Legionella effector LegA15/AnkH contains an unrecognized cysteine protease-like domain and displays structural similarity to LegA3/AnkD, but differs in host cell localization

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Ivy Yeuk Wah Chung ◽  
Lei Li ◽  
Miroslaw Cygler
Keyword(s):  
Cysteine Protease ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Structural Similarity ◽  
Catalytic Triad ◽  
Terminal Segment ◽  
Severe Form ◽  
Aquatic Environments ◽  
Cellular Functions ◽  
Lung Macrophage

Legionella pneumophila is a human pathogen that causes Legionnaires' disease, a severe form of pneumonia. It can be found in various aquatic environments ranging from cooling towers to ponds. In addition to causing disease in humans, it can also infect free-living amoebae commonly found in various aquatic environments. Once inside a human lung macrophage, it creates a niche called the Legionella-containing vacuole where it can evade phagolysosomal degradation and replicate. During infection, normal cellular functions are hijacked by proteins that are secreted by the pathogen, called bacterial effectors. Here, the structural characterization of the effector LegA15/AnkD is reported. The protein contains an ankyrin-repeat domain followed by a cysteine protease-like (CPL) domain with a putative catalytic triad consisting of His268–Asn290–Cys361. The CPL domain shows similarity to the CE clan in the MEROPS database, which contains ubiquitin-like hydrolases. The C-terminal segment of LegA15, including the CPL domain, shows structural similarity to another effector, LegA3/AnkH, while they share only 12% sequence identity. When expressed in mammalian cells, LegA15 is localized within the cytoplasm, in contrast to LegA3, which localizes to the nucleus.

scholarly journals Mitigation of Expression of Virulence Genes in Legionella pneumophila Internalized in the Free-Living Amoeba Willaertia magna C2c Maky

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 447 ◽  
Author(s):  
Rayane Mouh Mameri ◽  
Jacques Bodennec ◽  
Laurent Bezin ◽  
Sandrine Demanèche
Keyword(s):  
Gene Expression ◽  
Legionella Pneumophila ◽  
Virulence Genes ◽  
Natural Habitat ◽  
Acanthamoeba Castellanii ◽  
Severe Form ◽  
Aquatic Environments ◽  
Free Living ◽  
Intracellular Parasites ◽  
Free Living Amoeba

Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.

scholarly journals Evolution and Adaptation of Legionella pneumophila to Manipulate the Ubiquitination Machinery of Its Amoebae and Mammalian Hosts

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 112
Author(s):  
Christopher T.D. Price ◽  
Yousef Abu Kwaik
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Structural Similarity ◽  
Ubiquitin Pathway ◽  
Diverse Range ◽  
E3 Ligases ◽  
Environmental Bacterium ◽  
Natural Hosts

The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.

scholarly journals Model-guided design of mammalian genetic programs

2021 ◽  
Vol 7 (8) ◽  
pp. eabe9375
Author(s):  
J. J. Muldoon ◽  
V. Kandula ◽  
M. Hong ◽  
P. S. Donahue ◽  
J. D. Boucher ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Computational Models ◽  
Biological Complexity ◽  
Genetic Circuits ◽  
Cellular Functions ◽  
High Performing ◽  
Design Objective ◽  
Complex Functions ◽  
Mammalian Genetic ◽  
Analog Processing

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 707-720
Author(s):  
Jose Oca-Cossio ◽  
Lesley Kenyon ◽  
Huiling Hao ◽  
Carlos T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Mitochondrial Genes ◽  
Terminal Segment ◽  
Universal Genetic Code ◽  
Apocytochrome B ◽  
Dna Mutations ◽  
C Terminus ◽  
Cytoplasmic Compartment ◽  
Allotopic Expression

Abstract The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

scholarly journals Indispensable Role for the Eukaryotic-Like Ankyrin Domains of the Ankyrin B Effector of Legionella pneumophila within Macrophages and Amoebae

2010 ◽  
Vol 78 (5) ◽  
pp. 2079-2088 ◽  
Author(s):  
Christopher T. D. Price ◽  
Souhaila Al-Khodor ◽  
Tasneem Al-Quadan ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Molecular Mimicry ◽  
Ectopic Expression ◽  
Full Length ◽  
Null Mutant ◽  
Cell Plasma ◽  
Truncated Variants ◽  
Intracellular Proliferation ◽  
Ankyrin B

ABSTRACT The Dot/Icm-translocated ankyrin B (AnkB) effector of Legionella pneumophila exhibits molecular mimicry of eukaryotic F-box proteins and is essential for intracellular replication in macrophages and protozoa. In addition to two eukaryotic-like ankyrin (ANK) domains, AnkB harbors a conserved eukaryotic F-box domain, which is involved in polyubiquitination of proteins throughout the eukaryotic kingdom. We have recently shown that the F-box domain of the AnkB effector is essential for decoration of the Legionella-containing vacuole (LCV) with polyubiquitinated proteins within macrophages and protozoan hosts. To decipher the role of the two ANK domains in the function of AnkB, we have constructed in-frame deletion of either or both of the ANK domain-encoding regions (ankBΔA1, ankBΔA2, and ankBΔA1A2) to trans-complement the ankB null mutant. Deletion of the ANK domains results in defects in intracellular proliferation and decoration of the LCV with polyubiquitinated proteins. Export of the truncated variants of AnkB was reduced, and this may account for the observed defects. However, while full-length AnkB ectopically expressed in mammalian cells trans-rescues the ankB null mutant for intracellular proliferation, ectopic expression of AnkBΔA1, AnkBΔA2, and AnkBΔA1A2 fails to trans-rescue the ankB null mutant. Importantly, ectopically expressed full-length AnkB is targeted to the host cell plasma membrane, where it recruits polyubiquitinated proteins. In contrast, AnkBΔA1, AnkBΔA2, and AnkBΔA1A2 are diffusely distributed throughout the cytosol and fail to recruit polyubiquitinated proteins. We conclude that the two eukaryotic-like ANK domains of AnkB are essential for intracellular proliferation, for targeting AnkB to the host membranes, and for decoration of the LCV with polyubiquitinated proteins.

scholarly journals Roles of Emerging RNA-Binding Activity of cGAS in Innate Antiviral Response

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuying Ma ◽  
Xiaohui Wang ◽  
Weisheng Luo ◽  
Ji Xiao ◽  
Xiaowei Song ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Structural Similarity ◽  
Antiviral Response ◽  
Binding Activity ◽  
Type I Interferons ◽  
Type I ◽  
Innate Antiviral Response

cGAS, a DNA sensor in mammalian cells, catalyzes the generation of 2’-3’-cyclic AMP-GMP (cGAMP) once activated by the binding of free DNA. cGAMP can bind to STING, activating downstream TBK1-IRF-3 signaling to initiate the expression of type I interferons. Although cGAS has been considered a traditional DNA-binding protein, several lines of evidence suggest that cGAS is a potential RNA-binding protein (RBP), which is mainly supported by its interactions with RNAs, RBP partners, RNA/cGAS-phase-separations as well as its structural similarity with the dsRNA recognition receptor 2’-5’ oligoadenylate synthase. Moreover, two influential studies reported that the cGAS-like receptors (cGLRs) of fly Drosophila melanogaster sense RNA and control 3′-2′-cGAMP signaling. In this review, we summarize and discuss in depth recent studies that identified or implied cGAS as an RBP. We also comprehensively summarized current experimental methods and computational tools that can identify or predict RNAs that bind to cGAS. Based on these discussions, we appeal that the RNA-binding activity of cGAS cannot be ignored in the cGAS-mediated innate antiviral response. It will be important to identify RNAs that can bind and regulate the activity of cGAS in cells with or without virus infection. Our review provides novel insight into the regulation of cGAS by its RNA-binding activity and extends beyond its DNA-binding activity. Our review would be significant for understanding the precise modulation of cGAS activity, providing the foundation for the future development of drugs against cGAS-triggering autoimmune diseases such as Aicardi-Gourtières syndrome.

scholarly journals Oxygen radicals, nitric oxide, and peroxynitrite: Redox pathways in molecular medicine

2018 ◽  
Vol 115 (23) ◽  
pp. 5839-5848 ◽  
Author(s):  
Rafael Radi
Keyword(s):  
Nitric Oxide ◽  
Free Radical ◽  
Mammalian Cells ◽  
Oxygen Radicals ◽  
Biochemical Characterization ◽  
Cell Function ◽  
Molecular Medicine ◽  
Antioxidant Systems ◽  
Cellular Functions ◽  
Cell Degeneration

Oxygen-derived free radicals and related oxidants are ubiquitous and short-lived intermediates formed in aerobic organisms throughout life. These reactive species participate in redox reactions leading to oxidative modifications in biomolecules, among which proteins and lipids are preferential targets. Despite a broad array of enzymatic and nonenzymatic antioxidant systems in mammalian cells and microbes, excess oxidant formation causes accumulation of new products that may compromise cell function and structure leading to cell degeneration and death. Oxidative events are associated with pathological conditions and the process of normal aging. Notably, physiological levels of oxidants also modulate cellular functions via homeostatic redox-sensitive cell signaling cascades. On the other hand, nitric oxide (•NO), a free radical and weak oxidant, represents a master physiological regulator via reversible interactions with heme proteins. The bioavailability and actions of •NO are modulated by its fast reaction with superoxide radical (O2•−), which yields an unusual and reactive peroxide, peroxynitrite, representing the merging of the oxygen radicals and •NO pathways. In this Inaugural Article, I summarize early and remarkable developments in free radical biochemistry and the later evolution of the field toward molecular medicine; this transition includes our contributions disclosing the relationship of •NO with redox intermediates and metabolism. The biochemical characterization, identification, and quantitation of peroxynitrite and its role in disease processes have concentrated much of our attention. Being a mediator of protein oxidation and nitration, lipid peroxidation, mitochondrial dysfunction, and cell death, peroxynitrite represents both a pathophysiologically relevant endogenous cytotoxin and a cytotoxic effector against invading pathogens.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

2001 ◽  
Vol 114 (21) ◽  
pp. 3933-3942 ◽  
Author(s):  
Ana C. S. Monteiro ◽  
Magnus Abrahamson ◽  
Ana P. C. A. Lima ◽  
Marcos A. Vannier-Santos ◽  
Julio Scharfstein
Keyword(s):  
Cell Surface ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Tight Binding ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Reversible Inhibitor ◽  
Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

scholarly journals Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1403
Author(s):  
Maryia Karpiyevich ◽  
Katerina Artavanis-Tsakonas
Keyword(s):  
Mammalian Cells ◽  
Dynamic Balance ◽  
Fine Tuning ◽  
Protozoan Parasites ◽  
Cellular Functions ◽  
Parasitic Protozoa ◽  
Distinctive Features ◽  
Cellular Processes ◽  
Wide Range ◽  
Enzymatic Machinery

Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.

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