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...

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SummaryThe interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled

Twenty-two mutants of Mycoplasma pneumoniae spontaneously deficient in hemadsorption were isolated. Examination of mutant protein profiles by one- and two-dimensional polyacrylamide gel electrophoresis permitted the grouping of these mutants into four classes. The largest class of mutants was deficient in four high-molecular-weight proteins (215,000, 210,000, 190,000, and 140,000). A second class of mutants lacked three proteins previously designated A, B, and C (72,000, 85,000, and 37,000, respectively). A single mutant, in addition to lacking proteins A, B, and C, was missing a fourth protein of 165,000 molecular weight. The remaining mutants exhibited protein profiles apparently identical to that of the wild-type strain. All mutant strains attached to the respiratory epithelium of hamster tracheal rings in vitro at reduced levels; however, mutants lacking proteins A, B, and C recognized only neuraminidase-insensitive receptors. None of the mutants tested produced detectable pneumonia in intranasally inoculated hamsters, although one mutant class demonstrated low-level survival in vivo.

Hemadsorbing (HA') virulent Mycoplasma pneumoniae and spontaneously derived nonhemadsorbing (HA-) avirulent mutants were compared by biochemical and ultrastructural techniques in an attempt to understand the molecular basis for cytadsorption. Lactoperoxidase-catalyzed iodination of intact mycoplasmas indicated that both virulent and avirulent mycoplasmas displayed similar surface protein patterns. A specific external protein, P1 (molecular weight, 165,000), previously implicated as a major ligand mediating attachment, was readily detected in HA' and HA-mycoplasma strains. However, immunoferritin elec

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.Mycoplasmas are microorganisms derived from gram-positive bacteria with low GϩC content, and they are characterized by streamlined genomes and the absence of a cell wall. Their reduced metabolic abilities (32) lead them to a parasitic lifestyle, and many of them are pathogens for humans and animals (5). In particular, Mycoplasma genitalium is the leading cause of chlamydia-negative, nongonococcal urethritis (13), and it has been also associated with several extragenitourinary diseases (43). With just 517 genes (9), M. genitalium is considered one of the smallest self-replicating cells, and it is currently used as a model for the studies of the minimal gene complement (11,14). Despite its apparent simplicity, M. genitalium adheres to host cells via its complex tip structure, known as the attachment or terminal organelle. This polar membrane extension confers a flask-shaped appearance to the M. genitalium cells, and it is characterized by an electron-dense core that is a part of the mycoplasma cytoskeleton. The terminal organelle is present in several Mycoplasma species, and it has been extensively studied in Mycoplasma pneumoniae. In the lat...

The mechanisms by which virulent microorganisms mediate host cell injury in respiratory tissue are unclear. To better understand the cellular and subcellular events which accompany these infections requires the establishment of experimental models which permit monitoring of the infectious process under experimentally controlled conditions. During the past several years we have examined the capacity of virulent Mycoplasma pneumoniae organisms, human pathogens of the respiratory tract, to parasitize and produce cell injury in the respiratory epithelium (1-4). Tracheal organ culture served as the highly sensitive host indicator system and its feasibility for other models is recognized (5-9). Based upon our earlier studies we outlined a two step parasitism: (a) the specific attachment of virulent M. pneumoniae via their tip-like organelle to a sialic acid-associated receptor region on the respiratory epithelium and (b) early abnormal host cell function at the transcriptional or translational levels with subsequent tissue cytopathology resulting from the colonization, multiplication, and continued metabolic activity of attached virulent mycoplasmas. The success of this parasitism appeared initially and critically dependent upon the intimate physical association between the membranes ofM. pneumoniae and respiratory epithelial cells. However, the chemical nature of M. pneumoniae surfaces responsible for attachment was unknown.Limited information is available concerning external membrane components of M. pneumoniae. Razin et al. (10,11) extracted from M. pneumoniae a glycolipid which reacts with complement-fixing and metabolic-and growth-inhibiting antibodies raised against intact organisms suggesting a possible surface location of the glycolipid moiety. Additional nonprotein haptens have been demonstrated in other mycoplasma species (12, 13). Boatman and Kenny (14) reported that spherules of M. pneumoniae organisms which form during growth can be disaggregated into individual and viable mycoplasmas by treatment with crude lipase. This observation implied that a lipid moiety on the surface of M. pneumoniae might be responsible for their "stickiness." Other studies with Mycoplasma hominis indicated that membrane proteins exist as receptors for the adsorption of HeLa cells (15) and for indirect hemagglutination activity (16). In this report we examine the unique interaction between virulent M. pneumo-

dHydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice.