Mycoplasma pneumoniae CARDS toxin exploits host cell endosomal acidic pH and vacuolar ATPase proton pump to execute its biological activities

AbstractMycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalized children in the United States. It is also responsible for a spectrum of other respiratory tract disorders and extrapulmonary manifestations in children and adults. The main virulence factor of M. pneumoniae is a 591 amino acid multifunctional protein called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. The amino terminal region of CARDS toxin (N-CARDS) retains ADP-ribosylating activity and the carboxy region (C-CARDS) contains the receptor binding and vacuolating activities. After internalization, CARDS toxin is transported in a retrograde manner from endosome through the Golgi complex into the endoplasmic reticulum. However, the mechanisms and criteria by which internalized CARDS toxin is transported and activated to execute its cytotoxic effects remain unknown. In this study, we used full-length CARDS toxin and its mutant and truncated derivatives to analyze how pharmacological drugs that alter pH of intracellular vesicles and electrical potential across vesicular membranes affect translocation of CARDS toxin in mammalian cells. Our results indicate that an acidic environment is essential for CARDS toxin retrograde transport to endoplasmic reticulum. Moreover, retrograde transport facilitates toxin clipping and is required to induce vacuole formation. Additionally, toxin-mediated cell vacuolation is strictly dependent on the function of vacuolar type-ATPase.

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Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity

mBio ◽

10.1128/mbio.01663-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Kumaraguruparan Ramasamy ◽

Sowmya Balasubramanian ◽

Krishnan Manickam ◽

Lavanya Pandranki ◽

Alexander B. Taylor ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Amino Acid ◽

Mycoplasma Pneumoniae ◽

Retrograde Transport ◽

Airway Disease ◽

The United States ◽

Community Acquired Pneumonia ◽

Trauma Patients ◽

Airway Injury ◽

Cards Toxin

ABSTRACTMycoplasma pneumoniaeis an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization,M. pneumoniaeexpresses a unique ADP-ribosylating and vacuolating cytotoxin designatedcommunity-acquiredrespiratorydistresssyndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.IMPORTANCEMycoplasma pneumoniae, a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designatedcommunity-acquiredrespiratorydistresssyndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated withM. pneumoniaeinfection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminateM. pneumoniae-associated airway and extrapulmonary pathologies.

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Retrograde transport of toxins across the endoplasmic reticulum membrane

Biochemical Society Transactions ◽

10.1042/bst0311260 ◽

2003 ◽

Vol 31 (6) ◽

pp. 1260-1262 ◽

Cited By ~ 42

Author(s):

J.M. Lord ◽

E. Deeks ◽

C.J. Marsden ◽

K. Moore ◽

C. Pateman ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Retrograde Transport ◽

Proteasomal Degradation ◽

Degradation Pathway ◽

Endoplasmic Reticulum Membrane ◽

Cellular Processes ◽

A Chain ◽

Toxin Degradation ◽

Cellular Components

Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

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Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

Traffic ◽

10.1034/j.1600-0854.2001.21006.x ◽

2001 ◽

Vol 2 (10) ◽

pp. 717-726 ◽

Cited By ~ 77

Author(s):

Ferran Valderrama ◽

Juan M. Durán ◽

Teresa Babià ◽

Holger Barth ◽

Jaime Renau-Piqueras ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Mammalian Cells ◽

Retrograde Transport ◽

Actin Microfilaments

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Endoplasmic reticulum contains a common, abundant calcium-binding glycoprotein, endoplasmin

Journal of Cell Science ◽

10.1242/jcs.86.1.217 ◽

1986 ◽

Vol 86 (1) ◽

pp. 217-232 ◽

Cited By ~ 1

Author(s):

G. Koch ◽

M. Smith ◽

D. Macer ◽

P. Webster ◽

R. Mortara

Keyword(s):

Endoplasmic Reticulum ◽

Calcium Binding ◽

Mammalian Cells ◽

Terminal Sequence ◽

Binding Studies ◽

Amino Terminal ◽

Calcium Storage ◽

Amino Terminal Sequence ◽

Binding Glycoprotein ◽

In Cells

The most abundant protein in microsomal membrane preparations from mammalian cells has been identified as a 100 X 10(3) Mr concanavalin A-binding glycoprotein. The glycosyl moiety of the glycoprotein is completely sensitive to endoglycosidase H, suggesting a predominantly endoplasmic reticulum localization in the cell. Using a monospecific antibody it was shown by binding and immunofluorescence studies that the glycoprotein is intracellular. Immunoelectron microscopy showed that the glycoprotein was at least 100 times more concentrated in the endoplasmic reticulum than in any other cellular organelle. It was found to be substantially overexpressed in cells and tissues rich in endoplasmic reticulum. Since it is the major common protein component associated with the endoplasmic reticulum we refer to it as endoplasmin. Calcium-binding studies show that endoplasmin is a major calcium-binding protein in cells, suggesting that at least one of its roles might be in the calcium-storage function of the endoplasmic reticulum. The amino-terminal sequence of endoplasmin is identical to that of a 100 X 10(3) Mr stress-related protein.

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OSBP-Related Protein Family in Lipid Transport over Membrane Contact Sites

Lipid Insights ◽

10.4137/lpi.s31726 ◽

2015 ◽

Vol 8s1 ◽

pp. LPI.S31726 ◽

Cited By ~ 15

Author(s):

Vesa M. Olkkonen

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Binding Protein ◽

High Capacity ◽

Retrograde Transport ◽

Protein Family ◽

Oxysterol Binding Protein ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.

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Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity

Microbiology ◽

10.1099/mic.0.000928 ◽

2020 ◽

Vol 166 (7) ◽

pp. 629-640 ◽

Cited By ~ 3

Author(s):

Monica Feng ◽

Andrew C. Schaff ◽

Mitchell F. Balish

Keyword(s):

Mycoplasma Pneumoniae ◽

Type Species ◽

Cell Envelope ◽

Bacterial Pathogen ◽

Community Acquired Pneumonia ◽

Atypical Cell ◽

Content Type ◽

Link Type ◽

Cards Toxin

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H2S and H2O2, but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H2S and H2O2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

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Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase.

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2923 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2923-2931 ◽

Cited By ~ 321

Author(s):

S G Gould ◽

G A Keller ◽

S Subramani

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Firefly Luciferase ◽

Carboxy Terminus ◽

Peroxisomal Membrane ◽

Amino Terminal ◽

Peroxisomal Targeting ◽

Firefly Luciferase Gene ◽

Targeting Signal ◽

Peroxisomal Targeting Signal

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.

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Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.137.2.319 ◽

1997 ◽

Vol 137 (2) ◽

pp. 319-333 ◽

Cited By ~ 60

Author(s):

Jochen Scheel ◽

Rainer Pepperkok ◽

Martin Lowe ◽

Gareth Griffiths ◽

Thomas E. Kreis

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Transport ◽

Golgi Complex ◽

Mammalian Cells ◽

Brefeldin A ◽

Retrograde Transport ◽

Marker Proteins ◽

Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

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Molecular Detection and Characterization of Mycoplasma pneumoniae Among Patients Hospitalized With Community-Acquired Pneumonia in the United States

Open Forum Infectious Diseases ◽

10.1093/ofid/ofv106 ◽

2015 ◽

Vol 2 (3) ◽

Cited By ~ 28

Author(s):

Maureen H. Diaz ◽

Alvaro J. Benitez ◽

Kristen E. Cross ◽

Lauri A. Hicks ◽

Preeta Kutty ◽

...

Keyword(s):

United States ◽

Mycoplasma Pneumoniae ◽

Variable Number Tandem Repeat ◽

The United States ◽

Variable Number ◽

Community Acquired Pneumonia ◽

Molecular Characteristics

Abstract Background. Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP). The molecular characteristics of M pneumoniae detected in patients hospitalized with CAP in the United States are poorly described. Methods. We performed molecular characterization of M pneumoniae in nasopharyngeal/oropharyngeal swabs from children and adults hospitalized with CAP in the Centers for Disease Control and Prevention Etiology of Pneumonia in the Community (EPIC) study, including P1 typing, multilocus variable-number tandem-repeat analysis (MLVA), and macrolide susceptibility genotyping. Results. Of 216 M pneumoniae polymerase chain reaction-positive specimens, 40 (18.5%) were obtained from adults and 176 (81.5%) from children. P1 type distribution differed between adults (64% type 1 and 36% type 2) and children (84% type 1, 13% type 2, and 3% variant) (P < .05) and among sites (P < .01). Significant differences in the proportions of MLVA types 4/5/7/2 and 3/5/6/2 were also observed by age group (P < .01) and site (P < .01). A macrolide-resistant genotype was ide.jpegied in 7 (3.5%) specimens, 5 of which were from patients who had recently received macrolide therapy. No significant differences in clinical characteristics were ide.jpegied among patients with various strain types or between macrolide-resistant and -sensitive M pneumoniae infections. Conclusions. The P1 type 1 genotype and MLVA type 4/5/7/2 predominated, but there were differences between children and adults and among sites. Macrolide resistance was rare. Differences in strain types did not appear to be associated with differences in clinical outcomes. Whole genome sequencing of M pneumoniae may help ide.jpegy better ways to characterize strains.

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The Association of Shiga-like Toxin with Detergent-resistant Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the Endoplasmic Reticulum for a Cytotoxic Effect

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1035 ◽

2006 ◽

Vol 17 (3) ◽

pp. 1375-1387 ◽

Cited By ~ 69

Author(s):

Daniel C. Smith ◽

Daniel J. Sillence ◽

Thomas Falguières ◽

Rosemary M. Jarvis ◽

Ludger Johannes ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Cytotoxic Effect ◽

Retrograde Transport ◽

General Mechanism ◽

Complete Protection ◽

Essential Requirement ◽

Detergent Resistant Membranes ◽

Sequential Processes ◽

Toxin Sequestration

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb3) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.

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