iii Secretion ◽  
Inflammatory Responses ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Host Cells ◽  
Effector Protein ◽  
Dependent Manner ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.

scholarly journals Identification of a Twin-Arginine Translocation System in Pseudomonas syringae pv. tomato DC3000 and Its Contribution to Pathogenicity and Fitness

2005 ◽  
Vol 187 (24) ◽  
pp. 8450-8461 ◽  
Author(s):  
Philip A. Bronstein ◽  
Matthew Marrichi ◽  
Sam Cartinhour ◽  
David J. Schneider ◽  
Matthew P. DeLisa
Keyword(s):  
Pseudomonas Syringae ◽  
Virulence Factors ◽  
Fluorescent Protein ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Tomato Dc3000 ◽  
Phytopathogenic Bacterium ◽  
Type Iii ◽  
Twin Arginine Translocation ◽  
Tat System

ABSTRACT The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.

Role of the Active Site Cysteine of DpgA, a Bacterial Type III Polyketide Synthase†

Biochemistry ◽  
2004 ◽  
Vol 43 (4) ◽  
pp. 970-980 ◽  
Author(s):  
Claire C. Tseng ◽  
Shaun M. McLoughlin ◽  
Neil L. Kelleher ◽  
Christopher T. Walsh
Keyword(s):  
Active Site ◽  
Polyketide Synthase ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Active Site Cysteine ◽  
Bacterial Type

scholarly journals Role of Fraction 1 Antigen of Yersinia pestis in Inhibition of Phagocytosis

2002 ◽  
Vol 70 (3) ◽  
pp. 1453-1460 ◽  
Author(s):  
Yidong Du ◽  
Roland Rosqvist ◽  
Åke Forsberg
Keyword(s):  
Yersinia Pestis ◽  
Effector Proteins ◽  
Receptor Interaction ◽  
Virulence Plasmid ◽  
Protein Subunit ◽  
Bacterial Surface ◽  
Type Iii ◽  
F1 Antigen ◽  
J774 Cells

ABSTRACT Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37°C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.

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