ADP-ribosylating and vacuolating cytotoxin
of Mycoplasma pneumoniae represents unique
virulence determinant among bacterial pathogens
T. R. Kannan and Joel B. Baseman*
Department of Microbiology and Immunology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229
Edited by Stanley Falkow, Stanford University, Stanford, CA, and approved March 2, 2006 (received for review December 12, 2005)
Unlike many bacterial pathogens, Mycoplasma pneumoniae is not
known to produce classical toxins, and precisely how M. pneu-
moniae 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 pertus-
sis 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 vac-
uolization and ultimate cell death of mammalian cells, including
WT virulent M. pneumoniae. We observed dramatic seroconver-
sion to MPN372 in patients diagnosed with M. pneumoniae-asso-
ciated pneumonia, indicating that this toxin is synthesized in vivo
and possesses highly immunogenic epitopes.
ADP ribosylation ? community-acquired respiratory distress syndrome
toxin ? vacuolization
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 asso-
ciated with other airway disorders, such as tracheobronchitis and
pharyngitis. Extrapulmonary manifestations, such as hemato-
poietic, dermatologic, joint, central nervous system, liver, pan-
creas, kidney, and cardiovascular syndromes are considered
sequelae of primary M. pneumoniae infections. Also, M. pneu-
moniae has been linked to fulminant disease, with multiorgan
involvement (6). Therefore, M. pneumoniae causes a wide spec-
trum 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
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 the M. pneumoniae–host interplay revolve
around successful mycoplasma colonization of the respiratory
tract, facilitated by a specialized mycoplasma tip organelle that
he earliest reports of mycoplasmas as infectious agents in
humans appeared in the 1940s (1). Definitive studies in the
mediates surface parasitism (4, 5, 7). This distinct terminus is a
complex structure, composed of a network of interactive pro-
teins, designated adhesins, and adherence-accessory proteins (5,
7, 8). In addition, subpopulations of mycoplasma ‘‘cytoplasmic’’
proteins, specifically elongation factor-Tu (EF-Tu) and pyruvate
dehydrogenase ? subunit (PDH-B), are transferred to M. pneu-
moniae membrane surfaces and selectively bind fibronectin,
which further promotes mycoplasma interactions with respira-
tory mucosa (9). Although mycoplasmas are known primarily as
extracellular pathogens, recent sightings of intact mycoplasmas
distributed throughout the cytoplasm and perinuclear regions of
human cells, along with evidence that mycoplasmas are capable
of long-term intracellular survival and replication, provide ad-
ditional insights into their pathogenic potential (10).
However, the events in M. pneumoniae pathogenesis that
follow cytadherence are poorly understood, and no mycoplasma
products have been identified that exhibit classical toxin-like
activities. Therefore, the clinical course of mycoplasma infec-
inflammatory responses, rather than direct cytopathological
effects initiated by mycoplasmal cell components. In our search
to identify virulence factors of M. pneumoniae, we used the
human lung-enriched protein, surfactant protein A (SP-A), as
‘‘bait’’ to detect M. pneumoniae SP-A-binding proteins. SP-A is
synthesized primarily by type II pneumocytes and, to a lesser
extent, by nonciliated bronchioalveolar epithelial cells and other
cell types (11, 12). SP-A serves a number of diverse functions,
including facilitation of tubular myelin formation, reutilization
of surfactant phospholipids and proteins, and contribution to
innate immunity (13). SP-A affinity chromatography enabled us
to identify a prominent 68-kDa M. pneumoniae-binding protein,
which was subsequently sequenced and identified as MPN372
(14). In this study, we implicate MPN372, which we have
designated community-acquired respiratory distress syndrome
toxin (CARDS TX), as a virulence factor that exhibits ADP-
ribosyltransferase (ART) activity and elicits a distinct pattern of
cytopathology in mammalian cells.
Primary Sequence and Conserved Amino Acids of MPN372 (CARDS TX).
Primary amino acid sequence alignment revealed homologies
between the N terminus of MPN372 and pertussis toxin (PTX)
S1 subunit of Bordetella pertussis (27% identity over 239 resi-
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CARDS TX, community-acquired respiratory distress syndrome toxin;
rCARDS TX, recombinant CARDS TX; ART, ADP-ribosyltransferase; PTX, pertussis toxin;
EF-Tu, elongation factor-Tu; PDH-B, pyruvate dehydrogenase ? subunit; SP-A, surfactant
protein A; CFE, cell-free extract; CPE, cytopathic effect.
database (accession nos. DQ447746–DQ447750).
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
April 25, 2006 ?
vol. 103 ?
10 ng–50 ?g of filter-sterilized (0.22 ?m) rCARDS TX, rEF-
TuMp, or rPDH-BMp; the latter two served as negative controls.
After 2 h at 37°C, 5% FBS was added to each culture and
incubation continued for 16–72 h. CHO-K1, HeLa, and HEp-2
cell cultures were observed periodically for morphological
by determining incorporation of [32P]ADP-ribose moiety (from
[?-32P]NAD) into indicator mammalian cell proteins as de-
scribed for PTX (19) (see Supporting Materials and Methods).
Baboon Tracheal Organ Cultures. Tracheas from female baboons
(8–20 years old) were obtained from the Southwest Foundation
for Biomedical Research (San Antonio, TX). Excised tracheas
were placed in 50 mM Hepes-buffered DMEM (pH 7.5), sup-
plemented with 100 ?g?ml each of penicillin, streptomycin, and
gentamycin to control microbial contamination. Tracheal rings
of ?2–3 mm in thickness were prepared by transverse sectioning
between each cartilage, and single trachea yielded 14–16 rings
with an inner surface lined with ciliated epithelium. Up to three
tracheal rings were placed in plastic dishes containing 4.75 ml of
medium and incubated overnight at 37°C in 5% CO2and air to
assess the quality and integrity of individual rings. Ciliary
dish by using an inverted microscope at ?100 magnification.
Between 24 and 48 h after the immersion of tracheal rings in
antibiotic-containing medium, 0.25 ml of rCARDS TX (1.5, 5.0,
or 10.0 ?g?0.25 ml in DMEM) was added; equivalent concen-
trations of heat-inactivated rCARDS TX were included in
control cultures. Histopathology was performed by using stan-
dard protocols (see Supporting Materials and Methods).
Comparison of CARDS TX Genes Among M. pneumoniae Isolates. The
entire coding region of CARDS TX was PCR amplified by using
primers 1 and 2 (see Table 1) from the chromosomal DNAs of
reference strain M129 and clinical isolates with high fidelity Taq
polymerase (Sigma-Aldrich), cloned in pCR-II vectors (Invitro-
gen), and sequenced (Center for Advanced DNA Technologies,
University of Texas Health Science Center at San Antonio).
Sequences were analyzed by using the BLAST program available
in the National Center for Biotechnology Information database
Immune Assessment of M. pneumoniae-Infected Patient Sera to
rCARDS TX. Acute and convalescent phase sera were collected
from patients with M. pneumoniae-diagnosed respiratory infec-
tions that ranged from tracheobronchitis to bronchopneumonia.
These patients were diagnosed with mycoplasma infection based
upon ?4-fold increases in antibody titers to M. pneumoniae by
using ELISA and immunoblot criteria and, in some cases, by
direct isolation of M. pneumoniae from blood. Two or three
blood samples were obtained from each patient. The first blood
sample was collected during the acute phase of the disease, ?2
weeks after exposure to M. pneumoniae. The second and third
serum samples were obtained 14 and 28 days later, respectively.
Control baseline serum samples were obtained from healthy
women attending the University of Texas Health Science Center
at San Antonio Obstetrics and Gynecology Clinic. All serum
samples were assessed by immunoblotting against total M.
pneumoniae proteins and by ELISA for IgG reactivity by using
rCARDS TX (see Supporting Materials and Methods).
Statistical Analysis. DELTAGRAPH 4 (1999) and Microsoft EXCEL
software were used for analyses. Comparison of patients’ im-
mune responses was performed with Student’s t test, and results
are presented as means ? SD. The threshold for statistical
significance was P ? 0.05.
We thank Marianna Cagle and Pramod Gowda for technical assistance
and Drs. J. J. Coalson, R. L. Reddick, and A. M. Collier for interpre-
tation of histological data. This work was supported by National Insti-
tutes of Health Grant AI45737.
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