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

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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Elucidation of immunological response and its regulatory network by P-TUFT-ALT-2: a promising fusion protein vaccine for human lymphatic filariasis

Royal Society Open Science ◽

10.1098/rsos.172039 ◽

2018 ◽

Vol 5 (5) ◽

pp. 172039 ◽

Cited By ~ 3

Author(s):

Rajkumar Paul ◽

Sandeep Jaiswal ◽

Natarajan Mahalakshmi ◽

Perumal Kaliraj

Keyword(s):

Fusion Protein ◽

Lymphatic Filariasis ◽

T Cell Activation ◽

Cell Activation ◽

Vaccine Candidate ◽

Natural Infection ◽

Immunological Response ◽

Dependent Manner ◽

Th2 Response ◽

Enhanced Production

Human lymphatic filariasis, a mosquito-borne neglected tropical parasitic disease, needs an early development of prophylactic agents such as a vaccine for its successful elimination. Our earlier study suggested the enhanced immunological response by fusion protein (P-TUFT-ALT-2) of Tuftsin and ALT-2 in a mice model. We cultured human peripheral blood mononuclear cells (PBMCs) and treated cells with Escherichia coli -expressed ALT-2 (E-ALT-2) and P-TUFT-ALT-2. Real-time polymerase chain reaction was performed to identify the mRNA copy number of various cytokine and transcription factor genes. The recombinant vaccine candidate was also validated for humans by immunoreactivity with human sera samples of natural infection. In this study, P-TUFT-ALT-2 stimulated 12% higher PBMC proliferation in endemic normal (EN) individuals than E-ALT-2 alone. There was enhanced production of IFN γ , IL-2, IL-5 and IL-12, indicating a balanced Th1/Th2 response. However, higher expression of IL-5 and lower IL-4 validate the humoral response through an IL-5-dependent manner. Also, high level of IL-17 indicates a strong Th/Treg regulation over T-cell activation. The upregulated T-bet might have enhanced IFN-γ production, whereas GATA-3 was supposed to enhance IL-5 expression. The fusion protein also exhibited 15–16% higher reactivity with EN clinical sera, exposing the upregulation of IgG1 and IgM in natural infection. The higher reactivity of P-TUFT-ALT-2 with sera of natural infection (EN) was validated indirectly by B-cell activation through various cytokines and regulatory genes produced from different T cells. Thus, these findings endorse P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

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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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Characterization of lethal inhalational infection with Francisella tularensis in the common marmoset (Callithrix jacchus)

Journal of Medical Microbiology ◽

10.1099/jmm.0.020669-0 ◽

2010 ◽

Vol 59 (9) ◽

pp. 1107-1113 ◽

Cited By ~ 17

Author(s):

Michelle Nelson ◽

Mark S. Lever ◽

Rachel E. Dean ◽

Victoria L. Savage ◽

F. Javier Salguero ◽

...

Keyword(s):

Francisella Tularensis ◽

Immunological Response ◽

Common Marmoset ◽

Callithrix Jacchus ◽

Post Challenge ◽

Primate Model ◽

Immunological Parameters ◽

Suitable Animal Model ◽

The Common ◽

Schu S4

The intracellular Gram-negative pathogen Francisella tularensis is the causative agent of tularaemia and is prevalent in many countries in the northern hemisphere. To determine whether the common marmoset (Callithrix jacchus) would be a suitable non-human primate model of inhalational tularaemia, a pathophysiology study was undertaken. Ten animals were challenged with ∼102 c.f.u. F. tularensis strain SCHU S4 (F. tularensis subsp. tularensis). To look for trends in the infection, pairs of animals were sacrificed at 24 h intervals between 0 and 96 h post-challenge and blood and organs were assessed for bacteriology, pathology and haematological and immunological parameters. The first indication of infection was a raised core temperature at 3 days post-challenge. This coincided with a number of other factors: a rapid increase in the number of bacteria isolated from all organs, more pronounced gross pathology and histopathology, and an increase in the immunological response. As the disease progressed, higher bacterial and cytokine levels were detected. More extensive pathology was observed, with multifocal lesions seen in the lungs, liver and spleen. Disease progression in the common marmoset appears to be consistent with human clinical and pathological features of tularaemia, indicating that this may be a suitable animal model for the investigation of novel medical interventions such as vaccines or therapeutics.

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