Progression of primary pneumonic plague:
A mouse model of infection, pathology,
and bacterial transcriptional activity
Wyndham W. Lathem*, Seth D. Crosby†, Virginia L. Miller*‡, and William E. Goldman*§
Departments of *Molecular Microbiology and‡Pediatrics and†Genome Sequencing Center, Washington University School of Medicine,
660 South Euclid Avenue, St. Louis, MO 63110
Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved October 14, 2005 (received for review August 8, 2005)
Although pneumonic plague is the deadliest manifestation of
disease caused by the bacterium Yersinia pestis, there is surpris-
ingly little information on the cellular and molecular mechanisms
responsible for Y. pestis-triggered pathology in the lung. There-
fore, to understand the progression of this unique disease, we
characterized an intranasal mouse model of primary pneumonic
plague. Mice succumbed to a purulent multifocal severe exudative
bronchopneumonia that closely resembles the disease observed in
humans. Analyses revealed a strikingly biphasic syndrome, in
which the infection begins with an antiinflammatory state in the
first 24–36 h that rapidly progresses to a highly proinflammatory
state by 48 h and death by 3 days. To assess the adaptation of Y.
pestis to a mammalian environment, we used DNA microarray
technology to analyze the transcriptional responses of the bacteria
during interaction with the mouse lung. Included among the genes
up-regulated in vivo are those comprising the yop-ysc type III
secretion system and genes contained within the chromosomal
pigmentation locus, validating the use of this technology to iden-
tify loci essential to the virulence of Y. pestis.
inflammation ? microarray ? Yersinia pestis
pared with the bubonic form of plague, which is acquired by skin
penetration, primary pneumonic plague is highly contagious and
almost always fatal. The current worldwide incidence of plague is
low by historical standards, but the possible combination of wide-
spread aerosol dissemination and rapid disease progression are of
particular concern for defense against bioterrorism (1).
In cases of primary pneumonic plague in humans, microscopic
examination of lung tissue reveals multiple histological patterns,
including acute pneumonia, intraalveolar hemorrhage and edema,
and the presence of extracellular bacteria in the alveoli but not the
interstitium (2). In addition, extensive neutrophilic infiltrate and
loss of recognizable alveolar architecture results from the infection
(3, 4). Studies of experimental primary pneumonic plague in
monkeys, mice, and guinea pigs showed similar pathologic effects,
including extensive intraalveolar edema, massive bacterial prolif-
as are the bacterial responses to this dramatically changing host
environment. DNA microarray technology has been widely used to
assess transcriptional changes in bacterial gene expression in vitro,
but analyses of the bacterial transcriptome during host infection
nonspecific hybridization signals on the microarray, and the often-
would otherwise require large numbers of animals for analysis.
Progress has been made by multiple groups to overcome these
problems (10–12), although different pathogens, animal hosts, and
neumonic plague is the deadliest manifestation of disease
caused by the bacterium Yersinia pestis. Although rare com-
routes of infection necessitate empirically derived adaptations for
each study. Nonetheless, advances in microarray technology will
allow for more complete analyses of animal models of infection.
Although recent studies have examined the end result of pneu-
isolated from a fatal case of human pneumonic plague (3), in
combination with an intranasal inbred mouse model of infection to
more fully characterize the interaction between the bacterium and
the host during primary pneumonic plague. In addition, we present
compared with bacteria grown in vitro by use of DNA microarray
Materials and Methods
Bacterial Strains and Culture Conditions. The virulent wild-type Y.
pestis strain CO92 and its plasmid-cured derivative CO92 pCD1?
KIM6(pCD1Ap)? (13) was a kind gift from Robert Perry (Uni-
pMT1, pPCP1, and the pgm locus was confirmed by PCR for each
strain. Y. pestis was routinely grown on brain–heart infusion (BHI)
agar (Difco) at 26°C for 2–3 days. For liquid cultures, Y. pestis was
grown in BHI broth at 26°C for 6–8 h in a roller drum before being
diluted to an OD620of 0.05–0.1 in 10 ml of BHI broth with 2.5 mM
CaCl2in a 125-ml Erlenmeyer flask. Unless otherwise indicated,
bacteria were incubated at 37°C in a water bath shaker set at 250
rpm for 16–18 h.
Animals. Animal experiments were approved by the Washington
University Animal Studies Committee, protocol no. 20020257.
Pathogen-free 6- to 8-week-old female C57BL?6 mice were ob-
tained from The Jackson Laboratory and were housed in high-
efficiency particulate air-filtered barrier units kept inside biological
food and water ad libitum and were kept at 25°C with alternating
12-h periods of light and dark. Bacteria were grown in BHI broth,
washed in sterile PBS, and maintained at 37°C. Mice were lightly
anesthetized and inoculated by the intranasal route with 20 ?l of Y.
pestis in PBS. Numbers of colony-forming units (cfu) inoculated
an overdose of pentobarbital sodium (150 mg?kg).
LD50, Kinetics, and Survival Curves. Groups of four to five mice were
infected intranasally with serial 10-fold dilutions of Y. pestis strain
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: BHI, brain–heart infusion; cfu, colony-forming units; qRT-PCR, quantitative
RT-PCR; BAL, broncho-alveolar lavage; TTSS, type III secretion system.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
December 6, 2005 ?
vol. 102 ?
CO92, ranging from 10 to 1 ? 105cfu. Mice were monitored twice
daily for 7 days, and any surviving mice were killed with pentobar-
bital sodium (150 mg?kg). For experiments examining the kinetics
of infection, groups of five mice were infected intranasally with 1 ?
104cfu of Y. pestis strains CO92 or CO92 pCD1?. At various times
postinfection, mice were killed, the lungs and spleens surgically
removed and homogenized in 0.5 ml of sterile PBS, and serial
Histopathology. Groups of five mice were infected intranasally with
1 ? 104cfu of Y. pestis strain CO92. Uninfected mice and mice
infected for 24, 48, or 72 h were killed with pentobarbital sodium
(150 mg?kg) and their lungs inflated with 10% neutral buffered
formalin via cannulation of the trachea. Lungs and spleens were
removed and fixed in 10% formalin overnight before being em-
bedded in paraffin. Five-micrometer sections of tissue were stained
either with hematoxylin?eosin or by Steiner (silver) stain before
Cytokine Analysis. Levels of IL-10, IL-6, Il-12p70, TNF, IFN-?, and
monocyte chemoattractant protein (MCP-1) present in mouse
were infected intranasally with 1 ? 104cfu of Y. pestis strain CO92.
Lungs from uninfected mice and mice infected for 24, 48, or 72 h
were removed and homogenized in 0.5 ml of ice-cold PBS con-
taining protease inhibitors (Roche Diagnostics). Homogenates
by the manufacturer. Cytokine concentrations were determined by
using the cytometric BEAD ARRAY ANALYSIS software, Ver. 1.1.
RNA Isolation, T7-Based RNA Amplification, and Microarray Analysis.
A Y. pestis gene-specific microarray, consisting of 70-mer oligonu-
cleotides representing 100% of the ORFs of strain CO92, was
constructed (Supporting Text, which is published as supporting
information on the PNAS web site). Two independent identical
experiments were conducted as follows: 10 mice were infected with
Y. pestis strain CO92 and after 48 h, mice were killed, lungs were
lavaged with PBS, and the resulting material was pooled. RNA was
extracted from lavage-derived bacteria and from a culture of CO92
Text). To create technical replicates for each biological replicate,
RNAs were divided into two samples. These samples were inde-
pendently amplified, labeled, and hybridized to the arrays in such
a way that each of the two lavage samples was paired with each of
the two culture-derived samples. Thus four technical replicates
were tested, and each was dye-swapped for a total of eight DNA
microarrays that were used for each experiment (Supporting Text).
Slides were scanned immediately after hybridization and analyzed
with GENESPRING (Agilent Technologies, Palo Alto, CA). Within
each experiment genes that had signal-to-control or control-to-
signal ratios ?2.0 (P ? 0.05, Bonferroni Step-Down Multiple
Testing correction) in either the high- or low-photomultiplier scans
were considered to be significantly regulated (Supporting Text).
Only genes that were significantly regulated in all the technical
as supporting information on the PNAS web site).
Microarray Validation. Arandomlyselectedgroupofgenes(n?27)
that were differentially regulated as determined by microarray
hybridization were analyzed by quantitative RT-PCR (qRT-PCR)
(Supporting Text). Array data were considered valid if the fold
change of each gene tested by qRT-PCR was ?2.0 and in the same
direction as determined by microarray analysis.
Survival of C57BL?6 Mice Infected Intranasally with Y. pestis. The
progression of primary pneumonic plague in humans and other
animals is rapid; symptoms develop 24–48 h after exposure,
followed by death within 3–4 days (6, 14). To better understand the
course of primary pneumonic plague in the mouse model, we
inoculated C57BL?6 mice by the intranasal route with increasing
doses of Y. pestis from 10 to 1 ? 105cfu and monitored the
development of symptoms and survival of the animals. After 24 h,
signs of symptoms. By 36 h, however, mice receiving the highest
breathing rapidly, and had little reaction to being handled. By 48 h,
mice receiving 1 ? 104cfu exhibited similar symptoms, and by 60 h,
mice began to die from the infection (Fig. 4, which is published as
supporting information on the PNAS web site). By 3.5 days, 100%
of the mice receiving 105or 104cfu of Y. pestis had died, whereas
to succumb up to 4.5 days after inoculation, after which all
remaining mice survived. As expected, the percentage of surviving
mice depended on the dose of the inoculation, and there was a
direct correlation between the development of symptoms and the
progression to death (no mice that exhibited symptoms survived).
The LD50of Y. pestis strain CO92 administered intranasally for two
experiments was calculated as 2.8 ? 102and 2.4 ? 102cfu,
respectively. Mice infected with another strain of Y. pestis,
KIM6(pCD1Ap)?, showed similar results to CO92 in this model
the lowest dose tested that consistently gave a 100% mortality rate,
1 ? 104cfu.
We assessed the kinetics of Y. pestis CO92 infection after intranasal
inoculation by calculating the cfu present in the lungs and spleen at
or 10% of the bacteria, were recovered from the lungs of the mice
in other animal models (5, 15). Bacterial load proceeded to rise
rapidly, reaching 1010by day 3, with the greatest increase occurring
appeared in the spleens (Fig. 1B), indicative of dissemination from
the lungs into the peripheral tissues. Bacterial load in the spleens
also continued to increase with time, approaching 109cfu by 72 h.
In contrast, an isogenic strain of CO92 lacking the pCD1 plasmid
was unable to cause a lethal infection, was cleared from the lungs,
and was not detected in the spleen (Fig. 1 C and D).
Histopathology of Mouse Lungs Infected with Y. pestis. To compare
the effects of primary pneumonic plague on experimentally in-
in pathology of the lungs of C57BL?6 mice at various times after
infection with Y. pestis (Fig. 2). After 24 h, lungs looked unremark-
able compared with uninfected tissue. We observed normal
amounts of alveolar macrophages, although some appeared slightly
however, changes in the lung architecture and types of cells present
were visible compared with uninfected lungs. Although the larger
bronchi remained relatively unaffected, the smaller bronchi ap-
outward into the alveoli. Foci of neutrophils loosely packing the
a corresponding decrease in the number of alveolar macrophages
compared with 24 h. Some slight hemorrhage was also evident,
whereas other sections of lungs remained unremarkable, lacking
any visible damage or inflammatory effects. At 72 h postinocula-
bronchopneumonia. Entire lobes of the lungs appeared consoli-
Lathem et al.
December 6, 2005 ?
vol. 102 ?
no. 49 ?
some of the larger bronchi were still visible. Along with extensive
hemorrhage, large numbers of neutrophils were tightly packed
within what remained of the alveolar structure.
Localization of Y. pestis Within Infected Mouse Lungs.Inlungsections
from individuals who died of primary pneumonic plague, Y. pestis
is found mostly in the small airways and alveoli as extracellular
bacilli (16, 17). To determine the localization of Y. pestis after the
intranasal infection of mice, we used the Steiner silver stain to
visualize bacteria in the lungs at various times during the infection.
Consistent with the absence of pathological changes in the lungs at
24 h, we were unable to observe bacteria in the tissue at this point
during the infection (Fig. 5, which is published as supporting
clearly visible within the alveoli and small bronchioles, primarily
centered within and around the neutrophilic infiltrate. Overall, the
cases rings of bacteria appeared to encircle blood vessels. We did
not observe bacteria within the interstitium of the alveoli. At 72 h
after infection, large areas of the lungs were filled with masses of
darkly staining material, in which individual bacilli could not be
distinguished. These are likely large microcolonies of bacteria,
although the presence of bacterial debris could not be excluded. In
some sections, individual extracellular bacteria were visible. Al-
though the larger bronchi appeared hyperplastic, they remained
relatively free of bacteria.
Cytokine Production During Experimental Pneumonic Plague. The
production of pro- and antiinflammatory cytokines by immuno-
modulatory cells is a crucial component of the host response to
control bacterial infection. The pCD1-based type III secretion
system (TTSS) of Y. pestis, however, is thought to subvert the
immune response in part by altering the types and levels of
cytokines produced during plague infection. A survey of the
inflammatory cytokines produced in the lungs in response to Y.
pestis, particularly during primary pneumonic plague, has not been
reported. Therefore, we measured the levels of the cytokines
IL-12p70, TNF, IFN-?, monocyte chemoattractant protein (MCP-
1), IL-10, and IL-6 in lung homogenates of mice at various times
during infection with Y. pestis (Table 1). After 24 h, modest levels
of most cytokines tested were detected, although in the majority of
cases the amounts were not significantly different from uninfected
mice. The levels of most cytokines rose significantly by 48 h,
however, and continued to increase by day 3, particularly those of
the infection with the exception of one mouse. To confirm the lack
of IL-10 production during infection, we compared the mRNA
levels of this cytokine in RNA extracted from infected lung tissue
at 24 and 48 h with that present in uninfected lungs by qRT-PCR.
We did not detect any significant induction of IL-10 by this method
(data not shown).
In Vivo Transcriptional Profile of Y. pestis During Primary Pneumonic
Plague. The transmission of Y. pestis by an arthropod vector is
unique among the Enterobacteriaceae. As such, adaptation of the
bacterium from a flea to a mammalian host includes responses to
The transitions encountered in primary pneumonic plague are
quite different, because this represents a mammal-to-mammal
respiratory transition; the organisms are already adapted to 37°C,
and the first cells they encounter are those lining the airway. Thus,
an analysis of the global transcriptional responses of Y. pestis to a
how the bacterium causes pneumonic plague. To this end, we used
during experimental primary pneumonic plague using the intrana-
sal mouse model of infection.
We designed oligonucleotide-based arrays representing 100% of
the predicted ORFs of Y. pestis strain CO92 (18) and included
bacterial samples with eukaryotic RNA during host–pathogen
interactions, particularly in vivo, can interfere with microarray
hybridization and lead to false-positive signals (9). Therefore,
because of the observation that Y. pestis infection of lung tissue is
primarily extracellular, we compared the recovery of bacilli from
infected mouse lungs by bronchoalveolar lavage (BAL) with that
were either homogenized or lavaged with 3 ? 1 ml of PBS, and cfu
strain CO92 or CO92 pCD1?. Bacteria (1 ?
104) were introduced intranasally into
(A and C) and spleen (B and D) were deter-
mined every 12 h for 3 days (CO92, A and B)
or every 24 h for 4 days (CO92 pCD1?, C and
D). The limit of detection is indicated by a
dashed line. Each point indicates cfu recov-
ered from a single animal. Symbols below
the limit of detection represent mice that
survived but did not have detectable num-
cumbed to the infection. A solid line indi-
cates the median of cfu recovered.
Kinetics of infection with Y. pestis
www.pnas.org?cgi?doi?10.1073?pnas.0506840102Lathem et al.
were determined. There was no significant difference in the num-
bers of bacteria recovered by either method (Fig. 6A, which is
published as supporting information on the PNAS web site). RNA
levels of eukaryotic rRNA compared with those present from
homogenization, 10:1 bacterial to eukaryotic rRNA by BAL) (Fig.
6B). Therefore, RNA extracted from BAL fluid 48 h postinfection
was used for the microarray analysis.
RNA was isolated from BAL fluid recovered from two inde-
pendent replicates of intranasal infections of 10 C57BL?6 mice
each. Two sets of control RNA for array cohybridizations were
extracted from bacteria grown for 12 h at 37°C in BHI broth
containing 2.5 mM CaCl2, similar to conditions used to prepare Y.
pestis for the animal inoculations. To increase the total amount of
material available for hybridizations and downstream analyses,
RNA was linearly amplified (Supporting Text). This procedure
yielded, on average, ?50–75 ?g of antisense RNA from 0.5 ?g of
Of the 4,037 Y. pestis ORFs represented by oligonucleotides on
the DNA microarray, 405, or 10% of the genome, were differen-
tially regulated in the lungs of mice at 48 h (Table 2). Of these, 349
ORFs are encoded on the chromosome, whereas the remaining 56
are located on the three plasmids carried by Y. pestis strain CO92
(48 on pCD1; 4 on pMT1; 4 on pPCP1) (Fig. 3). Two hundred
thirty-four ORFs were up-regulated, and 171 ORFs were down-
regulated in vivo compared with bacteria grown in BHI broth at
37°C. Based on the functional classification of genes annotated by
the Sanger Institute (www.sanger.ac.uk?Projects?Y?pestis), the
greatest number of transcriptional changes occurred in genes
24 down-regulated), genes encoding transport and?or binding
proteins (17% of the total; 42 up-regulated, 27 down-regulated),
and those with unknown function (15% of the total, 24 up-
regulated, 37 down-regulated) (Table 3, which is published as
supporting information on the PNAS web site). Additionally, 27
genes involved in amino acid biosynthesis were up-regulated,
whereas only four were down-regulated in vivo. Among those
up-regulated were genes associated with the histidine, pyruvate,
glutamate, and aspartate families of amino acid biosynthesis. Also
of note is the down-regulation in vivo of genes involved with small
molecule degradation and energy metabolism, including those
associated with the tricarboxylic acid cycle and ATP-proton motive
Multiple virulence-associated loci were also differentially regu-
lated during the infection. Of the 234 Y. pestis ORFs up-regulated
in vivo, 33 genes, or 14% of the total, are associated with the
pCD1-based TTSS, essential to the virulence of Y. pestis. Both the
iron acquisition system yersiniabactin (ybtETPQXS, irp1, irp2, and
psn) and the hemin uptake operon (hmsHFRS), encoded by genes
contained within the 102-kb pgm locus necessary for virulence via
peripheral routes of infection (14), were up-regulated in the lungs
of infected mice. Interestingly, the hms operon is thought to be
posttranscriptionally regulated (19), although there may be envi-
ronmental signals that affect the transcription of this system in vivo.
Other virulence determinants, including the psa operon encoding
the pH 6.0 antigen fimbrial structure (psaABEF) and the plasmin-
ogen activator protease Pla, were down-regulated in vivo at 48 h
to be expressed during intracellular association with macrophages
(20) and is transcriptionally up-regulated in vitro at 37°C under low
pH, whereas Pla is essential for the dissemination of the plague
bacillus from peripheral sites of infection but unnecessary for
operon to pneumonic plague are unknown or unclear (22, 23), and
infection is warranted.
Genes involved in the detoxification of reactive oxygen species,
including katA, katY, and sodB, were also down-regulated in the
lungs, suggesting that Y. pestis may have reduced exposure to these
molecules at this point during the infection. Indeed, multiple genes
involved in the stress response were differentially regulated in vivo,
including the cold-shock responsive genes cspA1 and cspA2, both
homologues of cspB (YPO1398 and YPO2659), cspD, the global
stress responsive gene gsrA?htrA, and the flavohemoprotein gene
hmp. As expected, the F1 capsule, encoded by the caf operon
mouse lung, because capsule production is induced at 37°C, the
Lungs were inflated and fixed with 10% formalin, and 5-?m sections were
stained with hematoxylin?eosin. Lungs were examined from uninfected mice or
mice infected with 1 ? 104cfu Y. pestis for 24, 48, or 72 h. (Scale bars, 50 ?m.)
Histology of lungs after intranasal infection with Y. pestis strain CO92.
Table 1. Cytokine levels in lung homogenates
Mean cytokine level (pg?g of tissue) ? SEM at
0 h 24 h 48 h72 h
52 ? 5
31 ? 3
246 ? 28
40 ? 11
47 ? 5
46 ? 7
57 ? 30
128 ? 15
505 ? 293
137 ? 34
1031 ? 251
3715 ? 977
1099 ? 350
21,484 ? 5786
170 ? 23
1474 ? 174
2871 ? 462
22,056 ? 10,939
1 ? 1
126,132 ? 7015
ND, not detected.
Lathem et al.
December 6, 2005 ?
vol. 102 ?
no. 49 ?
Confirmation of Microarray Data by qRT-PCR. The validity of the
microarray results was assessed by qRT-PCR. The fold expression
change of 27 randomly selected, differentially regulated genes was
determined from the same antisense RNA samples used for
microarray hybridization. Overall, the change in expression of 89%
of the genes tested by qRT-PCR was in agreement with the
direction of fold change as determined by microarray analysis (Fig.
7, which is published as supporting information on the PNAS web
Although the incidence of plague has decreased substantially over
the last century, that Y. pestis infects humans via the inhalation of
aerosolized particles and causes the rapid and devastating disease
of the bacterium as a bioterror agent. In comparison with the more
common bubonic form of the disease, however, the molecular and
cellular mechanisms that are responsible for primary pneumonic
here provides information on the progression of primary pneumo-
in an inbred mouse model of infection using current technologies.
The pulmonary disease we observed in the C57BL?6 mouse
strain is remarkably similar to that seen in humans in both pro-
gression and severity. In the first 24 h after infection, mice remain
asymptomatic, although the bacterial load in the lungs increased
?10-fold every 12 h. Interestingly, early studies of pneumonic
plague in mice showed a reproducible decrease in cfu in the lungs
until ?24 h after inoculation (8, 24). This discrepancy with our
results may be caused by multiple factors, including differences in
the route of infection (aerosol vs. intranasal), strain of Y. pestis
(139L or EV-76 vs. CO92), strain of mouse (male Namru albino vs.
female C57BL?6), or bacterial growth condition for preparation of
the inoculum (room temperature vs. 37°C). Indeed, the tempera-
ture-dependent up-regulation of multiple antiphagocytic factors at
37°C may be the most likely explanation for these differences in the
kinetics of infection, because the bacteria prepared under our
conditions more closely mimics those in the mammal-to-mammal
transmission during primary pneumonic plague (37–37°C) and are
therefore primed to prevent phagocytosis.
The asymptomatic state of the infection in the first 24 h corre-
sponds with the absence of histological changes in the lungs or
dramatic alterations in inflammatory cytokine levels. The relative
quiescence at this point is likely because of the potent antiinflam-
matory activity of the pCD1-based TTSS, particularly against
alveolar macrophages. Indeed, Nakajima and Brubaker (25) ob-
served robust TNF and IFN-? production in the spleens of pCD1?
infected with pCD1?bacteria. The relatively high intranasal LD50
compared with the LD50via the s.c. route (one bacillus), however,
suggests that significant impediments within the respiratory tract,
whether immune response, mechanical interference, and?or clear-
that the mere introduction of the bacteria into the airway is not
sufficient to establish a lethal infection.
Although primary pneumonic plague results in pulmonary col-
lapse, dissemination of Y. pestis from the lungs to other tissues and
36 h after intranasal inoculation, we detected viable bacteria in the
spleens of infected mice, indicating that Y. pestis had escaped the
a 3-log increase in Y. pestis cfu in the lungs between 24 and 36 h
postinfection. The presence of bacteria surrounding blood vessels
within the lungs at 48 h suggests that the dramatic rise in cfu may
be caused by the recirculation of bloodborne Y. pestis back into the
lungs, creating additional foci of infection within previously un-
colonized areas. Thus, what we characterize as primary pneumonic
plague at this stage of the disease may in fact be a combination of
primary and secondary foci of infection.
By 48 h, the development of visible symptoms of pneumonic
plague corresponded with a shift to a more inflammatory state in
the lungs, in contrast to the relatively pre- or antiinflammatory
condition just 1 day earlier. Numerous extracellular bacilli in the
alveoli and small airways were often concomitant with an inflam-
matory infiltrate consisting primarily of neutrophils. Likewise, we
measured a significant rise in multiple proinflammatory cytokines
and chemokines at this time, including both TNF and IFN-?,
suggesting a vigorous (yet ultimately unsuccessful) host response to
the infection. Although we detected few intracellular bacteria
within neutrophils, the migration and activation of these immune
mary pneumonic plague. Genes differen-
tially regulated (48-h lungs vs. 37°C BHI
broth) 2-fold or more in both RNA amplifi-
cations were divided into 20 categories
based on the CO92 chromosomal annota-
tion; genes differentially regulated on the
three plasmids are indicated separately.
Functional classification of differ-
www.pnas.org?cgi?doi?10.1073?pnas.0506840102 Lathem et al.
cells, including the release of reactive nitrogen species and neutro-
phil elastase, may cause considerable damage to the host and may
and the resulting edema is likely to cause a significant impediment
in oxygen exchange.
The development of a proinflammatory state within the lungs of
Y. pestis-infected mice prompted us to begin studies to explore the
As an initial step toward this long-term goal, we chose to analyze
the transcriptional profile of the plague bacillus in the lungs of
obstacles to using DNA-based microarrays for the analysis of
bacterial transcription in vivo are the potential for excess amounts
of eukaryotic RNA to be copurified during the isolation procedure
the relatively low levels of bacterial RNA isolated from host tissue,
particularly early in the infection. We addressed the first problem
by isolating bacteria from the mouse lung by BAL, which was
essentially equivalent to homogenization of the tissue in terms of
bacterial recovery yet reduced the contamination of eukaryotic
RNA to ?10% of the total RNA purified. It is important to note,
however, that although similar numbers of bacteria were recovered
that remains in the lungs and is unrecoverable by BAL, thereby
altering the total transcriptional profile of the recovered bacteria
slightly. The second obstacle was overcome by in vitro transcription
(IVT)-mediated linear amplification of the purified RNA, a com-
monly used technique for the amplification of eukaryotic RNA
the enzyme poly(A) polymerase and then reverse-transcribing the
population with oligo(dT) primers containing a T7 RNA polymer-
ase promoter. Thus, the RNA extracted from Y. pestis isolated by
sufficient RNA from 10 mice per biological replicate for multiple
array hybridizations. In addition, random assessment of the differ-
ential expression of 27 genes by qRT-PCR showed 89% concor-
dance with the microarray data, validating the use of BAL in this
system to recover bacterial RNA relatively free of eukaryotic RNA
that might nonspecifically hybridize with the arrays.
The remarkable capacity of Y. pestis to replicate essentially
unchecked by the host immune response is attributed to the
antiphagocytic and antiinflammatory effects of the TTSS encoded
on pCD1. This system is expressed in vitro at 37°C in the absence
of calcium, and effector molecules are released into the culture
the system is ‘‘primed’’ in that the yop-ysc regulon is up-regulated
of the transcriptional response of Y. pestis grown in BHI medium
TTSS genes compared with 26°C (unpublished data). Our DNA
microarray analysis of bacteria grown in vivo, however, revealed
additional up-regulation of the pCD1-based TTSS when compared
contact, that stimulate a positive feedback mechanism (30). Inter-
estingly, lcrF, a transcriptional activator that controls expression of
the TTSS (31), was down-regulated 3-fold, suggesting that the
modulation of the system in vivo may be more sophisticated than
previously appreciated. Nonetheless, the observation that a major-
ity of these genes are up-regulated during pneumonic plague as
determined by DNA microarray analysis provides biologic valida-
delay the host inflammatory response and later contribute to the
survival of the bacteria in a highly proinflammatory environment.
Y. pestis causes a purulent multifocal severe exudative broncho-
pneumonia in mice when administered via the intranasal route.
Much like in humans, the disease is rapid and severe, resulting in
death of the animal within 3–4 days. Although bacterial prolifer-
ation is rapid, the initial stage of the infection is relatively quiescent
and antiinflammatory in nature. This develops rapidly into a highly
proinflammatory state as cytokine and chemokine levels rise,
dissemination of the bacterium to the spleen occurs, and mice
to measure the transcriptional profile of Y. pestis during infection
has provided a valuable tool to assess the global response of the
bacterium during the disease. Thus, this model system should be
useful for the continued study of primary pneumonic plague as a
distinct disease of mammals caused by Y. pestis, providing perspec-
tives from both the host and the bacterium.
We thank Drs. Craig Rubens and Marie LaRegina for their helpful
comments, Michael Heinz and Christopher Sawyer of the Washington
University Genome Sequencing Center Microarray Core for the mi-
croarray hybridizations, and the Washington University Digestive Dis-
eases Research Core (DDRCC) Facility for the histological sections.
This work was funded by National Institutes of Health (NIH) Grant U54
AI057160 to the Midwest Regional Center of Excellence for Biodefense
and Emerging Infectious Diseases Research (MRCE) and by NIH Grant
AI53298. The DDRCC core is supported by NIH Grant DK52574.
W.W.L. was supported by the Infectious Diseases Training Grant to
Washington University and by the Clinical?Translational Fellowship
Program of the MRCE.
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