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 ?
dues) (14). Although bacterial ADP-ribosylating enzymes do not
share extended amino acid conservation, especially relevant in
this case was the preservation of three motifs in MPN372
common to bacterial ADP-ribosylating toxins (ADPRTs) (15):
(i) potential catalytic glutamate as noted by Carroll and Collier
(16) observed at position 132; (ii) ??? region with a serine-
threonine-serine (STS) motif (residing at positions 49 to 51)
needed for structural integrity of the NAD-binding site; and (iii)
conserved arginine residue at position 10 necessary for NAD
binding in many ARTs (Fig. 1). Additionally, MPN372 contains
histidine 34, which corresponds to His 35 in PTX and His 44 in
two other ADP-ribosylating toxins, Escherichia coli heat-labile
enterotoxin and cholera toxin. As noted earlier, virulence fac-
tors, like classical bacterial toxins, have been heretofore unde-
tected among pathogenic mycoplasmas.
Site-Directed Mutagenesis, Expression, and Purification of Recombi-
nant CARDS TX (rCARDS TX). Due to inherent slow growth and
modest cell densities of M. pneumoniae in complex medium, it is
difficult to obtain sufficient amounts of nonabundant myco-
plasma proteins to permit functional studies and generate anti-
sera. This hurdle is further complicated by our observation that
very little CARDS TX is synthesized in mycoplasma broth
cultures. Therefore, it was necessary to express rCARDS TX in
E. coli to learn more about its biological properties. We used the
His-tag expression system and Ni (II)-NTA resin chromatogra-
phy to generate and purify rCARDS TX protein. Because
mycoplasmas use both UGA (universal stop codon) and UGG to
encode tryptophan, we analyzed the nucleotide and amino acid
sequences of CARDS TX for UGA-encoded tryptophan. The
gene encoding CARDS TX possesses eight UGA codons at
amino acid positions 148, 195, 233, 364, 392, 450, 462, and 508
that required PCR-mediated, site-directed mutagenesis to re-
place each UGA codon with UGG to express full-length
rCARDS TX (17) (Fig. 2 Upper). CARDS TX was predicted to
encode a protein of 591 aa. As appears in Fig. 2 Lower, the
complete CARDS TX gene was cloned, expressed as a His-10-
tagged protein and purified to homogeneity.
ART Activity of rCARDS TX. We examined the ability of rCARDS
TX to exhibit ART activity in CHO cells because of their
sensitivity to PTX activity (18, 19). Non-rCARDS TX-treated
CHO cell-free extracts (CFE) possessed weakly radiolabeled
protein bands with apparent molecular masses ranging from 26
to ?50 kDa, indicating normal mono-ADP-ribosylation events
that occur via intrinsic CHO-associated ARTs (Fig. 3a, lane 1).
However, CHO CFE treated with rCARDS TX (Fig. 3a, lane 2)
possessed additional and intensely radiolabeled ADP-
ribosylated proteins with apparent molecular masses of 45, 43,
28, 26, and 21 kDa, reinforcing the ability of CARDS TX to act
as an authentic ART. In addition, proteins with molecular
masses ?90 kDa were ADP-ribosylated (data not shown). We
further investigated whether sulfydryl agents influenced
rCARDS TX activity. Many bacterial ADP-ribosylating toxins
undergo enzymatic activation after reduction of a disulfide
bridge, and the primary structure of CARDS TX contains six
cysteine residues. Indeed, ADP-ribosylation activity was mark-
edly increased by the presence of DTT (Fig. 3a; compare lanes
B. pertussis pertussis toxin; LTX, E. coli heat-labile enterotoxin; CTX, cholera
toxin; EDIN, Staphylococcus aureus epidermal cell differentiation inhibitor;
C3bot, Clostridium botulinum C3 toxin; VIP2, Bacillus cereus vegetative insec-
Alignment of conserved residues between MPN372 and other ARTs.
tion of UGA codon within mpn372. The eight TGA codons within the coding
region of CARDS TX were modified into TGG codons (at nucleotide positions
shown in the schematic diagram) to express in E. coli. (Lower) CARDS TX gene
was cloned in pET19b vector and expressed in E. coli BL21(DE3). Recombinant
His-10-tagged protein was purified by using nickel affinity column chroma-
tography and eluted by imidazole. Proteins were resolved in 4–15% gradient
SDS?PAGE gel. Lane 1, overexpressed rCARDS TX in E. coli BL21(?DE3); lane 2,
purified rCARDS TX.
Expression and purification of CARDS TX protein. (Upper) Distribu-
ADP ribosylation of CHO cell-free extracts by rCARDS TX. CFEs were prepared
from confluent CHO cell monolayers and assayed for ADP ribosylation. CFEs
were incubated with and without rCARDS TX. The reaction mixture was
precipitated with trichloroacetic acid (TCA), and proteins were resolved by
gradient SDS?PAGE and transferred to nitrocellulose membrane for autora-
TX ? DTT; 4, CFE ? rCARDS TX ? ATP; 5, CFE ? rCARDS TX ? GTP. (b) ADP
were incubated with medium alone or in the presence of rCARDS TX or PTX
(holotoxin). Cells were washed and incubated with fresh medium, and CFEs
were prepared and assayed for ADP ribosylation. The reaction mixture was
precipitated with trichloroacetic acid (TCA), and proteins were resolved by
SDS?PAGE and transferred to nitrocellulose membrane for autoradiography.
Lanes: 1, cells in medium alone followed by preparation of CFE and addition
of rCARDS TX; 2, cells pretreated with rCARDS TX followed by preparation of
CFE and addition of rCARDS TX; 3, cells pretreated with PTX followed by
preparation of CFE and addition of rCARDS TX; 4, cells in medium alone
followed by preparation of CFE and addition of PTX.
Kannan and Baseman PNAS ?
April 25, 2006 ?
vol. 103 ?
no. 17 ?
2 and 3, with and without DTT, respectively), suggesting that
rCARDS TX-associated ART activity is sulfhydryl reduction-
dependent, similar to cholera and PTXs (20–22). The absence of
externally added GTP or ATP revealed less noticeable effects on
ADP ribosylation of target proteins (Fig. 3a, lanes 4 and 5,
We further examined rCARDS TX-associated ART activity
using human HEp-2 cells as targets (Fig. 3b). rCARDS TX-
treated HEp-2 CFE contained prominent radiolabeled proteins
with apparent molecular masses of 45, 43, 28, 26, and 21 kDa
(Fig. 3b, lane 1), similar to CHO cell protein patterns observed
in Fig. 3a, lane 2. When intact and viable HEp-2 cells were
preincubated with 5–50 ?g of rCARDS TX for 16 h and CFE
subsequently prepared and treated with additional rCARDS TX
plus [?-32P]NAD, marked decreases in radioactivity of ART-
targeted proteins were observed (Fig. 3b, lane 2). In other words,
HEp-2 cell proteins already modified as a result of their preex-
posure to rCARDS TX were no longer accessible to ADP
ribosylation, further reinforcing rCARDS TX-mediated ADP-
ribosylation events. In parallel experiments, similar results were
observed in CHO cells. When rCARDS TX was heat-inactivated
and added exogenously to intact HEp-2 or CHO cells, rCARDS
TX-mediated ADP ribosylation of target proteins in CFE was
abolished (data not shown). Importantly, substitution of the
CARDS TX-predicted catalytic glutamate (16) at position 132
with alanine (rCARDS TX132glu3ala) markedly reduced ADP-
ribosylation activity, reinforcing the categorization of CARDS
TX as a genuine ART-associated bacterial toxin.
To further delineate protein target specificity of ART-related
rCARDS TX activity, we compared ADP-ribosylation patterns
of rCARDS TX with the S1 subunit of PTX in HEp-2 cells (Fig.
3b, lanes 1 and 4). Similarities and differences among ART-
targeted proteins were observed. For example, proteins in the
range of 25–35 kDa differed between the two toxins whereas
other ribosylation patterns seemed to overlap [43 and 45 kDa
and ?90 kDa (latter not shown)]. Further, preincubation of
intact HEp-2 cells with PTX blocked ADP ribosylation of 45 and
43 kDa proteins, but not of lower molecular mass proteins
(ranging from 28–21 kDa) indicating that the latter were sub-
sequently accessible to CARDS TX-mediated ADP ribosylation
(Fig. 3b, lane 3).
Cytopathic Effects (CPEs) of rCARDS TX on Mammalian Monolayer Cell
Cultures. Because we and others reported that mammalian cells
parasitized by viable and cytadhering M. pneumoniae cells
exhibit numerous CPEs due to unknown mycoplasma factors
(23), we monitored the effect of rCARDS TX on intact and
viable mammalian cells (Fig. 4). CHO cells exposed to exoge-
nous rCARDS TX displayed distinct vacuolization and cell
rounding, with disruption of monolayer integrity. Cytopathology
was slow to develop at low concentrations of rCARDS TX
(10–50 ng?ml), requiring ?24–32 h, whereas higher concentra-
tions of rCARDS TX (10–50 ?g?ml; Fig. 4) elicited overt CPE
in 6–18 h. Heat inactivation of rCARDS TX preparations (15
min at 100°C) abolished CPE (Fig. 4, control), reinforcing the
cytotoxic properties of ‘‘heat labile’’ rCARDS TX and negating
the possible contribution of E. coli endotoxin in recombinant
protein preparations. In the latter case, all recombinant proteins
were expressed and purified from lpxM-inactivated E. coli BL21
(DE3) (24), which produces a nonmyristylated lipopolysaccha-
ride (nmLPS) with markedly reduced endotoxicity. Also, puri-
fied recombinant proteins were passed through sequential poly-
mixin columns to reduce remaining endotoxin contamination
before use, and we performed Limulus assays to determine
endotoxin concentrations in each recombinant preparation. In
all cases, recombinant test samples contained endotoxin levels at
or below the minimal detection levels of the assay (0.1 endotoxin
units?ml). Only full-length rCARDS TX induced distinct vacu-
olization of host cell cytoplasm. Interestingly, rCARDS
TX132glu3alaat 5 ?g?ml did not elicit vacuolization in CHO cells;
at 25 ?g?ml, vacuolization was discernible in only 5–10% of the
CHO cell population.
Intrigued by the vacuolating property of rCARDS TX on
CHO cells, we further tested the effect of rCARDS TX on HeLa
and HEp2 cells. As observed with CHO cell monolayers, HeLa
cells displayed a highly vacuolated phenotype, which was dose-
and time-dependent, followed by surface detachment (see Fig. 7,
which is published as supporting information on the PNAS web
site). HEp-2 cells demonstrated less pronounced CPE. Recom-
binant M. pneumoniae fibronectin-binding proteins rEF-TuMp
and rPDH-BMp did not elicit CPE under the same conditions
using similar or 10-fold higher molar concentrations.
CPEs of rCARDS TX on Baboon Tracheal Organ Cultures. Baboon
tracheal rings retain organized and synchronized ciliary activity
and respiratory epithelial integrity for at least 10 days when
Hepes-buffered DMEM (pH 7.5) is used as the fluid phase and
medium is changed every 2–3 days. However, the addition of 10
?g of rCARDS TX to tracheal rings caused noticeable slowing
and asynchronous movement of cilia within 24 h, followed by
dramatic reduction or cessation of ciliary movement, cilia dis-
organization, and possible ciliocytophoria at 48 h. rCARDS TX
at 5 and 1.5 ?g had similar effects, but the cellular changes were
delayed by at least 24–72 h, respectively. Thus, the time required
for reduction and disappearance of ciliary activity was CARDS
TX dose-dependent. Control cultures, which received similar
amounts of heat-inactivated rCARDS TX, exhibited normal
ciliary activity and respiratory cellular integrity throughout the
duration of the experiment.
To further determine the morphological changes in baboon
tracheal rings that accompanied treatment with CARDS TX, we
examined parallel tissue sections microscopically. Consistent
with reduced ciliary motion, histologic assessment revealed
extensive and sequential cytopathological changes, including
early events of marked thickening of the epithelial layer due to
cellular edema and cytoplasmic vacuolization (Fig. 5), nuclear
enlargement with chromatin margination and condensation,
disturbance of cellular polarity, disorganization in both airway
epithelial and submucosal cells, and foci of pyknotic nuclear
fragments. These observations were reinforced by using trans-
losses of tissue integrity, elimination of ciliated cells and mi-
crovilli from respiratory epithelium surfaces, and extensive
60% monolayer confluence before addition of 10 ?g of rCARDS TX for 16–40
h. Control CHO cells were treated with 10 ?g of heat-inactivated rCARDS TX
for 40 h. (Magnification: ?200.)
Effect of rCARDS TX on CHO cell morphology. Cells were grown to
www.pnas.org?cgi?doi?10.1073?pnas.0510644103 Kannan and Baseman
cytoplasmic vacuolization and nuclear fragmentation. These
pathological observations suggest a progression of cell injury,
degeneration, and death. Thus, within 48–72 h (Fig. 5), extensive
disorganization and disruption of respiratory epithelial integrity
were evident, responses directly attributable to active CARDS
TX and not heat-inactivated preparations.
Does M. pneumoniae Secrete CARDS TX? To further determine the
location of CARDS TX in M. pneumoniae cells (14), we per-
formed SDS?PAGE immunoblot analyses on mycoplasma cell
preparations from M. pneumoniae clinical isolate S1 and refer-
ence strain M129. Whole mycoplasma cell lysates and cytoplas-
mic, membrane, and culture supernatant fractions obtained
from each strain during mid-to-late exponential growth phase
were probed by using antiserum raised against rCARDS TX
(14). Immunoreactive CARDS TX was detected in total extracts
and cytoplasmic and membrane fractions, but not culture su-
pernatants. For example, during late log-phase growth of M.
pneumoniae, ?7% of CARDS TX was localized to the myco-
plasma membrane (9), with the majority of toxin detected in the
cytoplasm. There was no evidence of toxin release into the
Polymorphism in CARDS TX Sequence Among M. pneumoniae Clinical
isolates of M. pneumoniae, we characterized three additional
strains, designated L2, J1, and RJL1. In each case, CARDS TX
was detectable at very low levels by using immunoblot analysis
of concentrated mycoplasma cell-associated preparations. Inter-
estingly, when we compared mpn372 (cards tx) gene sequences of
reference strain M129 with recent clinical isolates, we observed
nucleotide polymorphisms reflected in amino acids at positions
38, 245, 308, 371, 391, and 392. For example, all clinical isolates
exhibited changes at amino acid position 371 (Ile to Ser). Only
strain JL possessed that single alteration. Strain RJL1 revealed
one additional change at position 392 (Trp to Arg). Strain L2
showed one additional change when compared with JL at amino
acid position 245 (Asp to Gly). Strain S1 had three additional
changes when compared with JL at amino acid positions 38 (Leu
to Pro), 308 (Ser to Pro), and 391 (Phe to Ser).
CARDS TX as Immunodominant Target in M. pneumoniae-Infected
Human. Because rCARDS TX exhibited ART and CPE activities
in mammalian cells, thereby displaying bona fide pathogenic
determinant characteristics, we screened acute- and convales-
cent-phase sera of nine documented M. pneumoniae-infected
individuals for CARDS TX-reactive antibodies. This study
would provide direct evidence for the synthesis of CARDS TX
during M. pneumoniae infection and lend credence to its immu-
nogenic properties and possible diagnostic, prognostic, and
vaccinogenic potential. Acute-phase sera, which were obtained
at the time of appearance of clinical symptoms, exhibited mild
reactivity to rCARDS TX, whereas sequential ‘‘convalescent’’
sera obtained at 14 and 28 days after the initial serum draw
demonstrated marked seroconversion to CARDS TX (Fig. 6).
Pooled sera from 20 healthy individuals possessed very low
reactivity to CARDS TX (Fig. 6, lane C).
Mycoplasma pneumoniae causes a wide spectrum of respiratory
and extrapulmonary pathologies (4, 6, 25), yet no single identi-
fiable virulence determinant has been associated with these
clinical signs and symptoms. Although cytadherence of M.
pneumoniae to the respiratory tract seems to be the initiating
event in the infectious process (26), it is not known how M.
pneumoniae injures respiratory epithelial cells after colonization
(4, 27). For example, it was reported in the 1960s and 1970s that
intimate contact and continued biochemical function of M.
pneumoniae during infection of host respiratory cells was essen-
tial for disruption of tissue integrity and cytotoxicity (23, 27–30).
These observations and the fact that tracheobronchitis is a
common manifestation of M. pneumoniae infections are partic-
ularly relevant to the studies described here. In those early
rings were incubated with 1.5, 5, or 10 ?g of CARDS TX for 24–48 h in 5 ml of
DMEM. Control baboon tracheal rings were treated with heat-inactivated
CARDS TX for 48 h. (Magnification: ?200.)
rCARDS TX and reacted with patient sera, which were collected at the onset of disease (I) and 14 (II) and 28 (III) days later.
ELISA-based screening of normal and M. pneumoniae-infected individuals for CARDS TX-reactive antibodies. Each well of ELISA plates was coated with
Kannan and BasemanPNAS ?
April 25, 2006 ?
vol. 103 ?
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reports, M. pneumoniae-infected hamster tracheal organ cultures
exhibited decreased rates of macromolecular synthesis, which
These events were followed by progressive coalescence of cyto-
plasmic vacuoles and distortion of the cytoplasm, resulting in
extensive cellular fragmentation and sloughing (28, 30–32).
Although it was proposed that mycoplasma adherence to host
target cells and active mycoplasma metabolic function, including
peroxide secretion, contributed to M. pneumoniae-mediated
pathogenicity (33), no differences in these properties were
demonstrable between virulent or attenuated mycoplasmas (28,
34). At that time, we postulated that unknown M. pneumoniae
‘‘dose-dependent toxic factors’’ mediated the observed host cell
dysfunction and CPE (27). Using tissue culture models and
baboon tracheal rings in organ cultures, we have demonstrated
that rMPN372 (rCARDS TX) possesses ART activity and elicits
characteristic and extensive CPE in mammalian cells. Therefore,
our earlier observations in the 1970s have been upheld and
extended by the identification of CARDS TX and its ADP-
ribosylating and vacuolating properties.
ADP ribosylation involves the transfer of the ADP-ribosyl
group from NAD?to specific amino acids in target proteins by
means of ART activity. Many classical bacterial exotoxins per-
form this reaction, leading to macromolecular dysfunction,
disruption of cellular homeostasis, initiation of CPE, and ulti-
mate cell death (35–37). Although ART-related toxins share
limited amino acid conservation within themselves and with
CARDS TX, they exhibit conserved catalytic domain-associated
amino acids (Fig. 1) (15, 16). Fully expressed rCARDS TX
ribosylates specific mammalian host proteins, and ART activity
is enhanced in the presence of DTT (Fig. 3a). The latter is likely
due to protection from oxidation of six cysteine residues that are
distributed throughout CARDS TX at positions 240, 257, 324,
406, 425, and 548. Because the predicted ART domain is
localized within the first 250 aa, the presence of DTT would
maintain the protein in reduced form, which apparently favors
Our data clearly implicate CARDS TX as the cause of
vacuolization and CPE in host target cells. Like purified VacA
protein of Helicobacter pylori (38), rCARDS TX causes the
formation of coalescent vacuoles in the cytoplasm of CHO and
HeLa cells. Early vacuoles seem to grow in size and fuse with
time until the intoxicated cell cytoplasm is occupied by mostly
large vacuoles (Fig. 4). Additional results are published in Fig.
7. This process is associated with the progressive addition of
membranes, resulting in the alteration of membrane traffic along
the endocytic–endosomal pathway (39). Whether a similar
mechanism is associated with CARDS TX is unknown. How-
ever, the abolition of ADP ribosylation and the dramatic de-
crease in vacuolization as a result of the CARDS TX glutamate-
to-alanine substitution underscore the close relationship
between CARDS TX-dependent ART activity, vacuolization,
and tissue disorganization. Further, the requirement of close
contact between M. pneumoniae and respiratory cells to elicit
cytopathology (29, 30) is consistent with our observations that a
subpopulation of CARDS TX is membrane-associated, trypsin-
sensitive, and SP-A-binding (14). Intimate contact between
mycoplasma and host cell could result in the release of CARDS
TX at membrane–membrane interfaces as well as the introduc-
tion of CARDS TX into target cells, leading to ADP ribosylation
and vacuolization (Fig. 4). Another possibility is that CARDS
TX remains associated with intact mycoplasmas during infection
and is released when extracellular mycoplasmas are degraded or
viable mycoplasmas invade target cells (10, 40).
Comparisons of mpn372 sequences among clinical isolates and
reference strain M129 revealed one consistent amino acid
change (371Ile3Ser) and several others, as described earlier. It is
unclear whether any of these amino acid differences alter
CARDS TX activities. However, they may serve to link CARDS
TX sequence polymorphisms to epidemiological and pathogenic
observations in geographically distinct infected populations.
The fact that patients seroconvert to CARDS TX during M.
TX as a fundamental virulence determinant because it is clear
that CARDS TX is synthesized in vivo and is highly immuno-
genic. It is particularly interesting that a ‘‘whoop’’ has been
described frequently in children infected with M. pneumoniae,
but no mycoplasma-associated factor has ever been correlated
with these clinical manifestations. The mode of action of PTX
S1 subunit, by means of its ADP-ribosylating properties, directly
leads to multiple alterations in protein targets, metabolic path-
ways, vascular permeability, and inflammation, contributing to
the characteristic sound of the whoop in whooping cough (18).
Based upon comparative ART profiles (Fig. 3b), CARDS TX
may elicit toxic activities and clinical symptoms that are both
TX is the only known ADP-ribosylating toxin discovered among
any pathogenic human or animal mycoplasmas and to our
knowledge, the only bacterial toxin to display both ADP-
ribosylating and vacuolating functions. Because ART bacterial
toxins play key roles in pathogenesis and are considered funda-
mental virulence factors, we hypothesize that the biological
properties of CARDS TX relate directly to M. pneumoniae-
mediated clinical symptoms and pathologies. In other words, we
consider CARDS TX to be an authentic pathogenic determinant
of M. pneumoniae. The long history of M. pneumoniae as a very
successful bacterial pathogen and its association with character-
istic CPE, inflammatory responses, and sequelae that lead to
acute and chronic airway diseases and extrapulmonary pathol-
ogies are well established (4, 6, 25). Therefore, the detection of
this unique ADP-ribosylating and vacuolating toxin in M. pneu-
moniae may help to explain the wide-ranging pathogenic capa-
bilities of M. pneumoniae and serve as a diagnostic and prog-
nostic indicator of infection and disease progression as well as a
vaccine and anti-drug target.
Materials and Methods
and clinical isolates S1 (14), L2, J1, and RJL1; and E. coli INV
alpha, E. coli BL21 (?DE3), and lipid A mutant of E. coli BL21
[?DE3, lpxM?(gift from J.-F. Gauchat, University of Montreal,
Montreal)] (24) strains were used in this study. Plasmids expressing
histidine tag (His-10) recombinant M. pneumoniae elongation
factor Tu (rEF-TuMp) and pyruvate dehydrogenase E1 ? subunit
(rPDH-BMp) fusion proteins have been described (9). Plasmids
expressing full-length rCARDS TX were constructed by site-
directed mutagenesis by using PCR (17) and expressed as His-10-
tagged protein by using primers presented in Table 1, which is
published as supporting information on the PNAS web site.
Expression and Purification of Recombinant Proteins. Using NdeI
and BamHI restriction sites incorporated in the oligonucleo-
tide primers, we cloned the entire fragment containing mpn372
(or cards tx) coding region into E. coli His-10-tagged expres-
sion vector, pET19b (Novagen) and purified rCARDS TX by
nickel and polymixin affinity chromatography (for endotoxin
analysis, see Supporting Materials and Methods, which is pub-
lished as supporting information on the PNAS web site).
rCARDS TX132glu3alawas generated by using PCR-directed
Mammalian Cell Lines. Monolayers of CHO-K1, HeLa, and HEp-2
cell (American Type Culture Collection) were grown to 60–75%
confluence in 25-ml flasks by using F12-K or MEM media
supplemented with 5% FBS. Depleted culture media were
replaced by fresh F12-K or MEM without serum but containing
www.pnas.org?cgi?doi?10.1073?pnas.0510644103 Kannan and Baseman
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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Kannan and BasemanPNAS ?
April 25, 2006 ?
vol. 103 ?
no. 17 ?