RESUMO
Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.
Assuntos
Proteínas de Bactérias , GTP Fosfo-Hidrolases , Legionella pneumophila , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação para Baixo , Células HEK293 , Doença dos Legionários/microbiologia , Doença dos Legionários/metabolismo , Vacúolos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.
Assuntos
Acanthamoeba castellanii , Legionella pneumophila , Doença dos Legionários , Humanos , Macrófagos , Legionella pneumophila/metabolismo , Acanthamoeba castellanii/genética , Virulência/genética , Proteínas de Bactérias/metabolismoRESUMO
Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.
RESUMO
Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.
Assuntos
Legionella , Doença dos Legionários , Humanos , Legionella/metabolismo , Vacúolos/metabolismo , Doença dos Legionários/microbiologia , Fosfolipases/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Lisofosfolipídeos/metabolismoRESUMO
Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage coculture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines. IMPORTANCE Multi-drug-resistant pathogens are an emerging threat to human health. Because conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogen's Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit toward precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.
Assuntos
Coxiella burnetii , Legionella pneumophila , Animais , Sistemas de Secreção Bacterianos/metabolismo , Legionella pneumophila/metabolismo , Camundongos , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Fatores de Virulência/genéticaRESUMO
Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.
Assuntos
Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Inativação Gênica , Legionella pneumophila/genética , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Estudo de Prova de Conceito , Células U937 , Virulência/genética , Fatores de Virulência/metabolismoRESUMO
AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/microbiologia , Legionella pneumophila/enzimologia , Doença dos Legionários/microbiologia , Monofosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/genética , Feminino , Complexo de Golgi/metabolismo , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Camundongos , Domínios ProteicosRESUMO
During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Legionella pneumophila/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Alostérica , Animais , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Doença dos Legionários/patologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Camundongos , Simulação de Dinâmica Molecular , Mimetismo Molecular , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPAssuntos
Interações Hospedeiro-Patógeno , Legionella/fisiologia , Legionella/patogenicidade , Doença dos Legionários/patologia , Doença dos Legionários/fisiopatologia , Fatores de Virulência/metabolismo , Acanthamoeba/microbiologia , Pesquisa Biomédica/tendências , Análise Mutacional de DNA , Legionella/genética , Viabilidade Microbiana , Virulência , Fatores de Virulência/genética , Microbiologia da ÁguaRESUMO
The intracellular pathogen Legionella pneumophila encodes translocated effector proteins that modify host cell processes to support bacterial survival and growth. Here, we show that the L. pneumophila effector protein LegK7 hijacks the conserved Hippo signaling pathway by molecularly mimicking host Hippo kinase (MST1 in mammals), which is the key regulator of pathway activation. LegK7, like Hippo/MST1, phosphorylates the scaffolding protein MOB1, which triggers a signaling cascade resulting in the degradation of the transcriptional regulators TAZ and YAP1. Transcriptome analysis revealed that LegK7-mediated targeting of TAZ and YAP1 alters the transcriptional profile of mammalian macrophages, a key cellular target of L. pneumophila infection. Specifically, genes targeted by the transcription factor PPARγ, which is regulated by TAZ, displayed altered expression, and continuous interference with PPARγ activity rendered macrophages less permissive to L. pneumophila intracellular growth. Thus, a conserved L. pneumophila effector kinase exploits the Hippo pathway to promote bacterial growth and infection.
Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Doença dos Legionários/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Legionella pneumophila/química , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Doença dos Legionários/genética , Doença dos Legionários/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , PPAR gama , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteólise , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. RESULTS: Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. CONCLUSIONS: Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.
Assuntos
Repetição de Anquirina/fisiologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Repetição de Anquirina/genética , Membrana Celular/metabolismo , Endocitose/fisiologia , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/patogenicidade , Lisossomos/metabolismo , Macrófagos/microbiologia , Proteínas Nucleares , Proteínas Recombinantes , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.
Assuntos
Legionella pneumophila/enzimologia , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , Células HEK293 , Humanos , Legionella pneumophila/genética , Doença dos Legionários/genética , Doença dos Legionários/microbiologia , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificação , Ubiquitinação/genéticaRESUMO
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/química , Multimerização Proteica , Fatores de Virulência/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu (J. Cell Sci.130, 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' (J. Cell Sci.130, 1981-1983).
Assuntos
Bactérias/enzimologia , Fenômenos Fisiológicos Bacterianos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Escherichia coli , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Legionella , Camundongos , Plantas/microbiologia , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Salmonella , Transdução de Sinais , Nicotiana , Ubiquitinação , Virulência , Xanthomonas campestrisRESUMO
We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc.
Assuntos
Ácidos Nucleicos/química , Análise Serial de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Vetores Genéticos/metabolismo , Células HeLa , HumanosRESUMO
AIM: According to the demographic development of our society, the numbers of octogenarians referred to cardiac surgery are continuously growing. Although the benefit of first-time cardiac procedures for these patients is well documented, the fate of octogenarians after redo-procedures, with special regard to long-term survival, functional status and quality of life, is poorly investigated. METHODS: We retrospectively identified 84 consecutive patients aged ≥80 years, who underwent a cardiac reoperation at the department for Cardiothoracic Surgery in the Heart & Vessel Center Bad Bevensen between January 2007 and 2013. Demographic profiles as well as operative data were analyzed, and the patients were prospectively followed. Patient's functional status and quality of life were assessed with the Barthel Index, New York Heart Association class and the short form-12 questionnaire. RESULTS: The mean age of the study group (61 men, 23 women) was 81.9 ± 1.9 years. Most redo-procedures were carried out after primary coronary artery bypass grafting (65%), primary aortic valve replacement (21%) and primary mitral valve replacement (6%). The most frequent actual surgical procedures were combined coronary artery bypass grafting and aortic valve replacement (26%), isolated coronary artery bypass grafting (19%), and isolated aortic valve replacement (19%). The mean length of hospital stay was 17 ± 15 days. In-hospital mortality counted for 32.1%. During follow up (29 ± 20 months) a further 19.0% of the patients died. The Barthel Index of the survivors was 89 ± 17 and their mean New York Heart Association class was 2 ± 1. A total of 93% of the patients were living at home. Summary scores of physical and mental health of the short form-12 questionnaire equalled those of an age- and sex-matched normative population. CONCLUSIONS: Despite high perioperative mortality, results document a sustainable recovery of the survivors offering the prospect of a highly independent and satisfying life. Therefore, advanced age alone should not be a contraindication for redo cardiac interventions. Geriatr Gerontol Int 2016; 16: 1138-1144.
Assuntos
Procedimentos Cirúrgicos Cardiovasculares/efeitos adversos , Qualidade de Vida , Reoperação/mortalidade , Fatores Etários , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos Cardiovasculares/métodos , Causas de Morte , Estudos de Coortes , Feminino , Idoso Fragilizado , Avaliação Geriátrica , Mortalidade Hospitalar/tendências , Humanos , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Reoperação/métodos , Estudos Retrospectivos , Medição de Risco , Fatores Sexuais , Análise de Sobrevida , Resultado do TratamentoRESUMO
The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.
Assuntos
Complexo de Golgi/metabolismo , Legionella pneumophila/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Biocatálise , Transporte ProteicoRESUMO
Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10â¯000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.