Novel class of OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection

2020 ◽  
Author(s):  
Donghyuk Shin ◽  
Anshu Bhattacharya ◽  
Yi-Lin Cheng ◽  
Marta Campos Alonso ◽  
Ahmad Reza Mehdipour ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Homology Detection ◽  
Host Pathogen Interactions ◽  
Catalytic Core ◽  
Ubiquitin Binding ◽  
Structural Differences ◽  
Host Pathogen ◽  
Ubiquitination System ◽  
Structural Insights

AbstractLegionella pneumophila is a gram-negative pathogenic bacterium that causes Legionaries’ disease. The Legionella genome codes more than 300 effector proteins able to modulate host-pathogen interactions during infection. Among them are also enzymes altering the host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on homology-detection screening on 305 Legionella effector proteins, we identified two LegionellaOTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was determined at 2.4 Å and compared with other OTU deubiquitinases, including LotB. Unlike the classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an extended helical lobe between the Cys-loop and the variable loop, which define a novel class of OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC interact with ubiquitin. LotB has an additional ubiquitin binding site (S1’) enabling specific cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC identified different categories of host-substrates for these two related DUBs. Together, our results provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of bacterial deubiquitinases in host-pathogen interactions.

scholarly journals Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Donghyuk Shin ◽  
Anshu Bhattacharya ◽  
Yi-Lin Cheng ◽  
Marta Campos Alonso ◽  
Ahmad Reza Mehdipour ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Host Cells ◽  
Catalytic Core ◽  
Polyubiquitin Chains ◽  
Ubiquitin Binding ◽  
Legionnaires Disease ◽  
Ubiquitination System ◽  
Structural Insights

Legionella pneumophila causes a severe pneumonia known as Legionnaires’ disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1’), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host–pathogen interactions.

scholarly journals The Ubiquitination System within Bacterial Host–Pathogen Interactions

2021 ◽  
Vol 9 (3) ◽  
pp. 638
Author(s):  
Vera Vozandychova ◽  
Pavla Stojkova ◽  
Kamil Hercik ◽  
Pavel Rehulka ◽  
Jiri Stulik
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Deubiquitinating Enzymes ◽  
Host Pathogen Interactions ◽  
Host Pathogen ◽  
Ubiquitination System

Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host–pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. Pathogenic bacteria (e.g., Salmonella enterica, Legionella pneumophila and Shigella flexneri) encode many effector proteins, such as deubiquitinating enzymes (DUBs), targeting the host ubiquitin machinery and thus disrupting pertinent ubiquitin-dependent anti-bacterial response. We focus here upon the host ubiquitination system as an integral unit, its interconnection with the regulation of inflammation and autophagy, and primarily while examining pathogens manipulating the host ubiquitination system. Many bacterial effector proteins have already been described as being translocated into the host cell, where they directly regulate host defense processes. Due to their importance in pathogenic bacteria progression within the host, they are regarded as virulence factors essential for bacterial evasion. However, in some cases (e.g., Francisella tularensis) the host ubiquitination system is influenced by bacterial infection, although the responsible bacterial effectors are still unknown.

scholarly journals Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jonas Kjellin ◽  
Maria Pränting ◽  
Frauke Bach ◽  
Roshan Vaid ◽  
Bart Edelbroek ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Legionella Pneumophila ◽  
Transcriptional Response ◽  
Infection Model ◽  
Intracellular Pathogens ◽  
Host Pathogen Interactions ◽  
Human Macrophages ◽  
Wide Range ◽  
Host Pathogen

Abstract Background During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

scholarly journals Gene expression profiling in human neutrophils after infection with Acinetobacter baumannii in vitro

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242674
Author(s):  
María Lázaro-Díez ◽  
Itziar Chapartegui-González ◽  
Borja Suberbiola ◽  
J. Gonzalo Ocejo-Vinyals ◽  
Marcos López-Hoyos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acinetobacter Baumannii ◽  
Protein Quantification ◽  
Effector Proteins ◽  
Human Neutrophils ◽  
Host Pathogen Interactions ◽  
Proinflammatory Response ◽  
Host Pathogen ◽  
Key Aspects

Acinetobacter baumannii is a Gram negative nosocomial pathogen that has acquired increasing worldwide notoriety due to its high antibiotic resistance range and mortality rates in hospitalized patients. Therefore, it is necessary to better understand key aspects of A. baumannii pathogenesis such as host-pathogen interactions. In this report, we analyzed both gene expression and cytokine production by human neutrophils infected with A. baumannii. Our assays reveal a proinflammatory response of neutrophils after A. baumannii infection, since intracellular transcription of effector proteins such as COX-2, transcription factors, and proinflammatory cytokines resulted significantly upregulated in neutrophils infected by A. baumannii, compared with unstimulated human neutrophils. Translation and release of CXCL-8, IL-1β and TNF-α by neutrophils was confirmed by protein quantification in culture supernatants. Results obtained in this report reinforce the importance of human neutrophils in controlling A. baumannii infections but also emphasize the proinflammatory nature of these host-pathogen interactions as a target for future immunomodulatory therapies.

scholarly journals Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions

2008 ◽  
Vol 72 (4) ◽  
pp. 765-781 ◽  
Author(s):  
Fiona M. Sansom ◽  
Simon C. Robson ◽  
Elizabeth L. Hartland
Keyword(s):  
Legionella Pneumophila ◽  
Purinergic Signaling ◽  
Extracellular Nucleotides ◽  
Microbial Pathogens ◽  
Nucleoside Triphosphate ◽  
Host Pathogen Interactions ◽  
Host Pathogen ◽  
Specific Receptors ◽  
Parasitic Pathogens

SUMMARYIn humans, purinergic signaling plays an important role in the modulation of immune responses through specific receptors that recognize nucleoside tri- and diphosphates as signaling molecules. Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) have important roles in the regulation of purinergic signaling by controlling levels of extracellular nucleotides. This process is key to pathophysiological protective responses such as hemostasis and inflammation. Ecto-NTPDases are found in all higher eukaryotes, and recently it has become apparent that a number of important parasitic pathogens of humans express surface-located NTPDases that have been linked to virulence. For those parasites that are purine auxotrophs, these enzymes may play an important role in purine scavenging, although they may also influence the host response to infection. Although ecto-NTPDases are rare in bacteria, expression of a secreted NTPDase in Legionella pneumophila was recently described. This ecto-enzyme enhances intracellular growth of the bacterium and potentially affects virulence. This discovery represents an important advance in the understanding of the contribution of other microbial NTPDases to host-pathogen interactions. Here we review other progress made to date in the characterization of ecto-NTPDases from microbial pathogens, how they differ from mammalian enzymes, and their association with organism viability and virulence. In addition, we postulate how ecto-NTPDases may contribute to the host-pathogen interaction by reviewing the effect of selected microbial pathogens on purinergic signaling. Finally, we raise the possibility of targeting ecto-NTPDases in the development of novel anti-infective agents based on potential structural and clear enzymatic differences from the mammalian ecto-NTPDases.

scholarly journals Proteomic identification of Coxiella burnetii effector proteins targeted to the host cell mitochondria during infection

2020 ◽  
Author(s):  
Laura F. Fielden ◽  
Nichollas E. Scott ◽  
Catherine S. Palmer ◽  
Chen Ai Khoo ◽  
Hayley J Newton ◽  
...  
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Effector Protein ◽  
Subcellular Localisation ◽  
Host Pathogen Interactions ◽  
Affinity Enrichment ◽  
Host Pathogen

AbstractModulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver a cohort of bacterial proteins, termed ‘effector proteins’ into the host cell during infection by sophisticated protein translocation systems which manipulate cellular processes and functions. Despite the importance of these proteins during infection the functional contribution of individual effectors is poorly characterised, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here we present development of a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify 4 novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins in epithelial cells confirmed the mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein is imported into mitochondria and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to the study of intracellular host-pathogen interactions occurring during infection, providing a robust strategy to examine the sub-cellular localisation of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

scholarly journals Intracellular Growth of Legionella pneumophila in Dictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions

2000 ◽  
Vol 68 (5) ◽  
pp. 2939-2947 ◽  
Author(s):  
Jonathan M. Solomon ◽  
Adam Rupper ◽  
James A. Cardelli ◽  
Ralph R. Isberg
Keyword(s):  
Genetic Analysis ◽  
Dictyostelium Discoideum ◽  
Legionella Pneumophila ◽  
Bacterial Pathogen ◽  
Intracellular Growth ◽  
Host Pathogen Interactions ◽  
Growth State ◽  
Myosin I ◽  
Host Pathogen ◽  
Endocytic Compartments

ABSTRACT Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible toL. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some knownD. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and themyoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a Gβ mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.

scholarly journals Proteomic identification of Coxiella burnetii effector proteins targeted to the host cell mitochondria during infection

2020 ◽  
pp. mcp.RA120.002370
Author(s):  
Laura F Fielden ◽  
Nichollas E. Scott ◽  
Catherine S Palmer ◽  
Chen Ai Khoo ◽  
Hayley J. Newton ◽  
...  
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Effector Protein ◽  
Subcellular Localisation ◽  
Host Pathogen Interactions ◽  
Affinity Enrichment ◽  
Host Pathogen

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed ‘effector proteins’ into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterised, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify 4 novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins confirmed their mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localises to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the sub-cellular localisation of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

scholarly journals Molecular and cellular pathobiology ofEhrlichiainfection: targets for new therapeutics and immunomodulation strategies

2011 ◽  
Vol 13 ◽  
Author(s):  
Jere W. McBride ◽  
David H. Walker
Keyword(s):  
New Technologies ◽  
Vaccine Development ◽  
Severe Disease ◽  
Acute Infection ◽  
Effector Proteins ◽  
Host Pathogen Interactions ◽  
Disease States ◽  
Recent Advances ◽  
Host Pathogen ◽  
Life Threatening

Ehrlichiaare small obligately intracellular bacteria in the order Rickettsiales that are transmitted by ticks and associated with emerging life-threatening human zoonoses. Vaccines are not available for human ehrlichiosis, and therapeutic options are limited to a single antibiotic class. New technologies for exploring host–pathogen interactions have yielded recent advances in understanding the molecular interactions betweenEhrlichiaand the eukaryotic host cell and identified new targets for therapeutic and vaccine development, including those that target pathogen virulence mechanisms or disrupt the processes associated with ehrlichial effector proteins. Animal models have also provided insight into immunopathological mechanisms that contribute significantly to understanding severe disease manifestations, which should lead to the development of immunomodulatory approaches for treating patients nearing or experiencing severe disease states. In this review, we discuss the recent advances in our understanding of molecular and cellular pathobiology and the immunobiology ofEhrlichiainfection. We identify new molecular host–pathogen interactions that can be targets of new therapeutics, and discuss prospects for treating the immunological dysregulation during acute infection that leads to life-threatening complications.

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