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

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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Respiratory Tract Infection with Mycoplasma pneumoniae in Interleukin-12 Knockout Mice Results in Improved Bacterial Clearance and Reduced Pulmonary Inflammation

Infection and Immunity ◽

10.1128/iai.01249-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 236-242 ◽

Cited By ~ 19

Author(s):

C. M. Salvatore ◽

M. Fonseca-Aten ◽

K. Katz-Gaynor ◽

A. M. Gomez ◽

A. Mejias ◽

...

Keyword(s):

Respiratory Disease ◽

Mycoplasma Pneumoniae ◽

Pulmonary Inflammation ◽

Airway Hyperreactivity ◽

Interleukin 12 ◽

Enzyme Linked Immunosorbent Assay ◽

Wild Type ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Ifn Γ

ABSTRACT Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 107 CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-γ than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-α (P = 0.078). No differences were found for IL-1β, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.

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CCR2 Deficiency Impairs Ly6Clo and Ly6Chi Monocyte Responses in Orientia tsutsugamushi Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.670219 ◽

2021 ◽

Vol 12 ◽

Author(s):

Michael Petermann ◽

Zacharias Orfanos ◽

Julie Sellau ◽

Mohammad Gharaibeh ◽

Hannelore Lotter ◽

...

Keyword(s):

Scrub Typhus ◽

Pulmonary Inflammation ◽

Plasma Concentrations ◽

Self Healing ◽

Cc Chemokine ◽

Mrna Induction ◽

Chemokine Ligand ◽

Monocytes And Macrophages ◽

And Function ◽

Deficient Mice

Orientia (O.) tsutsugamushi, the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi, we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6Chi monocytes was observed in CCR2-/- mice. In the lung, significantly higher numbers of Ly6Chi and Ly6Clo monocytes were found in the C57BL/6 mice compared to CCR2-/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos/arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6Chi and Ly6Clo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation.

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

PLoS ONE ◽

10.1371/journal.pone.0172447 ◽

2017 ◽

Vol 12 (2) ◽

pp. e0172447 ◽

Cited By ~ 8

Author(s):

Jorge L. Medina ◽

Edward G. Brooks ◽

Adriana Chaparro ◽

Peter H. Dube

Keyword(s):

Mycoplasma Pneumoniae ◽

Ige Response ◽

Cards Toxin

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Mycoplasma pneumoniae Induces Chronic Respiratory Infection, Airway Hyperreactivity, and Pulmonary Inflammation: a Murine Model of Infection-Associated Chronic Reactive Airway Disease

Infection and Immunity ◽

10.1128/iai.70.2.649-654.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 649-654 ◽

Cited By ~ 71

Author(s):

Robert D. Hardy ◽

Hasan S. Jafri ◽

Kurt Olsen ◽

Jeanine Hatfield ◽

Janie Iglehart ◽

...

Keyword(s):

Airway Obstruction ◽

Pulmonary Disease ◽

Mycoplasma Pneumoniae ◽

Respiratory Infection ◽

Murine Model ◽

Pulmonary Inflammation ◽

Lavage Fluid ◽

Airway Hyperreactivity ◽

Whole Body ◽

Chronic Respiratory Infection

ABSTRACT Because chronic Mycoplasma pneumoniae respiratory infection is hypothesized to play a role in asthma, the potential of M. pneumoniae to establish chronic respiratory infection with associated pulmonary disease was investigated in a murine model. BALB/c mice were intranasally inoculated once with M. pneumoniae and examined at 109, 150, 245, 368, and 530 days postinoculation. M. pneumoniae was detected in bronchoalveolar lavage fluid by culture or PCR in 70 and 22% of mice at 109 and 530 days postinoculation, respectively. Lung histopathology was normal up to 368 days postinoculation. At 530 days, however, 78% of the mice inoculated with M. pneumoniae demonstrated abnormal histopathology characterized by peribronchial and perivascular mononuclear infiltrates. A mean histopathologic score (HPS) at 530 days of 5.1 was significantly greater (P < 0.01) than that for controls (HPS score of 0). Serum anti-M. pneumoniae immunoglobulin G was detectable in all of the mice inoculated with M. pneumoniae and was inversely correlated with HPS (r = −0.95, P = 0.01) at 530 days postinoculation. Unrestrained whole-body plethysmography measurement of enhanced pause revealed significantly elevated airway methacholine reactivity in M. pneumoniae-inoculated mice compared with that in controls at 245 days (P = 0.03) and increased airway obstruction at 530 days (P = 0.01). Murine M. pneumoniae respiratory infection can lead to chronic pulmonary disease characterized by airway hyperreactivity, airway obstruction, and histologic inflammation.

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