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

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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Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical for Listeria monocytogenes Virulence

Infection and Immunity ◽

10.1128/iai.01001-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 44-51 ◽

Cited By ~ 27

Author(s):

P. S. Marie Yeung ◽

Yoojin Na ◽

Amanda J. Kreuder ◽

Hélène Marquis

Keyword(s):

Listeria Monocytogenes ◽

Phospholipase C ◽

Membrane Damage ◽

Bacterial Pathogen ◽

Host Cells ◽

Virulence Attenuation ◽

Infected Cells ◽

Host Cell Membranes ◽

Cell Spread

ABSTRACT Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBΔpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBΔpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

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

Infection and Immunity ◽

10.1128/iai.00778-16 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 22

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.

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Efficient Killing of Multidrug-Resistant Intracellular Bacteria by AIEgens in Vivo

10.26434/chemrxiv.12579611 ◽

2020 ◽

Author(s):

Ying Li ◽

Fei Liu ◽

Jiangjiang Zhang ◽

Xiaoye Liu ◽

Peihong Xiao ◽

...

Keyword(s):

Membrane Damage ◽

Multidrug Resistant ◽

Host Cells ◽

Comparable Efficacy ◽

Intracellular Bacteria ◽

New Agents ◽

Immune Clearance ◽

Infected Cells ◽

Dose Levels

<a>Bacteria infected cells acting as “Trojan horses” not only protect bacteria from antibiotic therapies and immune clearance, but also increase the dissemination of pathogens from the initial sites of infection. Antibiotics are hard and insufficient to treat such hidden intracellular bacteria, especially the multidrug</a>-resistant (MDR) bacteria. Herein, aggregation-induced emission luminogens (AIEgens) such as TBPs showed potent broad-spectrum bactericidal activity against both <a></a><a>extracellular and intracellular</a> Gram-positive pathogens at low-dose levels. TBPs triggered reactive oxygen species (ROS)-mediated membrane damage to kill bacteria, regardless of light irradiation. Additionally, such AIEgens activated mitochondria dependent autophagy to eliminate intracellular bacteria in host cells. Compared to the routinely used vancomycin in clinics, TBPs showed comparable efficacy against methicillin-resistant Staphylococcus aureus (MRSA) in vivo. Our studies demonstrate that AIEgens are promising new agents for the treatment of MDR bacteria associated infections.

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Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽

10.1128/msphere.00858-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 9

Author(s):

Alicja M. Cygan ◽

Terence C. Theisen ◽

Alma G. Mendoza ◽

Nicole D. Marino ◽

Michael W. Panas ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Translocation ◽

Affinity Purification ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Cyclin E1 ◽

Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

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Chemical Inhibitors of the Type Three Secretion System: Disarming Bacterial Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00975-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5433-5441 ◽

Cited By ~ 69

Author(s):

Miles C. Duncan ◽

Roger G. Linington ◽

Victoria Auerbuch

Keyword(s):

Bacterial Infections ◽

Pathogenic Bacteria ◽

Bacterial Pathogens ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Magic Bullet ◽

Antimicrobial Drugs ◽

Type Three Secretion System ◽

Type Three Secretion

ABSTRACTThe recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by “magic bullet” drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

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Extra! Extracellular Effector Delivery into Host Cells via the Type 3 Secretion System

mBio ◽

10.1128/mbio.00594-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 2

Author(s):

Melissa M. Kendall

Keyword(s):

Host Cell ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Alternative Mechanism ◽

Host Cell Membrane ◽

Type Three Secretion System ◽

Host Signaling ◽

Type 3 Secretion System ◽

Type 3

ABSTRACT The type three secretion system (T3SS) is critical for the virulence of diverse bacterial pathogens. Pathogens use the T3SS to deliver effector proteins into host cells and manipulate host signaling pathways. The prevailing mechanism is that effectors translocate from inside the T3SS directly into the host cell. Recent studies reveal an alternative mechanism of effector translocation, in which an effector protein located outside the bacterial cell relies on the T3SS for delivery into host cells. Tejeda-Dominguez et al. (F. Tejeda-Dominguez, J. Huerta-Cantillo, L. Chavez-Dueñas, and F. Navarro-Garcia, mBio 8:e00184-17, 2017, https://doi.org/10.1128/mBio.00184-17 !) demonstrate that the EspC effector of enteropathogenic Escherichia coli is translocated by binding to the outside of the T3SS and subsequently gains access to the host cell cytoplasm through the T3SS pore embedded within the host cell membrane. This work reveals a novel mechanism of translocation that is likely relevant for a variety of other pathogens that use the T3SS as part of their virulence arsenal.

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LcrG is Required for Efficient Translocation ofYersinia Yop Effector Proteins into Eukaryotic Cells

Infection and Immunity ◽

10.1128/iai.66.6.2976-2979.1998 ◽

1998 ◽

Vol 66 (6) ◽

pp. 2976-2979 ◽

Cited By ~ 42

Author(s):

Mahfuzur R. Sarker ◽

Marie-Paule Sory ◽

Aoife P. Boyd ◽

Maite Iriarte ◽

Guy R. Cornelis

Keyword(s):

Immune System ◽

Yersinia Enterocolitica ◽

Effector Proteins ◽

Host Cells ◽

Eukaryotic Cells

ABSTRACT Extracellular Yersinia disables the immune system of its host by injecting effector Yop proteins into host cells. We show that a Yersinia enterocolitica nonpolar lcrGmutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.

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Chlamydial Effector Proteins Localized to the Host Cell Cytoplasmic Compartment

Infection and Immunity ◽

10.1128/iai.00715-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 4842-4850 ◽

Cited By ~ 19

Author(s):

Betsy Kleba ◽

Richard S. Stephens

Keyword(s):

Bacterial Pathogens ◽

Eukaryotic Cell ◽

Effector Proteins ◽

Host Cells ◽

Developmental Cycle ◽

Secretion Systems ◽

Bacterial Proteins ◽

Cell Functions ◽

Infected Cells ◽

Cytoplasmic Compartment

ABSTRACT Disease-causing microbes utilize various strategies to modify their environment in order to create a favorable location for growth and survival. Gram-negative bacterial pathogens often use specialized secretion systems to translocate effector proteins directly into the cytosol of the eukaryotic cells they infect. These bacterial proteins are responsible for modulating eukaryotic cell functions. Identification of the bacterial effectors has been a critical step toward understanding the molecular basis for the pathogenesis of the bacteria that use them. Chlamydiae are obligate intracellular bacterial pathogens that have a type III secretion system believed to translocate virulence effector proteins into the cytosol of their host cells. Selective permeabilization of the eukaryotic cell membrane was used in conjunction with metabolic labeling of bacterial proteins to identify chlamydial proteins that localize within the cytosol of infected cells. More than 20 Chlamydia trachomatis and C. pneumoniae proteins were detected within the cytoplasmic compartment of infected cells. While a number of cytosolic proteins were shared, others were unique to each species, suggesting that variation among cytosolic chlamydial proteins contributes to the differences in the pathogenesis of the chlamydial species. The spectrum of chlamydial proteins exported differed concomitant with the progress of the developmental cycle. These data confirm that a dynamic relationship exists between Chlamydia and its host and that translocation of bacterial proteins into the cytosol is developmentally dependent.

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The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition

Frontiers in Immunology ◽

10.3389/fimmu.2021.714799 ◽

2021 ◽

Vol 12 ◽

Author(s):

Abigael Eva Chaouat ◽

Barbara Seliger ◽

Ofer Mandelboim ◽

Dominik Schmiedel

Keyword(s):

Immune System ◽

Immune Evasion ◽

Immune Cells ◽

Immune Escape ◽

Human Herpesvirus ◽

Immune Recognition ◽

Nkg2d Ligand ◽

Human Immune System ◽

Nkg2d Ligands ◽

Infected Cells

The coevolution of the human immune system and herpesviruses led to the emergence and diversification of both cellular danger molecules recognized by immune cells on the one hand and viral countermeasures that prevent the expression of these proteins on infected cells on the other. There are eight ligands for the activating receptor NKG2D in humans – MICA, MICB, ULBP1-6. Several of them are induced and surface-expressed on herpesvirus-infected cells to serve as danger signals to activate the immune system. Therefore, these ligands are frequently targeted for suppression by viral immune evasion mechanisms. Mechanisms to downregulate NKG2D ligands and thereby escape immune recognition have been identified in all other human herpesviruses (HHV), except for HHV-6A. In this study, we identify two HHV-6A encoded immunoevasins, U20 and U21, which suppress the expression of the NKG2D ligands ULBP1 and ULBP3, respectively, during infection. Additionally, MICB is targeted by a so far unexplored viral protein. Due to the diminished NKG2D ligand surface expression on infected cells, recognition of HHV-6A infected cells by innate immune cells is impaired. Importantly, our study indicates that immune escape mechanisms between the related herpesviruses HHV-6A and HHV-6B are evolutionary conserved as the same NKG2D ligands are targeted. Our data contribute an additional piece of evidence for the importance of the NKG2D receptor – NKG2D ligand axis during human herpesvirus infections and sheds light on immune evasion mechanisms of HHV-6A.

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Investigation of Bartonella quintana Host Immune Evasion

10.26686/wgtn.17131799.v1 ◽

2021 ◽

Author(s):

◽

Callum Lambert

Keyword(s):

Immune System ◽

Actin Cytoskeleton ◽

Host Cell ◽

Immune Evasion ◽

Mammalian Species ◽

Effector Proteins ◽

Host Cells ◽

Sand Fly ◽

Host Immune System ◽

Bartonella Quintana

Bartonella is a genus of gram-negative alphaproteobacteria that infect mammals, causing both acute and chronic disease. Bartonella are re-emerging infectious pathogens that cause a variety of clinical syndromes in humans worldwide, including cat scratch disease, trench fever, bacillary angiomatosis, and endocarditis. Bartonella spp. are spread by biting arthropods such as the sand fly, cat flea, and body louse, and have been isolated from almost all mammalian species tested. Bartonella are a re-emerging concern as the number of confirmed Bartonella diagnoses are increasing, primarily in immunocompromised groups, homeless populations, refugee camps, and in veterinary workers. The three primary human disease-causing Bartonella spp. are B. henselae, B. quintana, and B. bacilliformis. Bartonella are known to subvert the host immune system and persist within the host, often causing bacteraemia which is difficult to effectively diagnose and treat. B. quintana infects humans; after introduction to the skin the bacteria implement numerous immune evasion mechanisms to enter the bloodstream and invade erythrocytes. The mechanisms by which B. quintana modulates and evades the immune system during early infection are almost entirely unknown. Following exposure to B. quintana, the bacteria encounter host immune cells but survive, evading these cells and disseminating into the lymphatic system and eventually bloodstream. This thesis project aimed to dissect the interactions between B. quintana and the human innate immune system to better understand the early stages of infection. A gentamicin protection assay was developed to investigate the ability of THP-1 macrophages, representing human macrophages present in the skin, to internalise B. quintana. These data revealed THP-1 cells were unable to effectively internalise B. quintana, although the mechanism responsible was not determined. Subsequent experiments investigated the role of the B. quintana Type IV secreted effector protein BepA1 in the inhibition of internalisation. Bacterial effector proteins often pathogenically modulate host cell signalling to benefit the bacteria, i.e., altering the actin cytoskeleton to inhibit phagocytosis or supressing immune responses. It was hypothesised BepA1 could play a role in inhibiting phagocytosis; therefore, the host cell target of BepA1 was investigated with a yeast two-hybrid system assay. The human protein Myozap was uncovered as a potential protein that interacts with BepA1. Myozap is expressed in cardiac and lung tissue as well as epithelial and endothelial cells, where it modulates Rho-dependent actin signalling, potentially affecting the actin cytoskeleton and the transcription factor MRTF-A, which influences immune reaction through modulation of NF-κB. To investigate the functional effects of BepA1 activity in host cells, HeLa cells were transfected with BepA1; cell migration and cytokine secretion were assessed, revealing a decrease in pro-inflammatory cytokines in BepA1-transfected cells in response to TNF-a stimulation. These data suggest BepA1 may be deployed by B. quintana during infection to suppress the host immune response and avoid clearance from the site of infection. This research addressed a major gap in our understanding of B. quintana infections. Improving our understanding of the interactions between Bartonella and the host immune system is an essential first step in the development of improved diagnostic techniques and treatments.

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