scholarly journals Characterisation of the Bartonella quintana YopJ Homologue

2021 ◽  
Author(s):  
◽  
Alvey Little
Keyword(s):  
Immune Modulation ◽  
Effector Proteins ◽  
Host Protein ◽  
Host Cells ◽  
Host Immune System ◽  
Bartonella Quintana ◽  
Type Iv ◽  
Yersinia Species ◽  
System A ◽  
The Many

<p><b>Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function.</b></p> <p>Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria.</p> <p>My aim was to characterise the function of the B. quintana YopJ homologue.</p> <p>I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets.</p> <p>I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen.</p> <p>I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.</p>

scholarly journals Characterisation of the Bartonella quintana YopJ Homologue

2021 ◽  
Author(s):  
◽  
Alvey Little
Keyword(s):  
Immune Modulation ◽  
Effector Proteins ◽  
Host Protein ◽  
Host Cells ◽  
Host Immune System ◽  
Bartonella Quintana ◽  
Type Iv ◽  
Yersinia Species ◽  
System A ◽  
The Many

<p><b>Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function.</b></p> <p>Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria.</p> <p>My aim was to characterise the function of the B. quintana YopJ homologue.</p> <p>I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets.</p> <p>I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen.</p> <p>I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.</p>

scholarly journals Investigation of Bartonella quintana Host Immune Evasion

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

<p>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.   </p>

scholarly journals Investigation of Bartonella quintana Host Immune Evasion

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

<p>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.   </p>

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.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

scholarly journals MultipleLegionella pneumophilaeffector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

2017 ◽  
Vol 114 (48) ◽  
pp. E10446-E10454 ◽  
Author(s):  
Stephanie R. Shames ◽  
Luying Liu ◽  
James C. Havey ◽  
Whitman B. Schofield ◽  
Andrew L. Goodman ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Host Cells ◽  
Limiting Factor ◽  
Effector Protein ◽  
Host Immune System ◽  
Loss Of Function ◽  
High Throughput Analysis ◽  
Single Strain

Legionella pneumophilais the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain ofL. pneumophilaencodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number ofL. pneumophilaeffectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhancedL. pneumophilain the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute toL. pneumophilavirulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

scholarly journals DeepT3_4: A Hybrid Deep Neural Network Model for the Distinction Between Bacterial Type III and IV Secreted Effectors

2021 ◽  
Vol 12 ◽  
Author(s):  
Lezheng Yu ◽  
Fengjuan Liu ◽  
Yizhou Li ◽  
Jiesi Luo ◽  
Runyu Jing
Keyword(s):  
Neural Network ◽  
Neural Networks ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Sequence Motifs ◽  
Type Vi Secretion System ◽  
Type Iii ◽  
Type Iv

Gram-negative bacteria can deliver secreted proteins (also known as secreted effectors) directly into host cells through type III secretion system (T3SS), type IV secretion system (T4SS), and type VI secretion system (T6SS) and cause various diseases. These secreted effectors are heavily involved in the interactions between bacteria and host cells, so their identification is crucial for the discovery and development of novel anti-bacterial drugs. It is currently challenging to accurately distinguish type III secreted effectors (T3SEs) and type IV secreted effectors (T4SEs) because neither T3SEs nor T4SEs contain N-terminal signal peptides, and some of these effectors have similar evolutionary conserved profiles and sequence motifs. To address this challenge, we develop a deep learning (DL) approach called DeepT3_4 to correctly classify T3SEs and T4SEs. We generate amino-acid character dictionary and sequence-based features extracted from effector proteins and subsequently implement these features into a hybrid model that integrates recurrent neural networks (RNNs) and deep neural networks (DNNs). After training the model, the hybrid neural network classifies secreted effectors into two different classes with an accuracy, F-value, and recall of over 80.0%. Our approach stands for the first DL approach for the classification of T3SEs and T4SEs, providing a promising supplementary tool for further secretome studies.

scholarly journals Using an Optimal Set of Features with a Machine Learning-Based Approach to Predict Effector Proteins forLegionella pneumophila

2018 ◽  
Author(s):  
Zhila Esna Ashari ◽  
Kelly A. Brayton ◽  
Shira L. Broschat
Keyword(s):  
Machine Learning ◽  
Legionella Pneumophila ◽  
De Novo ◽  
Effector Proteins ◽  
Machine Learning Algorithms ◽  
Host Cells ◽  
Support Vector ◽  
Secretion Systems ◽  
Type Iv ◽  
Optimal Set

AbstractType IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This work focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogenLegionella pneumophilastrain Philadelphia-1, a cause of Legionnaires’ disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 760 effector proteins, more than any other study, 315 of which have been validated. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.

scholarly journals Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

2014 ◽  
Vol 82 (10) ◽  
pp. 4325-4336 ◽  
Author(s):  
Alan M. Copenhaver ◽  
Cierra N. Casson ◽  
Hieu T. Nguyen ◽  
Thomas C. Fung ◽  
Matthew M. Duda ◽  
...  
Keyword(s):  
Immune Response ◽  
Alveolar Macrophages ◽  
Legionella Pneumophila ◽  
Pulmonary Infection ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Content Type ◽  
Type Iv

ABSTRACTLegionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses itsdot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol,L. pneumophilaalso activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types thatL. pneumophilainfects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response againstL. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain ofL. pneumophilaproducing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viableL. pneumophiladuring pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient,L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir forL. pneumophilaand a source of proinflammatory cytokines that contribute to the host immune response againstL. pneumophiladuring pulmonary infection.

scholarly journals Postreplication Roles of theBrucellaVirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Erin P. Smith ◽  
Cheryl N. Miller ◽  
Robert Child ◽  
Jennifer A. Cundiff ◽  
Jean Celli
Keyword(s):  
Brucella Abortus ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Conditional Expression ◽  
Bacterial Replication

ABSTRACTBrucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, theBrucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by theBrucellaVirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of theBrucellaintracellular cycle. To address this issue, we have generated aB. abortusstrain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of thevirB11gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression ofvirB11prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in theBrucellaintracellular cycle.IMPORTANCEMany intracellular bacterial pathogens encode specialized secretion systems that deliver effector proteins into host cells to mediate the multiple stages of their intracellular cycles. Because these intracellular events occur sequentially, classical genetic approaches cannot address the late roles that these apparatuses play, as secretion-deficient mutants cannot proceed past their initial defect. Here we have designed a functionally controllable VirB type IV secretion system (T4SS) in the bacterial pathogenBrucella abortusto decipher its temporal requirements during the bacterium’s intracellular cycle in macrophages. By controlling production of the VirB11 ATPase, which energizes the T4SS, we show not only that this apparatus is required early to generate theBrucellareplicative organelle but also that it contributes to completion of the bacterium’s cycle and bacterial egress. Our findings expand upon the pathogenic functions of theBrucellaVirB T4SS and illustrate targeting of secretion ATPases as a useful strategy to manipulate the activity of bacterial secretion systems.

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