Mycoplasma pneumoniae CARDS toxin elicits a functional IgE response in Balb/c mice

ABSTRACTThe inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1β (pro-IL-1β) into its mature form. IL-1β is a critical proinflammatory cytokine that dictates the severity of inflammation associated with a wide spectrum of inflammatory diseases. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the current study, we uncovered a yet unknown mechanism of NLRP3 inflammasome activation by a pathogen-derived factor. We show that the unique bacterial ADP-ribosylating and vacuolating toxin produced byMycoplasma pneumoniaeand designated community-acquired respiratory distress syndrome (CARDS) toxin activates the NLRP3 inflammasome by colocalizing with the NLRP3 inflammasome and catalyzing the ADP-ribosylation of NLRP3. Mutant full-length CARDS toxin lacking ADP-ribosyltransferase (ADPRT) activity and truncated CARDS toxins unable to bind to macrophages and be internalized failed to activate the NLRP3 inflammasome. These studies demonstrate that CARDS toxin-mediated ADP-ribosylation constitutes an important posttranslational modification of NLRP3, that ADPRT activity of CARDS toxin is essential for NLRP3 inflammasome activation, and that posttranslational ADPRT-mediated modification of the inflammasome is a newly discovered mechanism for inflammasome activation with subsequent release of IL-1β and associated pathologies.IMPORTANCEInflammation is a fundamental innate immune response to environmental factors, including infections. The inflammasome represents a multiprotein complex that regulates inflammation via its ability to activate specific proinflammatory cytokines, resulting in an effective host protective response. However, excessive release of proinflammatory cytokines can occur following infection that skews the host response to “hyperinflammation” with exaggerated tissue damage.Mycoplasma pneumoniae, a common bacterial airway pathogen, possesses a unique protein toxin with ADP-ribosyltransferase and vacuolating properties capable of reproducing the robust inflammation and cytopathology associated with mycoplasma infection. Here, we show that the toxin uniquely activates the NLRP3 inflammasome by colocalizing with and ADP-ribosylating NLRP3, possibly leading to “hyperinflammation” and thus uncovering a novel target for therapeutic intervention.

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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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Mycoplasma pneumoniae CARDS Toxin Is Internalized via Clathrin-Mediated Endocytosis

PLoS ONE ◽

10.1371/journal.pone.0062706 ◽

2013 ◽

Vol 8 (5) ◽

pp. e62706 ◽

Cited By ~ 21

Author(s):

Manickam Krishnan ◽

T. R. Kannan ◽

Joel B. Baseman

Keyword(s):

Mycoplasma Pneumoniae ◽

Cards Toxin

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High co-expression of TNF-α and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia

Molecular Medicine ◽

10.1186/s10020-019-0105-2 ◽

2019 ◽

Vol 25 (1) ◽

Cited By ~ 3

Author(s):

Gang Li ◽

Liping Fan ◽

Yuqing Wang ◽

Li Huang ◽

Meijuan Wang ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Good Predictor ◽

Tnf Α ◽

Mycoplasma Pneumoniae Pneumonia ◽

Cards Toxin

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Analysis of Pulmonary Inflammation and Function in the Mouse and Baboon after Exposure to Mycoplasma pneumoniae CARDS Toxin

PLoS ONE ◽

10.1371/journal.pone.0007562 ◽

2009 ◽

Vol 4 (10) ◽

pp. e7562 ◽

Cited By ~ 55

Author(s):

R. Doug Hardy ◽

Jacqueline J. Coalson ◽

Jay Peters ◽

Adriana Chaparro ◽

Chonnamet Techasaensiri ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Pulmonary Inflammation ◽

And Function ◽

Cards Toxin

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Cellular Vacuoles Induced by Mycoplasma pneumoniae CARDS Toxin Originate from Rab9-Associated Compartments

PLoS ONE ◽

10.1371/journal.pone.0022877 ◽

2011 ◽

Vol 6 (7) ◽

pp. e22877 ◽

Cited By ~ 27

Author(s):

Coreen Johnson ◽

T. R. Kannan ◽

Joel B. Baseman

Keyword(s):

Mycoplasma Pneumoniae ◽

Cards Toxin

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Mycoplasma Pneumoniae Cards Toxin Regulates NLRP3 Inflammasome Activation

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2014.12.1439 ◽

2015 ◽

Vol 135 (2) ◽

pp. AB153

Author(s):

Jesus A. Segovia ◽

Santanu Bose ◽

Sudha R. Somarajan ◽

Te-Hung Chang ◽

Thirumalai Kannan ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Nlrp3 Inflammasome ◽

Inflammasome Activation ◽

Nlrp3 Inflammasome Activation ◽

Cards Toxin

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Immunological Response to Mycoplasma pneumoniae (Mp) and CARDS Toxin Is Related to Severe Histological Inflammation and a TH2 Response in a Primate Model

CHEST Journal ◽

10.1378/chest.1386649 ◽

2012 ◽

Vol 142 (4) ◽

pp. 192A

Author(s):

Diego Maselli ◽

Jay Peters ◽

Jorge Medina ◽

Oriol Sibila ◽

Jacqueline Coalson ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Immunological Response ◽

Th2 Response ◽

Primate Model ◽

Cards Toxin

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

Scientific Reports ◽

10.1038/s41598-021-90948-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kumaraguruparan Ramasamy ◽

Sowmya Balasubramanian ◽

Alejandra Kirkpatrick ◽

Daniel Szabo ◽

Lavanya Pandranki ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mycoplasma Pneumoniae ◽

Mammalian Cells ◽

Biological Activities ◽

Electrical Potential ◽

Retrograde Transport ◽

The United States ◽

Community Acquired Pneumonia ◽

Amino Terminal ◽

Cards Toxin

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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Neutrophil-Mediated Lung Injury Both via TLR2-Dependent Production of IL-1α and IL-12 p40, and TLR2-Independent CARDS Toxin after Mycoplasma pneumoniae Infection in Mice

Microbiology Spectrum ◽

10.1128/spectrum.01588-21 ◽

2021 ◽

Vol 9 (3) ◽

Author(s):

Shigeyuki Tamiya ◽

Eisuke Yoshikawa ◽

Monami Ogura ◽

Etsushi Kuroda ◽

Koichiro Suzuki ◽

...

Keyword(s):

Lung Injury ◽

Mycoplasma Pneumoniae ◽

Therapeutic Strategies ◽

Antibiotic Resistant ◽

Refractory Life ◽

Life Threatening ◽

Mycoplasma Pneumoniae Infection ◽

Cards Toxin

Although Mp-induced pneumonia is usually a self-limiting disease, refractory life-threatening pneumonia is often induced. In addition, the development of alternative therapeutic strategies for Mp is expected because of the emergence of antibiotic-resistant Mp.

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