Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem ß-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal ß-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.

Regulation of TLR3 Activation by S100A9.

Recognition of viral dsRNA by endosomal TLR3 activates innate immune response during virus infection. Trafficking of TLR3 to the endolysosomal compartment arising from fusion of late endosome (LE) with lysosome is required for recognition and detection of pathogen associated molecular patterns, which results in activation of the TLR3-dependent signaling cascade. Existing knowledge about the mechanism(s) and cellular factor(s) governing TLR3 trafficking is limited. In the current study, we identified intracellular S100A9 protein as a critical regulator of TLR3 trafficking. S100A9 was required for maturation of TLR3 containing early endosome (EE) into LE, the compartment that fuses with lysosome to form the endolysosomal compartment. A drastic reduction in cytokine production was observed in S100A9-knockout (KO) primary macrophages following RNA virus infection and treatment of cells with polyinosinic-polycytidylic acid (polyIC; a dsRNA mimetic that acts as a TLR3 agonist). Mechanistic studies revealed colocalization and interaction of S100A9 with TLR3 following polyIC treatment. S100A9-TLR3 interaction was critical for maturation of TLR3 containing EE into LE because TLR3 could not be detected in the LE of polyIC-treated S100A9-KO macrophages. Subsequently, TLR3 failed to colocalize with its agonist (i.e., biotin-labeled polyIC) in S100A9-deficient macrophages. The in vivo physiological role of S100A9 was evident from loss of cytokine production in polyIC-treated S100A9-KO mice. Thus, we identified intracellular S100A9 as a regulator of TLR3 signaling and demonstrated that S100A9 functions during pre-TLR3 activation stages by facilitating maturation of TLR3 containing EE into LE.

Evaluation of changes in tidal volume during mask ventilation following administration of neuromuscular blocking drugs.

The practice of checking the ability to mask ventilate before administering neuromuscular blocking drugs remains controversial. We prospectively evaluated the changes in the expired tidal volume during pressure-controlled ventilation (two-handed mask ventilation technique) as a surrogate marker to assess the ease of mask ventilation following administration of rocuronium. After informed consent, 125 patients were anaesthetised using a standard induction technique consisting of fentanyl, propofol and rocuronium, with anaesthesia then maintained with isoflurane in oxygen. The mean (SD) expired tidal volume before administration of rocuronium increased by 61 (13) ml at 2 min following onset of neuromuscular block (p < 0.001). This supports the concept that neuromuscular blockade induced by rocuronium facilitates mask ventilation.

Fatal outcomes in family transmission of Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and, on rare occasions, manifests as fulminant disease that leads to mortality, even in healthy individuals. METHODS: We conducted a retrospective study on members of a family who were quarantined by the Centers for Disease Control and Prevention in 2002 for respiratory failure and death of a 15-year-old brother (sibling 1) and a 13-year-old sister (sibling 2). Collected airway, cerebrospinal fluid (CSF), and serum samples from both deceased siblings and serum samples from both parents and the remaining 3 ill siblings (sibling 3-5) were tested using a range of diagnostic assays. Autopsy lung tissue samples from sibling 2 were also assessed using immunohistochemical and immunoelectron microscopic methods. RESULTS: Autopsy evaluation of sibling 1 revealed cerebral edema consistent with hypoxic ischemic encepatholopathy and pulmonary findings of bronchiolitis obliterans with organizing pneumonia (BOOP). Postmortem lung examination of sibling 2 revealed lymphoplasmacytic bronchiolitis with intraluminal purulent exudate, BOOP, and pulmonary edema. Results of diagnostic assays implicated the household transmission of M. pneumoniae among all 5 siblings and both parents. Further analysis of lung tissue from sibling 2 demonstrated the presence of M. pneumoniae organisms and community-acquired respiratory distress syndrome toxin. M. pneumoniae was cultured directly from sibling 2 autopsy lung tissue. CONCLUSION: Evidence is provided that M. pneumoniae was readily transmitted to all members of the household and that the resulting infections led to a spectrum of individual responses with variation in disease progression, including lymphoplasmacytic bronchiolitis, BOOP, and death.

Synthesis and distribution of CARDS toxin during Mycoplasma pneumoniae infection in a murine model.

Mice were infected with Mycoplasma pneumoniae and monitored for the synthesis and distribution of the unique adenosine diphosphate-ribosylating and vacuolating Community Acquired Respiratory Distress Syndrome (CARDS) toxin in bronchiolar lavage fluid (BALF) and lung. We noted direct relationships between the concentration of CARDS toxin and numbers of mycoplasma genomes in BALF and the degree of histologic pulmonary inflammation. Immunostaining of lungs revealed extensive colonization by mycoplasmas, including the detection of CARDS toxin in the corresponding inflamed airways. Lung lesion scores were higher during the early stages of infection, decreased gradually by day 14 postinfection, and reached substantially lower values at day 35. Infected mouse immunoglobulin (Ig) M and IgG titers were positive for CARDS toxin as well as for the major adhesin P1 of M. pneumoniae. These data reinforce the proposed pathogenic role of CARDS toxin in M. pneumoniae-mediated pathologies.

Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation.

Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.

Efficacy of increasing dosages of clarithromycin for treatment of experimental Mycoplasma pneumoniae pneumonia.

OBJECTIVES: Mycoplasma pneumoniae respiratory infection is a common cause of acute respiratory infection in children and adults. We evaluated the efficacy of increasing dosages of clarithromycin for the optimized therapy of M. pneumoniae respiratory infection in a mouse model. METHODS: BALB/c mice were intranasally inoculated once with M. pneumoniae or SP4 broth (control). Groups of mice were treated with increasing dosages of clarithromycin (10, 25 or 75 mg/kg/day) or placebo subcutaneously daily. Groups of mice were evaluated after 1, 2, 3, 6 and 12 days of therapy. Outcome variables included quantitative M. pneumoniae culture, histopathological score of the lungs, bronchoalveolar lavage (BAL) cytokine/chemokine/growth factor concentrations and plethysmography after aerosolized methacholine to assess airway hyperresponsiveness. RESULTS: Elevated dosages of clarithromycin resulted in greater antimicrobial efficacy with significantly reduced M. pneumoniae quantitative cultures (P < 0.05), as well as greater improvement in markers of disease severity with significantly reduced lung histopathology scores, BAL cytokine concentrations and airway hyperresponsiveness (P < 0.05). CONCLUSIONS: Escalated dosing of clarithromycin resulted in significantly greater therapeutic efficacy in the treatment of experimental M. pneumoniae respiratory infection.

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

Mycoplasma 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.

Molecular cloning, expression, and characterization of a Ca2+-dependent, membrane-associated nuclease of Mycoplasma genitalium.

In this study, we identified and characterized the enzymatic properties of MG_186, a calcium-dependent Mycoplasma genitalium nuclease. MG_186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG_186 (rMG_186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca2+ alone enhances its activity, which was inhibited by divalent cations, such as Zn2+ and Mn2+. Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG_186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG_186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG_186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG_186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG_186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.

Crystallization of community-acquired respiratory distress syndrome toxin from Mycoplasma pneumoniae.

Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 A, beta = 90.6 degrees. A complete 2.2 A data set was obtained from a single CARDS TX crystal.

Characterization of a unique ADP-ribosyltransferase of Mycoplasma penetrans.

Mycoplasma penetrans is a urogenital tract pathogen implicated in the deterioration of the immune system in human immunodeficiency virus-infected AIDS patients. Here, we describe a 78-kDa protein from M. penetrans, designated MYPE9110, that exhibits sequence similarity to known ADP-ribosyltransferases (ADPRTs) such as Bordetella pertussis pertussis toxin and Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin. MYPE9110 possesses key amino acid residues found in all ADPRTs that are essential for ADPRT activity. Several mammalian cell proteins are ADP-ribosylated by MYPE9110, and the full-length recombinant protein exhibits a strong auto-ADP-ribosylating activity. In the absence of target proteins, MYPE9110 demonstrates a NAD-glycohydrolase activity by hydrolyzing NAD. Furthermore, this toxin elicits cytopathology in HeLa cells by inducing cytoplasmic vacuolization in the presence of ammonium chloride. The deletion of the C-terminal region of MYPE9110 significantly diminishes its binding to host cells while still exhibiting an ADPRT activity, suggesting that MYPE9110 is a member of the family of A-B ADPRT toxins.

Amino acid changes in elongation factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium influence fibronectin binding.

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu(Mp)) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu(Mg)), in spite of sharing 96% identity with EF-Tu(Mp), does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu(Mp) that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu(Mg). Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu(Mp). Synthetic peptides corresponding to this region of EF-Tu(Mp) (EF-Tu(Mp) 340-358) blocked both recombinant EF-Tu(Mp) and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu(Mg) 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.

The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin.

Mycoplasma pneumoniae is a bacterial pathogen of the human respiratory tract that causes a wide range of airway diseases as well as extrapulmonary symptoms. It possesses a distinct, differentiated terminal structure, termed the attachment organelle, that mediates adherence to the host respiratory epithelium. Previously, we reported that surface-associated M. pneumoniae elongation factor Tu (EF-Tu, also called MPN665) serves as a fibronectin (Fn)-binding protein, facilitating interactions between mycoplasmas and extracellular matrix. In the present study, we determined that binding of M. pneumoniae EF-Tu to Fn is primarily mediated by the EF-Tu carboxyl region. A 179-amino-acid region spanning the carboxyl terminus (designated EC; amino acids 192 to 394) binds Fn in a dose-dependent manner. Further analysis of carboxyl constructs (ED3 and ED4) and their deletion truncations (ED3.1, ED3.2, and ED4.1) revealed that the carboxyl region possessed two distinct sites with different Fn-binding efficiencies. Immunogold electron microscopy using antibodies raised against recombinant ED3 and ED4 demonstrated the surface accessibility of the EF-Tu carboxyl region. Competitive binding assays using intact radiolabeled mycoplasmas and purified recombinant ED3 and ED4 proteins, along with antibody blocking assays, reinforced the role of the surface-exposed EF-Tu carboxyl region in Fn binding. Alkali and high-salt treatment of mycoplasma membranes and Triton X-114-partitioned mycoplasma fractions confirmed the stable association of EF-Tu within the mycoplasma membrane. These observations highlight the unique, multifaceted, and unpredictable role of the classically defined cytoplasmic protein EF-Tu relative to cellular function, compartmentalization, and topography.

Genomic analysis reveals Mycoplasma pneumoniae repetitive element 1-mediated recombination in a clinical isolate.

Mycoplasmas are cell wall-less bacteria that evolved by drastic reduction of the genome size. Complete genome analysis of Mycoplasma pneumoniae revealed the presence of numerous copies of four distinct large M. pneumoniae repetitive elements (RepMPs). One copy each of RepMP2/3, RepMP4, and RepMP5 are localized within the P1 operon (MPN140 to MPN142 loci), and their involvement in sequence variation in adhesin P1 and adherence-related protein B/C has been documented. Here we analyzed a clinical strain of M. pneumoniae designated S1 isolated from a 1993 outbreak of respiratory infections in San Antonio, TX. Based on the type of RepMPs within the P1 operon, we classified clinical isolate S1 as type 2 with unique minor sequence variations. Hybridization with oligonucleotide arrays revealed sequence divergence in two previously unsuspected hypothetical genes (MPN137 and MPN138 loci). Closer inspection of this region revealed that the MPN137 and MPN138 loci harbored previously unrecognized unique RepMP1 sequences found only in M. pneumoniae. PCR and sequence analyses revealed a recombination event involving three RepMP1-containing genes that resulted in fusion of MPN137 and MPN138 reading frames and loss of all but a short fragment of another RepMP1-containing locus, MPN130. The multiple copies of unique RepMP1 elements spread throughout the chromosome could allow vast numbers of sequence variations in clinical strains. Comparisons of amino acid sequences showed the presence of leucine zipper motifs in MPN130 and MPN138 proteins in reference strain M129 and the absence of these motifs in the fused protein of S1. The presence of tandem leucine and other repeats points to possible regulatory functions of proteins encoded by RepMP1-containing genes.

Characterization of a unique ClpB protein of Mycoplasma pneumoniae and its impact on growth.

Mycoplasma pneumoniae accounts for 20 to 30% of all community-acquired pneumonia and has been associated with other airway pathologies, including asthma, and a range of extrapulmonary manifestations. Although the entire genomic sequence of M. pneumoniae has been completed, the functions of many of these genes in mycoplasma physiology are unknown. In this study, we focused on clpB, a well-known heat shock gene in other bacteria, to examine its role in mycoplasma growth. Transcriptional and translational analyses of heat shock in M. pneumoniae indicated that clpB is significantly upregulated, reinforcing its status as a critical responder to heat stress. Interestingly, M. pneumoniae ClpB does not use dual translational start points for ClpB synthesis, like other ClpB-characterized bacteria. Biochemical characterization of purified M. pneumoniae recombinant ClpB revealed casein- and lysine-independent ATPase activity and DnaK-DnaJ-GrpE-dependent chaperone activity. An M. pneumoniae mini-Tn4001-integrated, clpB-null mutant was impaired in its ability to replicate under permissive growth conditions, demonstrating the growth-promoting status of ClpB.

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

Mycoplasma pneumoniae is 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. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated community-acquired respiratory distress syndrome (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, designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection 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 eliminate M. pneumoniae-associated airway and extrapulmonary pathologies.

Intravesical lidocaine for formalin-induced suprapubic pain: case report.

This case report describes the management of intravesical formalin induced suprapubic pain using intravesical lidocaine.

Propofol for pediatric radiotherapy.

OBJECTIVE: Pediatric radiotherapy is a day care procedure. In children, anaesthesia is necessary to prevent movement during the therapy. Traditionally intramuscular ketamine is used for these procedure because of its inherent safety in a child who used to be left alone in the cobalt room. METHODS: This study was designed to explore the efficacy of propofol and ketamine in pediatric radiotherapy in nineteen children. The inclusion criteria was a child fasting for six hours with no fever or URTI in the past week. A child coming to the radiotherapy (RT) unit without an intravenous cannula was given intramuscular ketamine 10 mg/kg and taken for the procedure. Before the child recovered from anaesthesia an intravenous cannula, 20-22G, Vasofix was inserted for subsequent sittings of RT. The child coming with an intravenous cannula was given propofol 2.5 mg/kg with xylocaine (0.1 mg/kg) without adrenaline. The parameters recorded were pulse rate, oxygen saturation and respiratory rate-baseline to every 30 seconds till five minutes. Onset time, recovery time, oral feeding time and any untoward effects like nausea, vomiting, nystagmus were also noted. RESULT: The drug was graded on a scale of 0-10 according to parental acceptability where 0 is the worst and 10 is the best acceptability. The mean (+/-SD) of all the measured parameters were calculated and compared between the two groups. CONCLUSION: Propofol was associated with faster onset, better recovery, early oral feeding time, no nausea and vomiting and better parental acceptability. There was no hypotension, bradycardia and oxygen saturation at 60 seconds, which was between 94-95%, was easily treatable with supplementation of oxygen by face mask.

Annexin A2 mediates Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin binding to eukaryotic cells.

UNLABELLED: Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells. IMPORTANCE: Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells.

ADP-ribosylation of NLRP3 by Mycoplasma pneumoniae CARDS toxin regulates inflammasome activity.

UNLABELLED: The inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1ß (pro-IL-1ß) into its mature form. IL-1ß 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 by Mycoplasma pneumoniae and 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-1ß and associated pathologies. IMPORTANCE: Inflammation 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.