ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens

Abstract

Unlike many bacterial pathogens, Mycoplasma pneumoniae is not known to produce classical toxins, and precisely how M. pneumoniae injures the respiratory epithelium has remained a mystery for >50 years. Here, we report the identification of a virulence factor (MPN372) possibly responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in humans. We show that M. pneumoniae MPN372 encodes a 68-kDa protein that possesses ADP-ribosyltransferase (ART) activity. Within its N terminus, MPN372 contains key amino acids associated with NAD binding and ADP-ribosylating activity, similar to pertussis toxin (PTX) S1 subunit (PTX-S1). Interestingly, MPN372 ADP ribosylates both identical and distinct mammalian proteins when compared with PTX-S1. Remarkably, MPN372 elicits extensive vacuolization and ultimate cell death of mammalian cells, including distinct and progressive patterns of cytopathology in tracheal rings in organ culture that had been previously ascribed to infection with WT virulent M. pneumoniae. We observed dramatic seroconversion to MPN372 in patients diagnosed with M. pneumoniae-associated pneumonia, indicating that this toxin is synthesized in vivo and possesses highly immunogenic epitopes.ADP ribosylation ͉ community-acquired respiratory distress syndrome toxin ͉ vacuolization T he earliest reports of mycoplasmas as infectious agents in humans appeared in the 1940s (1). Definitive studies in the early 1960s established Mycoplasma pneumoniae as the singular cause of cold agglutinin-associated primary atypical pneumonia (2, 3). Today, M. pneumoniae is the best known of the human mycoplasmas (4). These bacteria are most unusual, lacking typical cell walls possessed by other prokaryotes, using UGA to encode tryptophan, and requiring cholesterol for growth and maintenance of membrane function and integrity. Much has been learned about the role of M. pneumoniae as a respiratory tract pathogen (5). M. pneumoniae infections constitute 20-40% of all community-acquired pneumonia and are frequently associated with other airway disorders, such as tracheobronchitis and pharyngitis. Extrapulmonary manifestations, such as hematopoietic, dermatologic, joint, central nervous system, liver, pancreas, kidney, and cardiovascular syndromes are considered sequelae of primary M. pneumoniae infections. Also, M. pneumoniae has been linked to fulminant disease, with multiorgan involvement (6). Therefore, M. pneumoniae causes a wide spectrum of pathologies, with more extensive complications than previously recognized (6), yet no single virulence determinant has been associated with these clinical signs and symptoms. In addition, definitive diagnosis and therapeutic decisions relative to M. pneumoniae infections are often delayed or lacking because of the long incubation period (average 1-2 weeks) before clinical symptoms can be observed. Further, direct isolation of M. pneumoniae from patients frequently fails, and, when successful, broth or colony growth requires 10-21 days.The early stages of t...

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

“…The formation of a unique tip-like structure comprising a complex of adhesins and adhesion-related proteins was shown to be the basis for effective adherence of bacteria to the respiratory mucosa (Krause & Balish, 2004). The further colonization process is accompanied by the release of hydrogen peroxide (Hames et al, 2009;Schmidl et al, 2011) and of the pertussis toxin-like CARDS toxin (Kannan & Baseman, 2006), resulting in vacuolization and destruction of epithelial cells.…”

Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mediating interactions with the human extracellular matrix

“…The unique tip structure (attachment organelle) of these mycoplasmas comprises a complex network of adhesins and adherence-associated proteins that mediate the directed adhesion of the bacteria to the epithelial cells (Krause & Balish, 2004). The formation of superoxide radicals and the expression of the pertussis-like CARDS cytotoxin (Kannan & Baseman, 2006) were shown to disrupt the integrity of the respiratory epithelium.…”

“…The unique tip structure (attachment organelle) of these mycoplasmas comprises a complex network of adhesins and adherence-associated proteins that mediate the directed adhesion of the bacteria to the epithelial cells (Krause & Balish, 2004). The formation of superoxide radicals and the expression of the pertussis-like CARDS cytotoxin (Kannan & Baseman, 2006) were shown to disrupt the integrity of the respiratory epithelium.In recent years many reports have been published confirming the role of different glycolytic enzymes of bacteria, fungi and parasites as adhesion/virulence factors in a broader sense (Pancholi & Chhatwal, 2003). Besides their glycolytic activity in the cytosol, these enzymes have been found at the surface of micro-organisms, where they act as binding partners of proteins of the human Abbreviations: ECM, extracellular matrix; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMR, multiple mutation reaction.…”

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

“…Baboon tracheal organ cultures exposed to purified toxin demonstrate dose-dependent slowing and eventual cessation of cilia movement and disruption of respiratory cell integrity (12). Naive mice administered one dose respond with 30-to 80-fold increases in the expression of Th-2 cytokines and chemokines, a robust eosinophilia, accumulation of T and B cells, mucus metaplasia, and airway hyperreactivity, suggesting that an analogous allergic-type response in humans may play a causal role in Mp-associated asthma (16,17).…”

“…Their streamlined genome size imposes nutritional requirements that dictate a parasitic lifestyle. In this context, during 50 y of study, the organism was believed to be devoid of cytotoxins, a paradigm that shifted in 2005 when a 591-aa Mp protein with mono-ADP ribosyltransferase (mART) and vacuolating activities, termed Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX), was isolated through its ability to bind with high affinity to surfactant protein A (SP-A), the most abundant protein component of pulmonary surfactant (12)(13)(14). The discovery of a previously unknown Mp virulence factor cast Mp in a different light and opened the door for a new avenue of research on what has historically been an enigmatic organism.…”