Synthesis and distribution of CARDS toxin during Mycoplasma pneumoniae infection in a murine model.
ABSTRACT 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.
- SourceAvailable from: Krishnan Manickam[Show abstract] [Hide abstract]
ABSTRACT: Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.PLoS ONE 01/2013; 8(5):e62706. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: 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.mBio 08/2014; 5(4). · 5.62 Impact Factor
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ABSTRACT: The presence of Mycoplasma pneumoniae has been associated with worsening asthma in children. Sensitive assays have been developed to detect M pneumoniae-derived community-acquired respiratory distress syndrome (CARDS) toxin. To identify the frequency and persistence of M pneumoniae detection in respiratory secretions of children with and without asthma and to evaluate antibody responses to M pneumoniae and the impact of M pneumoniae on biological markers, asthma control, and quality of life. We enrolled 143 pediatric patients (53 patients with acute asthma, 26 patients with refractory asthma, and 64 healthy controls; age range, 5-17 years) during a 20-month period with 2 to 5 follow-up visits. We detected M pneumoniae using CARDS toxin antigen capture and polymerase chain reaction and P1 adhesin polymerase chain reaction. Immune responses to M pneumoniae were determined by IgG and IgM levels directed against CARDS toxin and P1 adhesin. pH was measured in exhaled breath condensates, and asthma control and quality of life were assessed using the Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire. M pneumoniae was detected in 64% of patients with acute asthma, 65% with refractory asthma, and 56% of healthy controls. Children with asthma had lower antibody levels to M pneumoniae compared with healthy controls. Exhaled breath condensate pHs and asthma control and quality of life scores were lower in M pneumoniae-positive patients with asthma. The results suggest that M pneumoniae detection is common in children, M pneumoniae detection is associated with worsening asthma, and children with asthma may have poor humoral immune responses to M pneumoniae.Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 05/2013; 110(5):328-334.e1. · 3.45 Impact Factor
M A J O R A R T I C L E
Synthesis and Distribution of CARDS Toxin
During Mycoplasma pneumoniae Infection in
a Murine Model
T. R. Kannan,1Jacqueline J. Coalson,2Marianna Cagle,1Oxana Musatovova,1R. Doug Hardy,3,aand Joel B. Baseman1
1Department of Microbiology and Immunology, and2Department of Pathology, the University of Texas Health Sciences Center at San Antonio; and
3Department of Internal Medicine and Pediatric Infectious Diseases, University of Texas Southwestern Medical Center, Dallas
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 is an atypical bacterium that
causes acute respiratory illnesses in humans, including
pneumonia [1–3]. It also has been directly linked to
reactive airway disease and asthma [4–8] and extra-
pulmonary manifestations [2, 9, 10]. M. pneumoniae has
been isolated from the respiratory tract of up to 20%–
25% of asthmatics experiencing acute exacerbations
[6, 11]. While the wide-ranging clinical significance of
M. pneumoniae infection is becoming more evident, the
mechanisms by which mycoplasma-mediated host cell
the years the pathogenic potential of M. pneumoniae has
been demonstrated in tracheal organ cultures and ham-
ster animal models [12–15]. Our earlier reports described
the specific attachment of virulent M. pneumoniae via a
constellation of mycoplasma tip organelle-associated
proteins to sialic acid–associated receptors on the re-
spiratory epithelium and via other mycoplasma surface
proteins that mediate binding to extracellular matrix
proteins, like fibronectin and surfactant protein A
[16–20]. We showed that viable and attached virulent
mycoplasmas elicited abnormal host cell reactions at
transcriptional and translational levels, with sub-
sequent interruption of host metabolic pathways and
generation of tissue cytopathology [13, 21]. In addi-
tion, microbiologic and histologic findings of experi-
mental murine M. pneumoniae pneumonia have been
Using hamster tracheal organ cultures and hamster
and murine animal models, we suggested that un-
identified virulence factor(s) associated only with viable
mycoplasmas mediates host cell injury [13, 21, 22,
27, 28]. Recently, we identified a novel M. pneumoniae
cell–associated adenosine diphosphate–ribosylating and
vacuolating cytotoxin, designated the Community Ac-
quired Respiratory Distress Syndrome (CARDS) toxin,
Received 5 May 2010; accepted 4 January 2011; electronically published 28
aPresent affiliation: I.D. Specialists, Forest Lane, Ste B-412, Dallas, TX.
Correspondence: Joel B. Baseman, PhD, Department of Microbiology and
Immunology, the University of Texas Health Science Center at San Antonio, 7703
Floyd Curl Dr, MC7758, San Antonio, TX 78229-3900 (firstname.lastname@example.org).
The Journal of Infectious Diseases
? The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
0022-1899 (print)/1537-6613 (online)/2011/20410-0017$14.00
d JID 2011:204 (15 November)
d Kannan et al
whichalone reproducedthe characteristic ciliostasis, cytoplasmic
and nuclear vacuolization, and extensive respiratory epithelial
cell fragmentation and sloughing  that had been observed
in M. pneumoniae–infected tracheal organ cultures [12–15].
In order to understand the possible relationship of CARDS
toxin in M. pneumoniae–mediated disease progression, we es-
tablished specific CARDS toxin assays to enable detection and
localization of mycoplasmas and CARDS toxin in biological
fluids. Specifically,we developeda toxin antigen captureassay to
quantify CARDS toxin protein levels in tissue samples ;
a CARDS toxin gene-based quantitative real-time polymerase
chain reaction (qPCR) assay to enable quantification of
M. pneumoniae genomes; and immunostaining methodology
that permitted identification and localization of mycoplasmas
and CARDS toxin in the lungs. This report focuses on CARDS
toxin–related events that for the first time to our knowledge
provide fundamental insights concerning the synthesis and
distribution of this unique toxin during M. pneumoniae air-
Organism and Growth Conditions
M. pneumoniae strain M129 (ATCC 29342) was grown in SP4
broth at 37?C for 72 hours and concentrated in 2 mL fresh SP4
to 7–8 log10 colony-forming units (CFU) per mL.
Two-month-old female BALB/c mice were intranasally (IN)
infected once (day 0) with 5.9–6.2 log10 CFU of M. pneumoniae
in 50 lL of SP4 broth. Control mice were inoculated with sterile
SP4 medium. Mycoplasma and murine virus–free mice (Charles
River andHarlan)werehoused in filter-topcages and allowed to
acclimate to their new environment for 1 week. Animal guide-
lines were followed in accordance with the Institutional Animal
Care and Research Advisory Committee at the University of
Texas Southwestern Medical Center at Dallas.
Sample Collection and Analysis
Mouse tissue samples were obtained at 1, 4, 7, 14, and 35 days
postinfection (PI). At each time point, 6 infected and 6 un-
infected control mice were sacrificed forbronchiolar lavage fluid
(BALF; 0.5 ml), serum samples, and lung specimens .
Whole-lung specimens, including trachea and both lungs, were
then collected and fixed with 10% neutral buffered formalin
solution for histologic evaluation. Following fixation, lungs
from each animal were cut coronally and processed for paraffin
embedment. Sections were prepared at 5 lm thickness and
stained with hematoxylin and eosin (H&E). Two control and 4
additional infected mice were sacrificed at 4, 7, and 14 days, and
the lungs were air inflated and frozen in liquid nitrogen. Cry-
stained using CD4 and CD19 biotinylated antibodies (1:25; BD
Pharmingen)with avidin-biotin–blockingreagents, streptavidin-
horseradish peroxidase conjugate, and diaminobenzidine (DAB)
chromogen (Vector Laboratories). Rabbit recombinant CARDS
(rCARDS) toxin antibodiesandrabbitwhole-cellM.pneumoniae
antibodies at 1:1000 and 1:1500 dilutions, respectively, were in-
cubated with representative lung sections, which were then
stained with DAB chromogen. Histopathological findings and
grading of lung lesions were performed by a pathologist (J. J. C.),
who was unaware of the infection status of animals from
which specimens were taken. Lesions of peribronchiolar and
bronchial infiltrates, bronchiolar and bronchial luminal exu-
dates, perivascular infiltrate, and parenchymal pneumonia
were evaluated . This method assigns values from 0 to 26
(the greater the score, the greater the inflammatory changes in
the lung). Inflammatory cell infiltrates of lymphocytes and
polymorphonuclear leukocytes were graded at few (grade 1),
numerous (grade 2), or abundant (grade 3) in peribronchial/
peribronchiolar, perivascular, and intra-alveolar pneumonitic
M. pneumoniae Quantification in SP4 Broth Culture and BALF
M. pneumoniae cells were quantified in SP4 cultures and BALF by
CFU and qPCR. In the latter case, 2 separate duplex assays (tar-
geting CARDS toxin [MPN372] and P1 adhesin [MPN141] gene
sequences) were compared. Primers and TaqMan probe for de-
previously . Standard curves were established using
M. pneumoniae chromosomal DNA serially diluted from 107to
5 copies per reaction.
Expression and Purification of M. pneumoniae Recombinant
Since CARDS toxin is expressed at low levels in mycoplasma
broth cultures , we used purified rCARDS toxin for com-
parative serological studies with the recombinant immunodo-
minant COOH epitope of adhesin P1 (rP1) . As described
before , proteins were expressed and purified by nickel-
affinity column chromatography and desalted in 50 mmol/L
Tris pH 7.4.
CARDS Toxin Capture Assay
CARDS toxin capture assay was performed as reported .
Extracted BALF samples were mixed with 1% bovine serum
albumin/phosphate-buffered saline with 0.05% Tween (BSA/
PBS-T) to a final volume of 50 lL and added to single wells. In
order to quantifythe amount ofCARDS toxin ina given sample,
known amounts of purified rCARDS toxin (ranging from 7 ng
to 7 fg per well) were diluted in 1% BSA/PBS-T to establish
a standard curve. To determine the sensitivity of the CARDS
toxin capture assay in the presence of BALF, we measured toxin
concentrations diluted in BALF derived from uninfected mice,
which were compared with the standard curve.
CARDS Toxin Synthesis in Murine Model
d JID 2011:204 (15 November)
Immunoglobulin (Ig) M and IgG antibodies to M. pneumoniae
CARDS toxin and adhesin P1 in infected mice were determined
by enzyme-linked immunosorbent assay. Ninety-six well micro-
titerplateswereindividuallycoatedwith 50 lL/well of equimolar
concentrations of recombinant proteins (P1 or CARDS toxin)
in carbonate-bicarbonate buffer (pH 9.6) overnight at 4?C and
washed 3 times with PBS. Plates were incubated 2 hours at
room temperature with individual mouse serum samples
(50 lL/well) at 1:200 dilution in 1% BSA/PBS, and experiments
were performed as reported . Values of uninfected control
mouse sera were compared with test samples.
Sigma Stat 2003 software (SPSS Science) was used. If data were
normally distributed, the t test compared values of different
groups of animals at identical points. When data were not
normally distributed, the Mann–Whitney rank-sum test was
applied for comparisons. Bonferroni correction was employed
where multiple comparisons were made. The Spearman rank-
order test was used for correlations, as all data taken together
were not normally distributed. A comparison was considered
statistically significant if the P value was , .05.
Quantification of M. pneumoniae Organisms and CARDS Toxin
Protein in BALF of Infected Mice
The number of M. pneumoniae cells in BALF was estimated by
CFU and real-time PCR. BALF was culture-positive in 100% of
IN-infected mice up to 14 days PI and in 75% of mice at 35 days
during the first 4 days of infection and 5.3 log10 CFU/mL at day
7 PI. At days 14 and 35 PI, mean CFU titers decreased to 2.95
and 1.95 log10 CFU/mL, respectively. All control mice had
negative BALF cultures. CARDS toxin real-time PCR values
paralleled CFU throughout the experiment (Figure 1). In addi-
tion, real-time PCR was 100% positive in infected mice and
negative in uninfected mice. To further analyze the sensitivity
and specificity of the CARDS toxin qPCR assay, we compared its
efficiency with adhesin P1–based qPCR and found that both
PCR targets were equally specific (data not shown).
Using the CARDS toxin capture assay, we detected
levelsobservedat day 1PI(3846 70pg/mL;Figure1) andlower
levels at days 4 and 7 PI (?293 6 42 and 303 6 37 pg/mL,
respectively). At days 14 and 35 PI, CARDS toxin values were
still detectable but at markedly reduced levels (4 6 1.54 and
0.18 6 0.1 pg/mL, respectively). We observed direct correlation
between the microbial loads and toxin concentration in BALF,
of M. pneumoniae .
Histopathology of M. pneumoniae–Infected Mouse Lungs
Histopathological scores (HPS) using the Cimoli et al 
methodology are shown in Figure 2A. Temporal shifts in lym-
phocyte and neutrophil populations at the different study times
over the 35-day infectious time frame are not well defined using
this grading method. As indicated in the Methods section, we
used an alternative grading system to define the lymphocytes
and neutrophil populations in the infected animals (Figure 2B).
H&E-stained lung specimens from sterile SP4 broth IN-
inoculated control mice were normal in appearance at all time
points. In infected mice at day 1 PI, bronchi and bronchioles
were lined by intact respiratory epithelium, but ulceration was
identified at terminal and respiratory bronchiole junctions
(Figure 3A, arrows). A mixed population of lymphocytes and
polymorphonuclear (PMN) cells was evident in the walls of
pulmonary vessels and airways, with numerous intra-alveolar
neutrophils and macrophages present in the pneumonitic exu-
dates in the alveolar spaces (Figure 3A). The graded cell findings
reflected in Figure 2B support the rapid recruitment of both
lymphocytic and PMN cells into the infected sites. By day 4 PI,
PMN recruitment had waned in the perivascular spaces, but the
PMN exudate in the pneumonitic response was at its peak
(Figure 2B). Multilayered collections of small lymphocytes with
thin rims of cytoplasm and abundant nuclear chromatin that
encircled large and small airways and pulmonary arteries and
veins also peaked in number at 4 days PI (Figure 3B). A con-
sistent finding at 4 days PI was the distinct formation of separate
aggregates of 2 subsets of lymphocytes (CD4 and CD19) in the
inflamed foci (Figure 4A and 4B). At day 7 PI, sites of
Community Acquired Respiratory Distress Syndrome (CARDS) toxin
concentration in mouse bronchiolar lavage fluid (BALF). Infected BALF
was collected and analyzed for the presence of M. pneumoniae genomes
and CARDS toxin molecules as described in Methods. Open circle,
M. pneumoniae genome copies per mL of BALF; open square, CARDS
toxin concentration in pg per mL of BALF. Note direct relationship
between mycoplasma genome number and toxin concentration.
Quantification of Mycoplasma pneumoniae organisms and
d JID 2011:204 (15 November)
d Kannan et al
pneumonia had largely disappeared, but lymphocytes remained
elevated in number (Figure 2B) and concentrated around peri-
vascular spaces, with fewer numbers in the walls of airways
(Figure 3C). Manylymphocytes were largerin size anddisplayed
abundant cytoplasm and enlarged nuclei, that is, more blastic in
appearance (Figure 3C). These reactive lymphocyteswere focally
embedded in infiltrates of small lymphocytes. Multiple and
comparable microscopic sites of these different lymphocyte
phenotypes were photographed on serial or sequentially cut
sections immunostained with CD4 and CD19. Although some
foci appeared to contain more cells of 1 phenotype, overall,
CD41T cells and CD191B cells were both evident in similar
numbers at all 3 time points. At day 14 PI, lymphoid infiltrates
in airway walls were decreased in number (Figure 2B), whereas
those in the perivascular spaces were persistently higher in
number throughout the 35-day study period (Figure 2B). At day
35 PI, perivascular lymphoid aggregates were infrequent and
showed depleted numbers of lymphocytes and occasional col-
lections of pigmented mononuclear cells (Figures 2B and 3D).
Detection of M. pneumoniae Cells and Localization of CARDS
Toxin in Infected Mouse Lungs
To localize M. pneumoniae cells during infection, we initially
used rabbit antisera raised against whole-cell lysates of broth-
grown M. pneumoniae. We identified mycoplasmas on the sur-
faces of respiratory epithelia at day 4 PI (Figure 5B, arrows) and
not in uninfected lung tissue (Figure 5A). However, we also
noted background staining below epithelial cell surfaces in both
infected and uninfected lung tissue (Figure 5A and 5B). This
staining pattern is likely due to the presence of antibodies to
mycoplasma membrane protein epitopes that cross-react with
mammalian proteins, such as fibrinogen, keratin, myosin, and
collagen . Importantly, when we used highly purified anti–
CARDS toxin antibodies, no background labeling occurred
(Figure 5C), and CARDS toxin, along with M. pneumoniae
organisms, was readily evident on respiratory epithelium sur-
faces of infected lungs (Figure 5D).
Closer inspection of infected lung tissues at day 4 PI revealed
mycoplasmas (Figure 6A) and CARDS toxin (Figure 6B) in
peribronchiolar alveolar spaces that contained inflammatory
exudates of edema, neutrophils, and alveolar macrophages/
monocytes. Notably, lungepithelialcells associated with CARDS
toxin distribution showed cellular damage and focal loss of cilia.
Alveolar walls in these sites revealed increased interstitial cellu-
larity, but necrosis was not observed. Some mycoplasmas ap-
peared in close proximity to both neutrophils and alveolar
of mycoplasmas, abundance of CARDS toxin, and co-localization
of mycoplasmas and toxin within alveolar spaces and inflam-
matory cells are clearly discernible (Figure 6A and 6B, arrows).
Seroconversion to M. pneumoniae CARDS Toxin and Adhesin P1
in Infected Mice
At days 1 and 4 PI, we observed little to no detectable levels of
antibodies reactive against CARDS toxin or adhesin P1 when
compared with SP4 broth–inoculated, uninfected controls.
However, serum IgM levels against CARDS toxin and P1 peaked
at day 7 in 75% of infected mice and approached background
values at 35 days PI (Figure 7). IgG titers to CARDS toxin and
P1 increased significantly between days 7 and 35 PI (Figure 7).
M. pneumoniae infection. A, Mean histopathology score (HPS) for mice
(n 5 5–8) inoculated with live M. pneumoniae. Time points are at 1, 4, 7,
14, and 35 days postinfection (PI). Values are expressed as mean 6 SD.
B, Temporal changes in the different inflammatory cell infiltrates are
depicted for the above group: a rapid polymorphonuclear and lymphocytic
recruitment into the different lung compartments at day 1, pneumonia
most severe at day 4 with abundant lymphocytes in vascular and airway
walls, loss of pneumonia by day 7, but a persistent presence of lymphoid
infiltrates throughout the 35-day study period (see text for details).
Open symbols, mice inoculated with SP4 medium; filled symbols, mice
inoculated with live M. pneumoniae; open and filled circles, peribron-
chial/bronchiolar lymphocytes; open and filled diamonds, perivascular
lymphocytes; open and filled squares, perivascular polymorphonuclear
leukocytes; open and filled triangles, polymorphonuclear leukocytes in
alveolar spaces/pneumonia. Values are expressed as mean 6 SD.
Quantitative analysis of murine lung histopathology during
CARDS Toxin Synthesis in Murine Model
d JID 2011:204 (15 November)
Using a murine M. pneumoniae acute pulmonary infection
model, we monitored the synthesis and distribution of CARDS
toxin following M. pneumoniae IN infection over a 35-day pe-
riod. We readily detected mycoplasmas and CARDS toxin in
BALF during the initial stages of M. pneumoniae colonization of
the airways (Figure 1). We observed a direct relationship be-
tween numbers of mycoplasmas, amounts of CARDS toxin, and
lung histopathology using CFU, real-time PCR, antigen capture
assay, and HPS (Figures 1–3). Immunostaining demonstrated
CD4 and CD19 positively stained subsets of lymphocytes in
respiratory bronchiolar level (arrows). The bronchiolar wall contains a pulmonary artery (a) and an inflammatory cellular infiltrate of predominantly
lymphocytes with scattered neutrophils (see insert). Conversely, within the alveolar spaces, exudates of numerous neutrophils and fewer alveolar
macrophages are evident (circle). (b) indicates bronchiole. Original magnification 203; insert 403. B, At day 4, abundant layers of small lymphocytes are
aggregated around large and small airways and pulmonary vessels. In the subjacent alveoli, intra-alveolar exudates of neutrophils, nuclear fragments,
and alveolar macrophages are evident (circle). Original magnification 203. C, At day 7, perivascular and peribronchiolar collections of lymphocytes are
still present, but as shown in this perivascular site, focal collections of lymphocytes have more cytoplasm and larger nuclei with a more lymphoblastic
appearance (left side of insert rectangles; small lymphocytes on right). Alveolar exudate is absent in this site. Original magnifications 203 and 603.
D, At day 35, a few residual foci of lymphocytic infiltrates persist, usually only in perivascular sites and more rarely in both perivascular and
peribronchiolar sites as depicted here. Control lungs at this same time period lack appreciable collections of lymphocytes. Original magnification 103.
Tissue sections were stained with hematoxylin and eosin.
Analysis of murine lung histopathology during M. pneumoniae infection. A, At day 1, there is an abrupt loss of bronchial epithelium at the
d JID 2011:204 (15 November)
d Kannan et al
inflamed foci (Figure 4) and localization of mycoplasmas and
CARDS toxin with respiratory epithelial cells and macrophages
(Figures 5 and 6). Also, the striking anti–CARDS toxin IgM
and IgG seroconversion not only reinforced the synthesis and
detection of CARDS toxin during airway infection but also es-
tablished its ability to elicit a strong immunogenic response
(Figure 7) consistent with observations in humans .
During the course of murine IN infection, M. pneumoniae
CFU in BALF were highest at days 1–4 PI (5.9 log10 CFU/mL)
and decreased to ?1.95 log10 CFU/mL at 35 days PI (Figure 1).
Similarly, CARDS toxin concentrations in BALF were highest
at day 1 PI and remained near this level during days 4 and 7
PI, indicating that CARDS toxin synthesis and stability are rel-
atively steady and sustainable during acute stages of infection.
However, toxin concentrations declined dramatically at 14 and
35 days PI, which paralleled reduced mycoplasma cell loads,
suggesting that CARDS toxin may serve as a surrogate marker to
track active M. pneumoniae infection and disease progression.
Interestingly, and in contrast to these in vivo dynamics, the
expression of CARDS toxin in broth-grown mycoplasmas falls
precipitously during the first 24–72 hours (during early-to-late
log phases) and remains at very low levels throughout in vitro
growth . This suggests that toxin expression is tightly reg-
ulated by host conditions that signal transcriptional activation
and synthesis of toxin, consistent with the important role that
CARDS toxin plays during infection.
Another fundamental finding was the apparent linkage be-
tween mycoplasma genome numbers, CARDS toxin levels, and
degree of histologic lung inflammation, which correlates well
with our recent report in which we compared 3 different strains
of M. pneumoniae and observed a direct link between load of
mycoplasma, CARDS toxin levels, and cytokine responses .
M. pneumoniae infection induced an inflammatory phase that
lasted ?3 weeks, with peak inflammatory changes occurring at
day 4 PI, consistent with early CARDS toxin levels in BALF.
Relatively low titers of mycoplasmas persisted, along with di-
minished amounts of CARDS toxin and pulmonary histologic
inflammation at days 14 and 35 PI (Figures 1–3). Interestingly,
mice that received IN rCARDS toxin alone elicited cellular
inflammation similar to M. pneumoniae infection, further sug-
gesting the central role of CARDS toxin in pulmonary in-
flammation . However, the induction of additional
cytokines in response to M. pneumoniae infection in BALF,
when compared with CARDS toxin alone [22, 26, 34, 35],
suggests that other M. pneumoniae factors contribute to the
Detailed microscopic analysis of lung sections confirmed the
development of pneumonia, alveolar wall and space edema,
inflammatory cell infiltrates, early and persistent lymphocyte-
and perivascular regions, and immunostained mycoplasmas
plus CARDS toxin in both airway and alveolar compartments
(Figures 2–6). The early neutrophilic pneumonitis induced in
this experimental infection model is accompanied by a robust
and simultaneous infiltration of perivascular lymphocytes, as we
reported with rCARDS toxin alone . Consistent features of
M. pneumoniae infection in humans are bronchiolar luminal
purulent exudates, lymphoplasmacytic infiltrate in bronchiolar
walls, peribronchiolar septal widening, and alveolar type 2 hy-
perplasia . In our mouse model, these histopathological fea-
tures are prominent. Additionally, the pneumonitis evident in
alveoli subjacent to infected bronchioles, which occurred pri-
marily in the first week, along with the subsequent retention of
the prominent perivascular lymphocytic infiltrates over time,
indicates that temporal changes occur in histopathology as in-
fection and intoxication events evolve (Figure 3A–D). In contrast
to mycoplasmal pneumonia, the dominant feature in most bac-
terial pneumonias is bronchiolar and alveolar filling with puru-
lent exudates, and bronchiolar lymphocytic infiltration is not
a prominent or diagnostic feature . M. pneumoniae is well
cytes associated with perivascular inflamed region of M. pneumoniae–
infected mouse lung at day 4 postinfection. The perivascular inflamed site
reveals the presence of both CD41cells (A) and CD191cells (B). Note the
distinctand separate aggregation of the 2 lymphocyte populations in each
micrograph. Original magnification 203.
Immunohistochemical characterization of infiltrating lympho-
CARDS Toxin Synthesis in Murine Model
d JID 2011:204 (15 November)
and autoantibodies [38, 39]. B cells, CD41T cells, and plasma
cells infiltrate the lungs, which is followed by further augmenta-
tion of the immune response, namely, abundance of lymphocytes,
production of immunoglobulins, and release of proinflammatory
cytokines [40, 41]. We observed considerable numbers of CD41
and CD191cells around the site of inflammation (Figure 4).
Previous studies have shown that the percentage of CD41and
CD191cells varies during M. pneumoniae infection. It was sug-
gested that the redistribution of CD41T cells to the site of in-
fection may explain the decreased proportion of these cells in the
blood [40, 42, 43]. However, further studies are needed to de-
termine how M. pneumoniae and CARDS toxin alter the immune
system and influence the concentration and distribution of CD4
and CD19 cells.
While we observed peribronchial and perivascular infiltration
in lungs of M. pneumoniae–infected mice, even at day 35, we
speculate that mycoplasmas persist and continue to synthesize
low levels of CARDS toxin, resulting in chronic infection and
inflammation that lead to other acute and chronic airway dis-
eases, like asthma, and extrapulmonary manifestations. In hu-
mans, M. pneumoniae is reported to persist in the respiratory
tract for many months, even after therapy with appropriate
antibiotics [44–48]. We have observed similar persistence of
M. pneumoniae in the mouse model and in mammalian cell
lines after prolonged antibiotic treatment [24, 49]. It will be
important to investigate the chronicity of M. pneumoniae
infection in our mouse model, along with CARDS toxin ex-
pression and stability, especially since chronic infections have
been postulated to lead to a range of pathologies accompanied
by dysfunctional changes and tissue remodeling in the re-
spiratory tract . Clearly, M. pneumoniae cells and CARDS
toxin localize to the ciliated respiratory epithelium (Figure 5)
and are also associated with lung macrophages (Figure 6). The
binding of CARDS toxin and mycoplasma cells selectively to
M. pneumoniae, lung samples were prepared as described in Methods and treated with rabbit anti–M. pneumoniae (1:1500 dilution) and affinity-purified
rabbit anti–rCARDS toxin (1:1000 dilution) antisera followed by Vector ABC and diaminobenzidine chromogen. A, Sterile SP4 broth–inoculated control
mouse lung with anti–M. pneumoniae antibodies. B, M. pneumoniae–infected mouse lung with anti–M. pneumoniae antibodies. C, Sterile SP4–
inoculated mouse lung with anti–rCARDS toxin antibodies. D, M. pneumoniae–infected mouse lung with anti–rCARDS toxin antibodies. Arrows indicate
immunostained aggregates of mycoplasma organisms (B) and CARDS toxin (D) that are intermeshed with remaining cilia along the surface of epithelium.
Original magnification 603.
Localization of M. pneumoniae and CARDS toxin in the ciliated bronchiolar epithelial region of mouse lung at day 4 postinfection. To localize
contain alveolar macrophages and neutrophils, both of which show immunostained intracytoplasmic aggregates. A, Although more background staining
is evident in the cytoplasm of lung cells treated with anti–M. pneumoniae rabbit antiserum, the positively labeled cytoplasmic aggregates are similar in
size and distribution with those more easily visualized in the cytoplasm of comparable cell types in the CARDS toxin antibody-stained preparation (B).
Samples were processed as described in Figure 5. Original magnification 603.
Localization of M. pneumoniae cells and CARDS toxin in the alveolar spaces of infected lungs at day 4 postinfection. Cellular exudates
d JID 2011:204 (15 November)
d Kannan et al
surfactant protein A probably facilitates these interactions
Seroconversion against CARDS toxin in experimentally
infected mice (Figure 7) parallels what is observed during
human M. pneumoniae infections . We also noted in
M. pneumoniae–infected mice that antibody titers against
CARDS toxin are at least equivalent to the ‘‘immunodo-
minant’’ M. pneumoniae adhesin P1, suggesting that CARDS
toxin may be used alone, or along with P1, for diagnostic and
prognostic applications. Also, these observations suggest that
CARDS toxin could serve as an effective vaccine candidate.
In summary, our studies provide unequivocal evidence that
CARDS toxin is synthesized during experimental infection,
which directly correlates with mycoplasma replication and
persistence, airway inflammation, and lung histopathology
[29, 30, 35]. We also demonstrate that CARDS toxin expression
colocalizes with mycoplasma cells that colonize respiratory ep-
ithelial cell surfaces. Further, we present additional evidence for
the immunodominant and diagnostic properties of CARDS
toxin. Other studies are in progress to advance our understand-
ing of the mechanisms that govern CARDS toxin expression and
its mode of action in vivo, which should aid in the development
of preventative therapies for the spectrum of acute and chronic
airway and extrapulmonary diseases associated with this com-
mon respiratory bacterial pathogen.
of the Department of Pathology for their technical assistance and Rose
Garza for her assistance in assembling the manuscript.
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institute of Allergy
and Infectious Diseases or the National Institutes of Health.
We thank Vicki T. Winter and Shellye R. Lampkin
of Allergy and Infectious Diseases, National Institutes of Health
(U19AI070412); and the Kleberg Foundation.
Potential conflicts of interest.All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
This work was supported by the National Institute
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