scholarly journals Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containing Legionella effectors

2019 ◽  
Author(s):  
Min Wan ◽  
Alan Sulpizio ◽  
Anil Akturk ◽  
Wendy H.J. Beck ◽  
Michael Lanz ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Posttranslational Modification ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Physiological Processes ◽  
Ubiquitination Pathway ◽  
Posttranslational Protein Modification

SummaryPosttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these two genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella containing vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.Statement of significanceUbiquitination is a vital posttranslational modification in eukaryotes. A variety of microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the causative bacterial pathogen of Legionnaires’ disease, has been show to hijack host ubiquitination pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we report two new players, DupA and DupB, involved in this unconventional pathway. We found that DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination pathways in eukaryotes.

scholarly journals Deubiquitination of phosphoribosyl-ubiquitin conjugates by phosphodiesterase-domain–containing Legionella effectors

2019 ◽  
Vol 116 (47) ◽  
pp. 23518-23526 ◽  
Author(s):  
Min Wan ◽  
Alan G. Sulpizio ◽  
Anil Akturk ◽  
Wendy H. J. Beck ◽  
Michael Lanz ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Host Cells ◽  
Serine Residue ◽  
Physiological Processes ◽  
Ubiquitination Pathway ◽  
Exogenous Expression ◽  
Golgi Fragmentation ◽  
Posttranslational Protein Modification

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.

scholarly journals Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer

2005 ◽  
Vol 187 (22) ◽  
pp. 7716-7726 ◽  
Author(s):  
Karim Suwwan de Felipe ◽  
Sergey Pampou ◽  
Oliver S. Jovanovic ◽  
Christopher D. Pericone ◽  
Senna F. Ye ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Open Reading Frames ◽  
Host Cells ◽  
Dependent Manner ◽  
Major Mechanism ◽  
Cell Functions

ABSTRACT Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the γ-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.

scholarly journals The Dot/Icm Type IV Secretion System of Legionella pneumophila Is Essential for the Induction of Apoptosis in Human Macrophages

2002 ◽  
Vol 70 (3) ◽  
pp. 1657-1663 ◽  
Author(s):  
Steven D. Zink ◽  
Lisa Pedersen ◽  
Nicholas P. Cianciotto ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Ii Secretion ◽  
Type Iv ◽  
Induction Of Apoptosis

ABSTRACT We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.

scholarly journals Legionella pneumophilaIs Directly Sensitive to 2-Deoxyglucose-Phosphate via Its UhpC Transporter but Is Indifferent to Shifts in Host Cell Glycolytic Metabolism

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Jordan V. Price ◽  
Kallie Jiang ◽  
Abigail Galantowicz ◽  
Alana Freifeld ◽  
Russell E. Vance
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Phosphate Transporter ◽  
Host Cells ◽  
Growth Defect ◽  
Toxic Metabolite ◽  
Glycolytic Metabolism ◽  
Content Type ◽  
Hexose Phosphate

ABSTRACTToll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection withLegionella pneumophila, but the role of host macrophage glycolysis in terms of intracellularL. pneumophilareplication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocksL. pneumophilareplication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG onL. pneumophilain macrophages requires theL. pneumophilahexose-phosphate transporter UhpC. Experiments with UhpC-deficientL. pneumophilarevealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG onL. pneumophilain mammalian cells requires 2DG phosphorylation. UhpC-deficientL. pneumophilareplicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellularL. pneumophilareplication. These results are consistent with the view thatL. pneumophilacan employ diverse metabolic strategies to exploit its host cells.IMPORTANCEWe explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen,Legionella pneumophila. Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication ofL. pneumophiladuring infection of macrophages, leading to speculation thatL. pneumophilamay exploit macrophage glycolysis. We isolatedL. pneumophilamutants resistant to the inhibitory effect of 2DG in macrophages, identifying aL. pneumophilahexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication ofL. pneumophila, which is consistent with a view ofL. pneumophilaas a metabolic generalist.

scholarly journals Legionella Subvert the Functions of Rab1 and Sec22b to Create a Replicative Organelle

2004 ◽  
Vol 199 (9) ◽  
pp. 1201-1211 ◽  
Author(s):  
Jonathan C. Kagan ◽  
Mary-Pat Stein ◽  
Marc Pypaert ◽  
Craig R. Roy
Keyword(s):  
Endoplasmic Reticulum ◽  
Legionella Pneumophila ◽  
Molecular Mechanisms ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Intracellular Growth ◽  
Host Proteins ◽  
Morphological Studies ◽  
Bacterial Replication ◽  
Inhibition Studies

Legionella pneumophila is a bacterial pathogen that infects eukaryotic host cells and replicates inside a specialized organelle that is morphologically similar to the endoplasmic reticulum (ER). To better understand the molecular mechanisms governing transport of the Legionella-containing vacuole (LCV), we have identified host proteins that participate in the conversion of the LCV into a replicative organelle. Our data show that Rab1 is recruited to the LCV within minutes of uptake. Rab1 recruitment to the LCV precedes remodeling of this compartment by ER-derived vesicles. Genetic inhibition studies demonstrate that Rab1 is important for the recruitment of ER-derived vesicles to the LCV and that inhibiting Rab1 function abrogates intracellular growth of Legionella. Morphological studies indicate that the Sec22b protein is located on ER-derived vesicles recruited to the LCV and that Sec22b is delivered to the LCV membrane. Sec22b function was found to be important for biogenesis of the specialized organelle that supports Legionella replication. These studies demonstrate that Legionella has the ability to subvert Rab1 and Sec22b function to facilitate the transport and fusion of ER-derived vesicles with the LCV, resulting in the formation of a specialized organelle that can support bacterial replication.

scholarly journals Identification of a Gene That Affects the Efficiency of Host Cell Infection by Legionella pneumophila in a Temperature-Dependent Fashion

2003 ◽  
Vol 71 (11) ◽  
pp. 6256-6263 ◽  
Author(s):  
Dennis A. Ridenour ◽  
Suat L. G. Cirillo ◽  
Sheng Feng ◽  
Mustapha M. Samrakandi ◽  
Jeffrey D. Cirillo
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Growth Conditions ◽  
Host Cells ◽  
Secretion Systems ◽  
Cell Infection ◽  
Type Iv ◽  
Low Copy Number ◽  
Type Gene

ABSTRACT The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans. We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions. We have developed an approach using expression of L. pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection. Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L. pneumophila infection of mammalian cells. The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems. We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L. pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene. The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37°C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30°C. These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L. pneumophila grown at lower temperatures.

scholarly journals A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

2021 ◽  
Author(s):  
Thomas E. Wood ◽  
Jessica M. Yoon ◽  
Heather D. Eshleman ◽  
Daniel J. Slade ◽  
Cammie F. Lesser ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Inositol Phosphates ◽  
Bacterial Pathogen ◽  
Cysteine Proteases ◽  
Effector Proteins ◽  
Host Cells ◽  
Expression Library ◽  
Inositol Pyrophosphates ◽  
Type 3

Shigella spp. cause diarrhea by invading human intestinal epithelial cells. Effector proteins delivered into target host cells by the Shigella type 3 secretion system modulate host signaling pathways and processes in a manner that promotes infection. The effector OspB activates mTOR, the central cellular regulator of growth and metabolism, and potentiates the inhibition of mTOR by rapamycin. The net effect of OspB on cell monolayers is cell proliferation at infectious foci. To gain insights into the mechanism by which OspB potentiates rapamycin inhibition of mTOR, we employ in silico analyses to identify putative catalytic residues of OspB and show that a conserved cysteine-histidine dyad is required for this activity of OspB. In a screen of an over-expression library in Saccharomyces cerevisiae, we identify a dependency of OspB activity on inositol pyrophosphates, a class of eukaryotic secondary messengers that are distinct from the inositol phosphates known to act as cofactors for bacterial cysteine proteases. We show that inositol pyrophosphates are required for OspB activity not only in yeast, but also in mammalian cells - the first demonstration of inositol pyrophosphates being required for virulence of a bacterial pathogen in vivo.

scholarly journals Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

2021 ◽  
Author(s):  
Rebecca R. Noll ◽  
Colleen M. Pike ◽  
Stephanie S. Lehman ◽  
Chad Williamson ◽  
Ramona Neunuebel
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Pathogen ◽  
Nutrient Release ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Wild Type ◽  
Cellular Processes ◽  
Type Iv

Autophagy is a fundamental eukaryotic process that mediates clearance of unwanted molecules and facilitates nutrient release. The bacterial pathogen Legionella pneumophila establishes an intracellular niche within phagocytes by manipulating host cellular processes, such as autophagy. Effector proteins translocated by L. pneumophila's Dot/Icm type IV secretion system have been shown to suppress autophagy. However evidence suggests that overall inhibition of autophagy may be detrimental to the bacterium. As autophagy contributes to cellular homeostasis and nutrient acquisition, L. pneumophila may translocate effectors that promote autophagy for these benefits. Here, we show that effector protein Lpg2411 binds phosphatidylinositol-3-phosphate lipids and preferentially binds autophagosomes. Translocated Lpg2411 accumulates late during infection and co-localizes with the autophagy receptor p62 and ubiquitin. Furthermore, autophagy is inhibited to a greater extent in host cells infected with a mutant strain lacking Lpg2411 compared to those infected with wild-type L. pneumophila, indicating that Lpg2411 stimulates autophagy to support the bacterium's intracellular lifestyle.

scholarly journals Posttranslational Protein Modification in Archaea

2005 ◽  
Vol 69 (3) ◽  
pp. 393-425 ◽  
Author(s):  
Jerry Eichler ◽  
Michael W. W. Adams
Keyword(s):  
Disulfide Bond ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Protein Modification ◽  
Bond Formation ◽  
The Other ◽  
Extreme Conditions ◽  
Disulfide Bond Formation ◽  
Domains Of Life ◽  
Posttranslational Protein Modification

SUMMARY One of the first hurdles to be negotiated in the postgenomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various posttranslational modifications that proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation, and proteolytic cleavage. Whereas these and other posttranslational protein modifications have been well characterized in Eucarya and Bacteria, posttranslational modification in Archaea has received far less attention. Although archaeal proteins can undergo posttranslational modifications reminiscent of what their eucaryal and bacterial counterparts experience, examination of archaeal posttranslational modification often reveals aspects not previously observed in the other two domains of life. In some cases, posttranslational modification allows a protein to survive the extreme conditions often encountered by Archaea. The various posttranslational modifications experienced by archaeal proteins, the molecular steps leading to these modifications, and the role played by posttranslational modification in Archaea form the focus of this review.

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.

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