scholarly journals Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components

2017 ◽  
Vol 85 (10) ◽  
Author(s):  
Erin E. Zwack ◽  
Eric M. Feeley ◽  
Amanda R. Burton ◽  
Baofeng Hu ◽  
Masahiro Yamamoto ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Membrane Damage ◽  
Effector Proteins ◽  
Host Cells ◽  
Target Cells ◽  
Inflammasome Activation ◽  
Secretion Systems ◽  
Content Type ◽  
Endosomal Membrane ◽  
Galectin 3

ABSTRACT Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (Gbp Chr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

scholarly journals Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Maarten F. de Jong ◽  
Neal M. Alto
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
E Coli ◽  
Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

scholarly journals Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Erez Mills ◽  
Kobi Baruch ◽  
Gili Aviv ◽  
Mor Nitzan ◽  
Ilan Rosenshine
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Public Health Problem ◽  
Effector Proteins ◽  
Host Cells ◽  
Global Public Health ◽  
Secretion Systems ◽  
Comprehensive Description ◽  
Content Type ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

scholarly journals Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Ahalieyah Anantharajah ◽  
Julien M. Buyck ◽  
Charlotta Sundin ◽  
Paul M. Tulkens ◽  
Marie-Paule Mingeot-Leclercq ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Transcriptional Activation ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Inflammasome Activation ◽  
Protective Effects ◽  
Secretion Systems ◽  
Content Type

ABSTRACT Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosa in vitro. Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa. Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa. Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

scholarly journals Epistatic Interplay between Type IV Secretion Effectors Engages the Small GTPase Rab2 in the Brucella Intracellular Cycle

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Erin P. Smith ◽  
Alexis Cotto-Rosario ◽  
Elizabeth Borghesan ◽  
Kiara Held ◽  
Cheryl N. Miller ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Bacterial Pathogens ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iv ◽  
Infectious Cycle

ABSTRACT Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors. IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

scholarly journals Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems

2017 ◽  
Vol 114 (9) ◽  
pp. E1698-E1706 ◽  
Author(s):  
Eric M. Feeley ◽  
Danielle M. Pilla-Moffett ◽  
Erin E. Zwack ◽  
Anthony S. Piro ◽  
Ryan Finethy ◽  
...  
Keyword(s):  
Host Defense ◽  
Binding Proteins ◽  
Host Cells ◽  
Carbohydrate Binding ◽  
Bacterial Protein ◽  
Secretion Systems ◽  
Defense Proteins ◽  
Galectin 3 ◽  
Protein Secretion Systems ◽  
Bacterial Secretion

Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as “patterns of pathogenesis” associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3–deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3–dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

scholarly journals Role of Bacterial Secretion Systems and Effector Proteins – Insight into the Pathogenicity Mechanisms in Aeromonas

2020 ◽  
Author(s):  
Joanna Matys ◽  
Anna Turska-Szewczuk ◽  
Anna Sroka-Bartnicka
Keyword(s):  
Virulence Factors ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Target Cells ◽  
Virulence Determinants ◽  
Secretion Systems ◽  
Variable Expression ◽  
Type Iv ◽  
T3ss Effector

Gram-negative bacteria have developed several nanomachine channels known as type II, III, IV and VI secretion systems that enable export of effector proteins/toxins from the cytosol across the outer membrane to target host cells. Protein secretion systems are critical to bacterial virulence and interactions with other organisms. Aeromonas utilize various secretion machines e.g. two-step T2SS, a Sec-dependent system as well as one-step, Sec-independent T3SS and T6SS systems to transport effector proteins/toxins and virulence factors. Type III secretion system (T3SS) is considered the dominant virulence system in Aeromonas. The activity of bacterial T3SS effector proteins most often leads to disorders in signalling pathways and reorganization of the cell cytoskeleton. There are also scientific reports on the pathogenicity mechanism associated with host cell apopotosis/pyroptosis resulting from secretion of a cytotoxic enterotoxin, i.e. the Act protein, by the T2SS secretion system and an effector protein Hcp by the T6SS system. Type IV secretion system (T4SS) is the system which translocate protein substrates, protein-DNA complexes and DNA into eukaryotic or bacterial target cells. In this paper, the contribution of virulence determinants involved in the pathogenicity potential of Aeromonas is discussed. Considering that the variable expression of virulence factors has a decisive impact on the differences observed in the virulence of particular species of microorganisms, it is important to assess the correlation between bacterial pathogenicity and their virulence-associated genes.

scholarly journals Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Vera Göser ◽  
Carina Kommnick ◽  
Viktoria Liss ◽  
Michael Hensel
Keyword(s):  
Type Iii Secretion ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
Superresolution Microscopy ◽  
Type Iii ◽  
T3ss Effector ◽  
Novel Approach ◽  
Temporal And Spatial

ABSTRACTType III secretion systems (T3SS) are molecular machines in Gram-negative pathogens that translocate effector proteins with central roles in virulence. The analyses of the translocation, subcellular localization, and mode of action of T3SS effector proteins are of central importance for the understanding of host-pathogen interaction and pathogenesis of bacterial infections. The analysis of translocation requires dedicated techniques to address the temporal and spatial dynamics of translocation. Here we describe a novel approach to deploy self-labeling enzymes (SLE) as universal tags for localization and tracking of translocated effector proteins. Effector-SLE fusion proteins allow live-cell imaging of translocation by T3SS, superresolution microscopy, and single-molecule tracking of effector motility in living host cells. We describe the application of the approach to T3SS effector proteins for invasion and intracellular lifestyle ofSalmonella entericaserovar Typhimurium and to a T3SS effector ofYersinia enterocolitica. The novel approach enables analyses of the role of T3SS in host-pathogen interaction at the highest temporal and spatial resolution, toward understanding the molecular mechanisms of their effector proteins.IMPORTANCEType III secretion systems mediate translocation of effector proteins into mammalian cells. These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. The new approach of self-labeling enzyme tags fusions will enable analyses of type III secretion system effector proteins with new dimensions of temporal and spatial resolution.

scholarly journals Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

2019 ◽  
Vol 87 (3) ◽  
Author(s):  
Natasha P. Medici ◽  
Maheen Rashid ◽  
James B. Bliska
Keyword(s):  
Immune Responses ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Cell Physiology ◽  
Inflammasome Activation ◽  
Wild Type ◽  
Content Type

ABSTRACT Pathogenic Yersinia species deliver Yop effector proteins through a type III secretion system into host cells. Among these effectors, YopE and YopT are Rho-modifying toxins, which function to modulate host cell physiology and evade immune responses. YopE is a GTPase-activating protein (GAP) while YopT is a protease, and they inhibit RhoA by different modes of action. Modifications to RhoA are sensed by pyrin, which, once activated, assembles a caspase-1 inflammasome, which generates cytokines such as interleukin-1β (IL-1β) and cell death by pyroptosis. In Yersinia-infected macrophages, YopE or YopT triggers inflammasome assembly only in the absence of another effector, YopM, which counteracts pyrin by keeping it inactive. The glucosyltransferase TcdB from Clostridium difficile, a well-studied RhoA-inactivating toxin, triggers activation of murine pyrin by dephosphorylation of Ser205 and Ser241. To determine if YopE or YopT triggers pyrin dephosphorylation, we infected lipopolysaccharide (LPS)-primed murine macrophages with ΔyopM Yersinia pseudotuberculosis strains expressing wild-type (wt) or YopE mutant variants or YopT. By immunoblotting pyrin after infection, we observed that wt YopE triggered dephosphorylation of Ser205 and inflammasome activation. Pyrin dephosphorylation was reduced if a YopE variant had a defect in stability or RhoA specificity but not membrane localization. We also observed that wt YopT triggered pyrin dephosphorylation but more slowly than YopE, suggesting that YopE is dominant in this process. Our findings provide evidence that RhoA-modifying toxins trigger activation of pyrin by a conserved dephosphorylation mechanism. In addition, by characterization of YopE and YopT, we show that different features of effectors, such as RhoA specificity, affect the efficiency of pyrin dephosphorylation.

scholarly journals The Salmonella Effector SteA Contributes to the Control of Membrane Dynamics of Salmonella-Containing Vacuoles

2014 ◽  
Vol 82 (7) ◽  
pp. 2923-2934 ◽  
Author(s):  
Lia Domingues ◽  
David W. Holden ◽  
Luís Jaime Mota
Keyword(s):  
Systemic Disease ◽  
Bacterial Pathogen ◽  
Membrane Dynamics ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
Microtubule Motors ◽  
Serovar Typhimurium ◽  
Microtubule Motor

ABSTRACTSalmonella entericaserovar Typhimurium is a bacterial pathogen causing gastroenteritis in humans and a typhoid-like systemic disease in mice.S. Typhimurium virulence is related to its capacity to multiply intracellularly within a membrane-bound compartment, theSalmonella-containing vacuole (SCV), and depends on type III secretion systems that deliver bacterial effector proteins into host cells. Here, we analyzed the cellular function of theSalmonellaeffector SteA. We show that, compared to cells infected by wild-typeS. Typhimurium, cells infected by ΔsteAmutant bacteria displayed fewerSalmonella-induced filaments (SIFs), an increased clustering of SCVs, and morphologically abnormal vacuoles containing more than one bacterium. The increased clustering of SCVs and the appearance of vacuoles containing more than one bacterium were suppressed by inhibition of the activity of the microtubule motor dynein or kinesin-1. Clustering and positioning of SCVs are controlled by the effectors SseF and SseG, possibly by helping to maintain a balanced activity of microtubule motors on the bacterial vacuoles. Deletion ofsteAinS. Typhimurium ΔsseFor ΔsseGmutants revealed that SteA contributes to the characteristic scattered distribution of ΔsseFor ΔsseGmutant SCVs in infected cells. Overall, this shows that SteA participates in the control of SCV membrane dynamics. Moreover, it indicates that SteA is functionally linked to SseF and SseG and suggests that it might contribute directly or indirectly to the regulation of microtubule motors on the bacterial vacuoles.

scholarly journals Live imaging of Yersinia translocon formation and immune recognition in host cells

2022 ◽  
Author(s):  
Maren Rudolph ◽  
Alexander Carsten ◽  
Martin Aepfelbacher ◽  
Manuel Wolters
Keyword(s):  
Immune System ◽  
Membrane Damage ◽  
Effector Proteins ◽  
Host Cells ◽  
Immune Recognition ◽  
Type Three Secretion System ◽  
Galectin 3 ◽  
Infected Cells ◽  
Host Cell Membranes ◽  
Phagosomal Membrane

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia -containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

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