scholarly journals The TIR-domain containing effectors BtpA and BtpB from Brucella abortus block energy metabolism

2019 ◽  
Author(s):  
Julia María Coronas-Serna ◽  
Arthur Louche ◽  
María Rodríguez-Escudero ◽  
Morgane Roussin ◽  
Paul R.C. Imbert ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Interleukin 1 ◽  
Effector Proteins ◽  
Host Cells ◽  
Yeast Cells ◽  
Tir Domain ◽  
Tir Domains

ABSTRACTBrucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

scholarly journals Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Plinio S. Vieira ◽  
Isabela M. Bonfim ◽  
Evandro A. Araujo ◽  
Ricardo R. Melo ◽  
Augusto R. Lima ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Model Organism ◽  
Citrus Canker ◽  
Effector Proteins ◽  
Glycoside Hydrolases ◽  
Host Cells ◽  
System A ◽  
Enzymatic Machinery

AbstractXyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

scholarly journals Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

2016 ◽  
Vol 29 (8) ◽  
pp. 651-660 ◽  
Author(s):  
Georgy Popov ◽  
Malou Fraiture ◽  
Frederic Brunner ◽  
Guido Sessa
Keyword(s):  
Pseudomonas Syringae ◽  
Map Kinases ◽  
Molecular Mechanisms ◽  
Inducible Promoter ◽  
Effector Proteins ◽  
Host Cells ◽  
In Planta ◽  
Callose Deposition ◽  
Type Iii ◽  
Xanthomonas Euvesicatoria

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast–based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid–inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

scholarly journals Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of Rickettsia typhi

2008 ◽  
Vol 190 (18) ◽  
pp. 6234-6242 ◽  
Author(s):  
Nicole C. Ammerman ◽  
M. Sayeedur Rahman ◽  
Abdu F. Azad
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Mammalian Host ◽  
Signal Peptides ◽  
Cell Infection ◽  
Rickettsia Typhi ◽  
Sec Translocon

ABSTRACT As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

scholarly journals Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer

2005 ◽  
Vol 187 (22) ◽  
pp. 7716-7726 ◽  
Author(s):  
Karim Suwwan de Felipe ◽  
Sergey Pampou ◽  
Oliver S. Jovanovic ◽  
Christopher D. Pericone ◽  
Senna F. Ye ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Open Reading Frames ◽  
Host Cells ◽  
Dependent Manner ◽  
Major Mechanism ◽  
Cell Functions

ABSTRACT Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the γ-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.

scholarly journals A tractable Drosophila cell system enables rapid identification of Acinetobacter baumannii host factors

2020 ◽  
Author(s):  
Qing-Ming Qin ◽  
Jianwu Pei ◽  
Gabriel Gomez ◽  
Allison Rice-Ficht ◽  
Thomas A. Ficht ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Model System ◽  
Host Factors ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Organelle Biogenesis ◽  
Drosophila Cell

AbstractAcinetobacter baumannii is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported. A. baumannii is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that Drosophila S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the Drosophila system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the Drosophila system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between A. baumannii and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.

scholarly journals NAD+ cleavage activity by animal and plant TIR domains in cell death pathways

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 793-799 ◽  
Author(s):  
Shane Horsefield ◽  
Hayden Burdett ◽  
Xiaoxiao Zhang ◽  
Mohammad K. Manik ◽  
Yun Shi ◽  
...  
Keyword(s):  
Cell Death ◽  
Nicotinamide Adenine Dinucleotide ◽  
Interleukin 1 ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Effector Proteins ◽  
Nicotinamide Adenine ◽  
Cleavage Activity ◽  
Pathogen Effector ◽  
Cell Death Signaling ◽  
Tir Domains

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association–dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

scholarly journals Peptide-mediated Interference of TIR Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-κB

2005 ◽  
Vol 280 (16) ◽  
pp. 15809-15814 ◽  
Author(s):  
Maria Loiarro ◽  
Claudio Sette ◽  
Grazia Gallo ◽  
Andrea Ciacci ◽  
Nicola Fantò ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Cell System ◽  
Tir Domain ◽  
Loop Region ◽  
Good Target ◽  
A Cell ◽  
Tir Domains ◽  
Myeloid Differentiation Factor

Myeloid differentiation factor 88 (MyD88) plays a crucial role in the signaling pathways triggered by interleukin (IL)-1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the Toll/IL-1 receptor (TIR) domain in MyD88 homodimerization in cell-free andin vitroexperimental settings by using epta-peptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified glutathioneS-transferase-MyD88 TIR or co-immunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 epta-peptide inhibits homodimerization of MyD88 TIR domains in anin vitrocell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-κB. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signalingin vivo.

scholarly journals Anti-Inflammatory Effects of Analogues of N-Acyl Homoserine Lactones on Eukaryotic Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9448
Author(s):  
Agathe Peyrottes ◽  
Garance Coquant ◽  
Loïc Brot ◽  
Dominique Rainteau ◽  
Philippe Seksik ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Model ◽  
Homoserine Lactone ◽  
Biological Properties ◽  
Host Cells ◽  
Medical Community ◽  
Eukaryotic Cells ◽  
Reporter Strain ◽  
Ifn Γ ◽  
Anti Inflammatory

Background: Since acyl-homoserine lactone (AHL) profiling has been described in the gut of healthy subjects and patients with inflammatory bowel disease (IBD), the potential effects of these molecules on host cells have raised interest in the medical community. In particular, natural AHLs such as the 3-oxo-C12-HSL exhibit anti-inflammatory properties. Our study aimed at finding stable 3-oxo-C12-HSL-derived analogues with improved anti-inflammatory effects on epithelial and immune cells. Methods: We first studied the stability and biological properties of the natural 3-oxo-C12-HSL on eukaryotic cells and a bacterial reporter strain. We then constructed and screened a library of 22 AHL-derived molecules. Anti-inflammatory effects were assessed by cytokine release in an epithelial cell model, Caco-2, and a murine macrophage cell line, RAW264.7, (respectively, IL-8 and IL-6) upon exposure to the molecule and after appropriate stimulation (respectively, TNF-α 50 ng/mL and IFN-γ 50 ng/mL, and LPS 10 ng/mL and IFN-γ 20 U/mL). Results: We found two molecules of interest with amplified anti-inflammatory effects on mammalian cells without bacterial-activating properties in the reporter strain. The molecules furthermore showed improved stability in biological medium compared to the native 3-oxo-C12-HSL. Conclusions: We provide new bio-inspired AHL analogues with strong anti-inflammatory properties that will need further study from a therapeutic perspective.

scholarly journals Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ

Science ◽  
2020 ◽  
Vol 370 (6521) ◽  
pp. eabd9993 ◽  
Author(s):  
Raoul Martin ◽  
Tiancong Qi ◽  
Haibo Zhang ◽  
Furong Liu ◽  
Miles King ◽  
...  
Keyword(s):  
Active Site ◽  
Nicotiana Benthamiana ◽  
Interleukin 1 ◽  
Leucine Rich Repeat ◽  
Tir Domain ◽  
Pathogen Effectors ◽  
Cryo Electron Microscopy ◽  
Pathogen Effector ◽  
Tir Domains ◽  
Direct Recognition

Plants and animals detect pathogen infection using intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8-angstrom-resolution cryo–electron microscopy structure of the activated ROQ1 (recognition of XopQ 1), an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain bound to the Xanthomonaseuvesicatoria effector XopQ (Xanthomonas outer protein Q). ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.

scholarly journals A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System

2010 ◽  
Vol 192 (12) ◽  
pp. 3114-3122 ◽  
Author(s):  
Loren Rodgers ◽  
Romila Mukerjea ◽  
Sara Birtalan ◽  
Devorah Friedberg ◽  
Partho Ghosh
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii ◽  
Potential Association ◽  
Chaperone Binding ◽  
Bacterial Type ◽  
Characteristic Manner

ABSTRACT Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the Yersinia pseudotuberculosis chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation.

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