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Infection and Immunity, January 2001, p. 97-107, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.97-107.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Time between Inoculations and Karyotype Forms of
Pneumocystis carinii f. sp. carinii Influence
Outcome of Experimental Coinfections in Rats
Melanie T.
Cushion,1,2,*
Sally
Orr,1,2
Scott P.
Keely,3 and
James R.
Stringer3
Department of Internal Medicine, Division of
Infectious Diseases,1 and Department of
Molecular Genetics, Biochemistry, and
Microbiology,3 University of Cincinnati College
of Medicine, and the Cincinnati VA Medical
Center,2 Cincinnati, Ohio
Received 11 April 2000/Returned for modification 4 July
2000/Accepted 27 September 2000
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ABSTRACT |
The prevalence of Pneumocystis carinii pneumonia (PCP)
in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary
rather than recurrent disease. To better understand the factors
influencing the development of coinfections, the time periods between
inoculations and the genotype of the infecting organisms were evaluated
in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats
differentiated by karyotypic profiles exhibit the same low level of
genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual
combinations, spaced 0, 10, and 20 days apart. Infections comprised of
both organism forms resulted from admixtures inoculated at the same
time. In contrast, coinfections did not develop in most rats, where a
10- or 20-day gap was inserted between inoculations; only the first
organism form inoculated was detected by pulsed-field gel
electrophoresis in the resultant infection. Organism burdens were
reduced with combinations of forms 1 and 2 spaced 20 days apart but not
in rats inoculated with forms 1 and 6. A role for the host response in
the elimination of the second population and in reduction of the
organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by
autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were
associated with differences in biological responses. This model
provides a useful method for the study of P. carinii
coinfections and their transmission in humans.
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INTRODUCTION |
Organisms termed Pneumocystis
carinii were placed in the Fungal kingdom more than 10 years ago
on the basis of gene sequence comparisons (14, 29), yet
much of their basic biological processes remains poorly understood, due
in large part to their poor growth outside the mammalian lung
environment (11). Recent genetic comparisons
(32), as well as animal inoculation studies
(16) and antigenic analyses (2, 15), provide
strong evidence that "P. carinii" is actually a family
of organisms that, although related, exhibit species specificity among
its mammalian hosts.
Comparisons at five genetic loci suggest three levels of genetic
divergence are present among P. carinii organism populations (30). The highest level of divergence, class III, was
observed among Pneumocystis populations isolated from
different mammalian hosts and ranged from 5 to 50% divergence in
nucleotide sequence at these selected loci, with the internal
transcribed sequence (ITS) regions being most divergent. Class II
divergence ranged from 4 to 7% for all genes, with a 20 to 30%
difference at the ITS regions. Organisms demonstrating class II
divergence have been found to infect the same mammalian host but, only
two mammalian hosts, the ferret and the rat, have been shown to harbor
such divergent populations to date (12). Class I sequences
differed by 0 to 0.8% in the four gene sequences and by 2 to 4% in
the ITS regions. This low level of divergence has been described for all P. carinii f. sp. hominis populations found
in human beings and among several subpopulations of P. carinii f. sp. carinii infecting rats ((12,
30, 32); see also below).
The genetic divergence observed for Pneumocystis organisms
within each of the class III and class II levels is equivalent to those
differences seen among bona fide species of other microbes (29). Based on these genotypic differences and phenotypic
differences (2, 15, 16), the community of
Pneumocystis investigators voted to move toward a
standardized system of nomenclature recognizing these differences
(28). The nomenclature follows the recommendations of the
Botanical Code for special physiological forms of fungi and uses a
tripartite naming convention (21). Thus, for example, the
term P. carinii f. sp. hominis specifies
organisms from human beings, the term P. carinii f. sp.
mustelae specifies organisms from ferrets, and the term
P. carinii f. sp. mus specifies organisms from
mice. This convention is followed in the present publication.
P. carinii f. sp. carinii and P. carinii f. sp. ratti are both found in rats.
Nevertheless, the two special forms exhibit class II genetic divergence
and markedly different serologic responses to anti-P.
carinii monoclonal antibodies and polyclonal sera (12, 31), suggesting they may be distinct species (9).
Previous karyotyping studies found that these two populations could
either coexist within the same rat lung or exist as apparent individual infections, although infection by P. carinii f. sp.
ratti alone was far less frequent than infection by P. carinii f. sp. carinii (8, 9).
Subpopulations of P. carinii f. sp. carinii,
defined by karyotypic profiles on pulsed-field gels, exhibit a
low-level genetic divergence (11, 23) similar to that seen
in P. carinii f. sp. hominis isolates (24,
25, 30). In the present study, we used three of these
subpopulations to investigate the influence of genetic identity on the
establishment of coinfections in immunosuppressed rats. Surveys of
several commercial rat colonies identified eight distinct populations
of P. carinii f. sp. carinii based on karyotype profiles (8, 9, 11). We have recently identified two
additional karyotype forms, forms 9 and 10, from other commercial rat
colonies (unpublished data). Forms 1, 2, and 6 were chosen for these
experiments because of their distinct karyotypic profiles, facilitating
the recognition of coinfections on pulsed-field gels.
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MATERIALS AND METHODS |
Induction of experimental P. carinii f. sp.
carinii infections.
Viral-antibody-negative CD male
rats were received in filtered containers at 140 to 160 g from
Charles River Breeding Laboratories, Hollister, Calif. Upon receipt,
the rats were immediately placed under barrier isolation consisting of
sterile polycarbonate shoebox cages with sterile bedding; the cages had
been fitted with microisolator tops placed on stainless steel racks
within a horizontal flow hood. To reduce the occurrence of infection
with other microbial pathogens, the rats received irradiated food
(TekLab Irradiated Chow; Harlan Industries, Indianapolis, Ind.) and
autoclaved water, into which a sterile solution of cephadrine (Velosef;
E. R. Squibb & Sons, Inc., Princeton, N.J.) was injected to
achieve a final concentration of 0.200 mg/ml, as described in detail
elsewhere (7, 8). After 7 days of acclimation, the rats
were started on a regimen of immunosuppression consisting of weekly
injections of methylprednisolone acetate (Depo Medrol; The Upjohn Co.,
Kalamazoo, Mich.) at 4 mg/week. After two injections of
methylprednisolone, the rats received intratracheal inoculations of
P. carinii organisms or sham inoculations as described
below. Immunosuppression was continued throughout the course of the
experiment. The time course of the experiment included the initial
2-week immunosuppression, the first inoculation, and an additional 8 weeks of immunosuppression with the second inoculations administered 10 and 20 days after the initial inoculation during this period.
Preparation of inocula.
Cryopreserved and enumerated
P. carinii f. sp. carinii preparations were used
in these studies. P. carinii f. sp. carinii populations were isolated from individual rats and frozen in RPMI 1640 supplemented with 10% fetal bovine serum (Sigma Chemical Co., St.
Louis, Mo.) and 7.5% dimethyl sulfoxide (tissue culture grade; Sigma)
as previously described (6). Populations were characterized by electrophoretic karyotyping (8, 9) and sequencing of the mitochondrial large-subunit rRNA (27)
and the nuclear small-subunit rRNA (29). Form 1 originated
from a Sprague-Dawley rat (2669, O'Fallon Colony; Sasco, Inc., St. Louis, Mo.) which had been cryopreserved for 5 months prior to usage.
Form 2 originated from a Sprague-Dawley rat (T5-967; Hilltop Laboratories, Scottsdale, Pa.) and was cryopreserved 17 months prior to
usage. Form 6 originated from a Sprague-Dawley rat (T5-999; Charles
River Breeding Laboratories [room 064]) and was cryopreserved for 17 months prior to usage. Cryovials containing the populations were
removed from liquid nitrogen, rapidly thawed in a 37°C water bath,
centrifuged, and reconstituted in 1.0 ml of prewarmed RPMI 1640 tissue
culture medium per vial. A 5-µl sample was removed for evaluation of
viability by the calcein AM-ethidium homodimer dual fluorescent
staining assay (22). Organism preparations were diluted in
RPMI 1640 supplemented with 10% fetal bovine serum to a parasite
density required for the experimental groups (see below). The fetal
bovine serum served to help stabilize the thawed organisms. It did not
participate in the reduction of organism burden observed for forms 1 and 2 after a 20-day gap or in the elimination of the second inoculated
population since it was common to all inoculations and such reductions
did not occur in all cases (e.g., forms 1 and 6) nor in the elimination
of second populations (i.e., form 1 was inoculated 10 days after form
2). The target number of organisms was delivered in a volume of 0.2 ml.
The numbers of organisms were adjusted to compensate for the loss of
viability, as determined by the dual fluorescent assay. The percentage
of organisms exhibiting bright green fluorescence without nuclear staining (live) varied between 70 and 90% in the preparations used.
After preparation, the inocula were stored on ice prior to
inoculations. Inoculations were administered after light anesthesia with halothane using a feeding cannula as described by Boylan and
Current (6).
Experimental design.
Two inoculation studies were performed
using six rats per group, which provides a power of 0.80, an 
value
of 0.05 assuming a standard deviation of 0.5, and an expected
difference of 1.0 in the organism burden (see the grading system
below). Prior to the initiation of the inoculations, all rats had
received 2 weeks of immunosuppression. In study 1, for the single
inoculation, rats were each inoculated with preparations of 5 × 107 nuclei of P. carinii f. sp.
carinii forms 1, 2, or 6. For the simultaneous-coinoculation, two groups received mixtures of 2.5 × 107 nuclei each of forms 1 and 2 or forms 1 and 6. For the
10- day or 20-day delays between inoculations, rats received an initial inocula of 2.5 × 107 nuclei of form 1, 2 or 6 and
then a second inocula of either form 1, 2, or 6 after 10 or 20 days.
After inoculation, all rats remained on immunosuppression for the
duration of the 10-week study. All rats were sacrificed at a single
terminal time point 8 weeks after the first inoculation.
A second, more-limited study was conducted to verify the form 1 and 2 results observed in the previous study, to assess the distribution of
the forms throughout the lobes of the rat lungs, and to increase the
sensitivity of detection of low organism numbers. Study 2 was conducted
as described above, except that 2.5 × 107 P. carinii f. sp. carinii were used for all inoculations.
A control group of six rats that received the full regimen of steroids,
but no
P. carinii f. sp.
carinii inoculation, was
included in each study. These rats were sacrificed at the completion
of
each study to ensure that the rats were not latently infected
with
P. carinii f. sp.
carinii (or
P. carinii f. sp.
ratti) or
did not become contaminated
during the course of the study. The
PCR-based studies (described below)
were conducted on the rat
lungs from study
2.
Preparation of P. carinii f. sp. carinii
organisms from rats.
Rats were sacrificed by carbon dioxide
inhalation, and the lungs were removed and processed for pulsed-field
gel electrophoresis (PFGE) as described in detail elsewhere (8,
9). Briefly, the lungs from individual rats were homogenized
with a laboratory blender (Stomacher 80; Tekmar, Inc., Cincinnati,
Ohio). Large particles were removed by sieving the material through
sterile gauze, and the homogenates were treated with aqueous ammonium chloride (0.85%, pH 6.8) to lyse the erythrocytes and some host cells.
Host cell numbers were further reduced by at least two passes through
10-µm-pore-size filters (Mitex; Millipore Corp., Bedford, Mass.). For
the PCR studies described below, a small portion (ca. 0.05 g) of
each of the five lobes of the lungs was excised prior to homogenization.
Estimation of organism burden and statistical analyses.
To
evaluate the organism burden in the inoculated rats, we used a
modification of semiquantitative methods used by other investigators (1, 6). Slides with touch impressions of a medial section of the left lung were stained with a rapid variant of the Giemsa stain
(LeukoStat; Fisher Scientific, Cincinnati, Ohio) and evaluated according to the following scale: 0.5+ (at least 1 organism was seen in
30 oil immersion fields and up to an average of 2.2/field, a value
corresponding to approximately 1.3 × 104 to 9.7 × 105 organisms/ml); 1+ (2.3 to 22.7 organisms/field or up
to 107 organisms/ml); 2+ (23 to 227 organisms/field; up to
108/ml); 3+ (228 to 2,268 organisms/field; up to
109 organisms/ml); and 4+ (>2,269 organisms/field;
>1010/ml). Although this system was only semiquantitative,
it could differentiate between heavy, moderate, and light infections. A one-way analysis of variance was performed on the mean and standard error of the mean from rats in each inoculation group, and the results
were evaluated for significance by the Student Newman-Keuls multiple-comparison test using the GraphPad INSTAT v.3 program. The
-value was set at 0.05; a P value of <0.05 was
considered significant.
Preparation of organisms for PFGE.
After release from the
lungs, the P. carinii f. sp. carinii organisms
were treated with DNase I (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) at 10 µg/ml in a solution of 150 mM NaCl-10 mM
MgCl2-10 mM Tris at pH 7.2 for 30 min at 37°C to digest
extracellular DNA and then washed once in 250 mM EDTA and twice in 125 mM EDTA (5, 6). Organisms were embedded in
low-melting-point agarose (Boehringer Mannheim) at a final
concentration of 0.8% in disposable plug molds (Bio-Rad, Hercules,
Calif.) or in disposable spectrophotometric cuvettes (Fisher
Scientific), depending on the organism densities. Gel-embedded
organisms were digested with 0.25 mg of proteinase K (Boehringer
Mannheim) per ml in a solution of 1% N-lauroylsarcosine (Sigma)-0.45 M EDTA-0.01 M Tris in a 55°C water bath for 24 to 48 h. Digested samples were stored at 4°C in 0.5 M EDTA.
CHEF conditions.
Gels for contour-clamped homogeneous
electrical-field (CHEF) electrophoresis contained 1% FMC SeaKem
GTG-agarose (SeaKem, Rockland, Maine) prepared in 0.5× TBE (45 mM Tris
HCl, 45 mM boric acid, 1.25 mM EDTA) for a total volume of 200 ml and
final dimensions of 14 by 21 cm. Electrophoresis was performed using a
Bio-Rad CHEF DR II or CHEF DR III apparatus. Gels were run for 104 to 144 h at 14°C in 0.5× TBE at 3.8 V/cm with a 50-s initial pulse that was gradually increased to 100 s (8, 9).
Chromosome-sized DNA bands were visualized by the nucleic acid stain,
SYBR-Gold (Molecular Probes, Inc., Eugene, Oreg.).
PCR conditions.
The PCR was performed on lung tissue samples
(~0.05 g) taken from each of the five lung lobes of rats inoculated
with forms 1 and 2 individually and in combination at the different
time periods (see above) and from uninoculated immunosuppressed rats. The lung lobes were numbered as follows: 1, left lung; 2, cranial lobe
of the right lung; 3, medial lobe of the right lung; 4, caudal lobe of
the right lung; and 5, accessory lobe. Tissue samples were prepared for
the PCR by digestion in lysis buffer (1% sodium dodecyl sulfate, 25 mg
of proteinase K per ml) at 55°C overnight and precipitation of the
resultant DNA with isopropanol at 
20°C. Primers directed to a
region upstream of the -tubulin gene p10 (5'-AAAGATGGTGAATTGTAACTC-3') and pIV (36) were
used in 25-µl reactions amplified under the following conditions: a
hot start of 95°C for 5 min, then 40 cycles of incubation at 95°C
for 60 s and at 42°C for 60 s, and finally elongation at
72°C for 60 s (23). Amplicons were electrophoresed
through 0.7% gels at 60 V for 1 h and visualized by ethidium
bromide staining under UV light. Under these conditions, P. carinii f. sp. carinii form 1 DNA produced a product of
ca. 520 bp, while form 2 DNA had an approximate size of 600 bp due to
the presence of multiple repeats of TAACCCTAA sequences
(23).
Evaluations to determine the efficiency of the PCR for form 1 and form
2 templates were conducted. Dilutions of DNA extracted
from the same
numbers of each organism population were individually
amplified with
the primers described above and with primers directed
to a region in
the mitochondrial large ribosomal subunit that
is the identical size
(340 bp) in each genome (
27). Amplicons
were visualized by
ethidium bromide staining of 1% agarose gels,
quantified by
densitometric analysis, converted to pixels, and
graphed against the
number of
P. carinii f. sp.
carinii in the
reaction.
Computer simulation of form 1 and form 2 trajectories.
A
program written in Fortran 77 was used to simulate different growth
models of form 1 and form 2 populations to determine if the lack of the
presence of a population could be simply due to differences in growth
rates (Jonathan Arnold, Department of Genetics and Computational
Biology, University of Georgia). The trajectories of the numbers of
form 1 and form 2 were calculated in a simple competition model
(Lotka-Volterra) (20) with a common carrying capacity for
each form. Doubling times were varied from 6.98 to 698 days, rates of
increases ranged from 0.001 to 0.1, lag times were set at 0, 10, and 20 days, and the length of the experiment was set at 10-day increments
beginning 10 days and continuing through 60 days.
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RESULTS |
Electrophoretic karyotypes of P. carinii f. sp.
carinii produced by single and coinoculations.
All
rats inoculated with form 1, 2, or 6 produced P. carinii f.
sp. carinii with the same karyotype profile as that
inoculated (Fig. 1 and
2),
showing that these inocula were viable and stable upon passage.
Inoculation of equal mixtures of P. carinii f. sp. carinii forms 1 and 2 administered at the same time produced
infections containing both forms (Fig. 1, lanes 1+2; interval, 0 days).
Fewer form 2 organisms were present than form 1 organisms, as evidenced by the bands of lighter intensity of <600 and 500 kb corresponding to
the same sized bands in the form 2 control lane (arrows). Forms 1 and 6 administered at the same time and in equivalent numbers produced
karyotypes that contained similar numbers of both forms, as evidenced
by bands of equal intensity corresponding to each P. carinii
f. sp. carinii form in these rats (Fig. 2, lanes 1+6; interval, 0 days). Note the doublet bands at 600 and 550 kb (arrows).
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FIG. 1.
Karyotypic profiles of P. carinii f. sp.
carinii from rats inoculated with forms 1 and 2 individually
and in combinations. The karyotype forms inoculated and the times
between inoculations in days (d) are shown at the top of the gel and
correspond to the lanes immediately below. Lambda concatamers (48.5-kb
increments) were used as size markers (not shown). Lanes: 1, form 1 individually inoculated; 2, form 2 individually inoculated; 3 and 4, forms 1 and 2 inoculated at the same time, arrows indicate bands
belonging to form 2; 5 and 6, forms 1 and 2 inoculated 10 days apart; 7 and 8, forms 2 and 1 inoculated 10 days apart, arrows indicate bands of
form 1; 9 and 10, forms 2 and 1 inoculated 20 days apart.
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FIG. 2.
Karyotypic profiles of P. carinii f. sp.
carinii from rats inoculated with forms 1 and 6 individually
and in combinations. The karyotype forms inoculated and the times
between inoculations in days (d) are shown at the top of the gel and
correspond to the lanes immediately below. Lambda concatamers (48.5-kb
increments) were used as size markers (not shown). Lanes: 1, form 1 individually inoculated; 2, form 6 individually inoculated; 3 and 4, forms 1 and 6 inoculated at the same time, arrows indicate bands
belonging to form 1; 5 and 6, forms 1 and 6 inoculated 10 days apart; 7 and 8, forms 6 and 1 inoculated 10 days apart; 9 and 10, forms 1 and 6 inoculated 20 days apart.
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The karyotypes produced from inoculations spaced 10 days apart were
that of the first form to be inoculated, with one exception
(Fig.
1 and
2, Tables
1 and
2). The
single exception occurred
in rats that received an initial inoculation
of form 2, followed
10 days later by an inoculation of form 1 (Fig.
1,
lanes 2+1;
10-day interval). In these rats,
P. carinii f.
sp.
carinii forms
1 and 2 were present in apparently equal
amounts despite the gap
between inoculations. Note the presence of
doublet bands at ca.
700 kb, the seven bands between 680 and 580 kb
versus only four
bands in this region in the single form 1 or 2 karyotypes, and
the bands of 450 and <400 kb, belonging to form 1 (arrows). In
rats receiving form one organism followed by a second dose
of
form 1, 10 or 20 days later, form 1 karyotypes were produced as
expected (data not shown). Rats inoculated first with killed form
1 organisms, followed 10 days later by inoculation of form 2,
did not
produce karyotypes due to the low numbers of organisms.
Although
sufficient organism numbers were present in rats inoculated
first with
the live form 1
P. carinii f. sp.
carinii,
followed
by the killed form 2, karyotypes were not discernible due to
the
abundance of degraded DNA (data not shown). Thus, although the
organism burdens in rats coinoculated with live
P. carinii
f.
sp.
carinii 10 days apart were equal to those inoculated
with
a single form of
P. carinii f. sp.
carinii
or with the admixtures
(see below), the karyotypes of the resultant
organisms causing
the infection in most of these rats were comprised of
the
P. carinii f. sp.
carinii organisms first
inoculated.
Karyotypes from rats that received inoculations 20 days apart followed
the same pattern as most of the infections resulting
from the 10-day
gap (Fig.
1 and
2). The initial
P. carinii f.
sp.
carinii form inoculated was the only form detected by PFGE
in all cases, even with the combination of form 2 followed by
form 1 that produced the mixed karyotype when administered 10
days apart. Two
inoculations with live form 1 organisms produced
karyotypes of form 1 (data not shown), but those rats receiving
killed form 1 organisms
prior to form 2 did not produce a karyotype
due to the low organism
number. Form 1 karyotypes were produced
from rats that received live
form 1 as the initial inocula, followed
20 days later by autoclaved
form 2 organisms (data not shown).
Most of the rats inoculated with
combinations of live forms 1
and 2 had reduced organism burdens but
nonetheless were able to
produce discernible
karyotypes.
Organism burdens resulting from single and coinoculations.
Rats inoculated with equivalent numbers of P. carinii f. sp.
carinii form 1, 2, or 6 developed fulminant infection with
no significant differences in organism burden (Fig.
3), indicating that the inocula used in
these studies were similar in viability and did not have dramatically
disparate growth rates. Coinoculations of forms 1 and 2 or forms 1 and 6 at the same number of total organisms as the individual inocula
(5 × 107) produced infections with the same organism
burdens as those produced by inoculation with a single form, showing
that these admixtures had no deleterious effect on organism burdens
(Fig. 3, Tables 1 and 2).
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FIG. 3.
Organism burdens from rats inoculated with individual
populations and admixed combinations of P. carinii f. sp.
carinii karyotype forms. Semiquantitative estimates of
organism burdens from rats inoculated with individual karyotype forms
1, 2, or 6 and combinations of forms 1 and 2 and forms 1 and 6 administered at the same time and in equal numbers. Organism burdens
were determined as averages and standard errors of the mean of
infections scored according to a semiquantitative system described in
Materials and Methods.
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No significant differences in the infection scores were observed among
the combinations when a gap of 10 days was introduced
between
inoculations (Fig.
4, Table
1). However,
spacing of the
inoculations 20 days apart produced lower organism
burdens in
rats receiving a combination of form 1 followed by form 2 (Fig.
5, form 1+2; Table
1) but not in
rats receiving form 1 followed
by form 6 (Fig.
5, form 1+6; Table
2).
Although a trend toward
reduction was observed in rats inoculated first
with form 2 and
then form 1, it did not reach significance. Thus,
differences
in organism burdens were associated with the different
P. carinii f. sp.
carinii karyotype combinations
inoculated.
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FIG. 4.
Organism burdens from rats inoculated with combinations
of karyotype forms 10 days apart. Semiquantitative estimates of
organism burdens from rats inoculated with combinations of karyotype
forms in which the second population was given 10 days after the first
population. Asterisks indicate significant differences as detailed in
Table 2. Organism burdens were determined as averages and standard
errors of the mean of infections scored according to a semiquantitative
system described in Materials and Methods.
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FIG. 5.
Organism burdens from rats inoculated with combinations
of karyotype forms 20 days apart. Semiquantitative estimates of
organism burdens from rats inoculated with combinations of karyotype
forms in which the second population was given 20 days after the first
population. Asterisks indicate significant differences as detailed in
Table 2. Organism burdens were determined as averages and standard
errors of the mean of infections scored according to a semiquantitative
system described in Materials and Methods.
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A role for the host immune response in the reductions was suggested by
the results of control groups. Three control groups
were included for
inoculations spaced 10 and 20 days apart: (i)
two inoculations of live
P. carinii f. sp.
carinii form 1 organisms
from
the same cryopreserved batch at each interval (1+1); (ii)
inoculation
of autoclaved form 1 organisms prior to that of live
form 2
P. carinii f. sp.
carinii (1A+2); and (iii) inoculation
of
autoclaved form 2 organisms after live form 1
P. carinii f.
sp.
carinii (1+2A). These three groups were included to
evaluate
the specificity of any observed host response and the
requirement
of live or killed organisms in this response. Inoculation
of live
form 1 into rats inoculated with form 1 organisms 10 days
beforehand
produced no difference in the organism burden (Fig.
4, form
1+1),
yet the spacing of this same inoculation 20 days apart resulted
in decreased organism burdens (Fig.
5, form 1+1). Significant
decreases
in the level of infection also were observed in rats
receiving
autoclaved form 1 organisms 10 and 20 days prior to
the live form 2 inocula (Fig.
4 and
5, form 1A+2) and in rats
receiving the killed form
2 organisms 10 days after the live form
1 inocula (Fig.
4, form 1+2A).
The administration of killed form
2 organisms 20 days after live form 1 inoculation also resulted
in a trend toward decreased burdens. Recall
that reductions in
organism burden only occurred when live inoculations
were spaced
20 days apart and only with specific pairs of populations,
either
forms 1 and 2 or forms 1 and 1. Thus, killed organisms were able
to elicit protection more rapidly than were live
organisms.
Detection of forms 1 and 2 by PCR.
Previous studies in our
laboratories have shown the lower limit of detection for PFGE to be
5 × 106 P. carinii f. sp.
carinii nuclei per lane by visualization of the bands
stained with SYBR-Gold. To detect the presence of lower numbers of
P. carinii f. sp. carinii in the lungs of
inoculated rats and to assess the distribution of organisms within the
lungs, a PCR-based assay was used in a second inoculation experiment. Forms 1 and 2 were chosen for this analysis because of the availability of a rapid PCR method that could differentiate the two forms and because of the unique interactions demonstrated by karyotyping. Products derived from amplification of an area upstream of the -tubulin gene have previously been shown to differ in size for P. carinii f. sp. carinii form 1 and 2 populations due to several copies of a telomere-like sequence repeat in
form 2 that is not present in form 1 (23). The sequences
of this region in the genomes of the two forms are shown in Fig.
6. All five lobes of the infected rat
lungs from at least two rats per time point were sampled to provide
information on the distribution of the populations. In rats that were
coinoculated with an admixture of forms 1 and 2, amplicons
corresponding to products from both forms were present in each of the
five lobes (Fig. 7A, lanes 1 to 5 [each
lane corresponds to a rat lung lobe as defined in Materials and Methods
and in the figure legend]). Form 2 amplicons were less intense than
those from form 1, suggesting that there may have been fewer of the form 2 organisms, similar to results from the first experiment revealed
by CHEF gel analyses (Fig. 1, lanes 1+2; 0-day interval). Amplicons
from form 2 populations were not detected in rat lungs inoculated with
form 1 followed 10 days later by form 2 inoculations (Fig. 7B). The
lack of detection of form 2 populations by the PCR suggests that the
organisms were in much lower abundance than were the form 1 organisms,
if at all present. In contrast, amplicons corresponding to forms 1 and
2 were apparent in rats that were inoculated with form 2 followed 10 days later by form 1 (Fig. 7C), which again corresponded to the results
of the karyotypic profiles.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 6.
Nucleotide alignment of the upstream region of the
-tubulin gene, the "-repeat." F1, form 1; F2, form 2 (P. carinii f. sp. carinii). Dots indicate the
identity, and dashes indicate the gaps that were introduced for maximal
alignment. The underline indicates the start codon of the -tubulin
gene (23).
|
|
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 7.
Distribution of P. carinii f. sp.
carinii forms 1 and 2 in the lobes of rats detected by
amplification of the -repeat region. (A) F1, amplicon from rat lung
inoculated with form 1; F2, amplicon from rat lung inoculated with form
2; lanes 1 to 5, amplicons from individual lobes of a single rat
inoculated with forms 1 and 2 at the same time. In all panels, the lane
numbers correspond to the different lung lobes as described in
Materials and Methods. (B) Lanes 1 to 5, amplicons from individual
lobes of a single rat inoculated with form 1, followed 10 days later
with form 2, and from rats inoculated with individual populations of
form 1 (F1) or form 2 (F2). (C) Lanes 1 to 5, amplicons from individual
lobes of a single rat inoculated with form 2, followed 10 days later by
form 1, and from rats inoculated with individual populations of form 1 (F1) or form 2 (F2).
|
|
Although PCR is expected to have a much greater sensitivity than many
detection methods, including CHEF analysis, some factors,
such as
differences in amplification efficiency, can reduce the
sensitivity of
the reaction. Since both the PCR data and the CHEF
karyotypes predicted
that form 2 organisms would be absent in
some rats, we conducted
experiments to evaluate the amplification
efficiency of form 1 and form
2 templates with the primers directed
to the
-repeat region and the
mitochondrial large ribosomal subunit.
At nuclei numbers of 10,000, the
form 2 efficiency of amplification
was only 70% of that for form 1. More-dramatic decreases were
observed with numbers lower than 10,000. In contrast, primers
directed to the mitochondrial large ribosomal
subunit did not
produce a difference in efficiency. The lower
efficiency of the
-repeat primers was likely due to the addition of
the ~80-bp
telomere-like repeat in the form 2 genome, making it 20%
longer
than the form 1 product. Approximately 2% of the infected rat
lung was sampled for PCR processing (0.05 g of a 2.50-g lung).
At least
10,000 form 2 organisms would have to be present to be
detected if an
equal or greater number of form 1 organisms were
also present. If
equally dispersed throughout the lung at these
levels, then a total
estimated organism burden of form 2 in these
lungs would be 500,000; a
number insufficient to be detected by
CHEF gel analysis. Thus, we can
assume that the form 2 organisms,
if present, were present in numbers
no greater than 500,000 per
lung, a number far lower than the
25,000,000 organisms
inoculated.
ATP levels of form 1 and 2 organisms mixed in vitro.
In some
fungal systems, the addition of a second fungal population to a host or
artificial medium colonized by another population with a similar
genetic background results in a destructive process causing organism
loss in one or both populations (4, 17). Since most
P. carinii f. sp. carinii forms inoculated 10 or
20 days after the first inoculation were not detectable by PFGE or PCR
(form 2), we postulated that there may be a direct inhibitory effect of
one organism population on the other. Experiments were performed to
assess the effect on the viability of 2 forms of P. carinii
f. sp. carinii mixed in vitro. The viability was determined by measurement of cellular ATP using a bioluminescent assay (7, 10). There is no long-term culture in which detectable and
consistent ATP levels of P. carinii f. sp.
carinii can be measured over time. The ATP bioluminescent
assay is a short-term cell-free assay routinely used in our laboratory
for the screening of candidate anti-P. carinii agents and
has been shown to be of predictive value for efficacy in animal models
(10). Spacing of the introduction of one form onto another
was reduced to a 3-day gap since the ATP levels gradually decrease
after 7 days. However, the same organism numbers as those inoculated in
vivo were used in the in vitro study. Forms 1 and 2 were selected for
the in vitro study since both PCR and karyotype data on both forms were
available from the in vivo studies. Form 1 and form 2 inoculated
individually at these organism densities increased their ATP levels
from 35 to 50% throughout 5 days with a slight decrease beginning at
day 6 (Table 3, groups 1 to 4).
Statistical analyses revealed no difference in the slopes of these
lines. Mixing equal numbers of form 1 and 2 prior to culture (group 5)
did not result in any decrease in ATP, a result which would have been
expected if there were a deleterious effect of one form on another.
Rather, this group achieved ATP levels almost identical to the
mathematical sums of groups 1 and 2 and to the ATP measurements of
groups 3 and 4 inoculated with 5 × 107 organisms
each, thus indicating a simple additive effect of the two forms in
group 5. The addition of form 2 to cultures of form 1 after 3 days
resulted in an increase of ATP concordant with an increase in organisms
(group 6). Likewise, when form 1 organisms were added to cultures of
form 2, a similar increase in ATP was observed (group 7). Thus, a
direct effect on the viability of one form on another was not observed
in this in vitro situation.
| |
DISCUSSION |
Two general conclusions can be drawn from these studies. First,
the time between inoculations of the two P. carinii f. sp. carinii forms was critical in determining the organism
population(s) that ultimately caused the pneumonia. Coinfections were
only established when equal numbers of the two organism forms were
administered at the same time. When gaps of 10 or 20 days were
introduced between these inoculations, the first organism form
inoculated became the causative agent of infection in all but one case.
Second, reductions in organism burdens resulted from a variety of
factors, including the viability of organism inoculations, the
karyotypes of the inoculated organisms, and the amount of time between inoculations.
The preponderance of the first population inoculated was not due to
differential growth rates or to a direct effect of one population on
the other.
A 10-day gap between all but one set of paired
inoculations was sufficient to produce an end infection consisting only
of the first population inoculated, as detected by karyotypic analysis. These results showed that the second inocula did not contribute to the
organism burden and in fact were in numbers below 5 million, the
minimum number of organisms needed for visualization with nucleic acid
stains on the pulsed-field gel. These results cannot be explained by a
simple difference in viability among the inocula, since burdens were
similar in rats receiving live inoculations of forms 1, 2, and 6 as
individual inocula and as paired admixtures. Likewise, it is unlikely
that the 10- or 20-day delay in growth contributed to the absence of
the second population. Each of the inocula contained 2.5 × 107 organisms, sufficient in itself to produce a visible
karyotype. If one assumes for argument's sake that only 10% of the
organisms were able to grow, due to host response or the loss of
viability, and a lengthy doubling time of 1 week, the elimination of 10 days from the total 8-week run of the experiment would produce
approximately 3 × 108 P. carinii f. sp.
carinii, an amount more than sufficient to produce a visible
karyotype. Elimination of 20 days of growth time would permit
replication to a level of 8 × 107, which would also
be detectable on the pulsed-field gels. The same argument can be made
for disparate growth rates of the different populations. Even if the
growth rates were disparate, the slower-replicating population should
still be able to be visualized on the pulsed-field gels since the band
patterns are sufficiently discriminating. In addition, the growth rates
could not be dramatically different, since the P. carinii f.
sp. carinii populations administered as individual
inoculations produced equivalent infection burdens after a growth
period of 8 weeks.
A mathematical model was constructed to further validate this
conclusion. Using form 1 and form 2 as the test populations,
a model
based on the Lotka-Volterra paradigm (
20) was used to
calculate the resultant population numbers of forms 1 and 2 from
logistic growth curves as trajectories, assuming uniform competition
between the forms and similar carrying capacities of each population.
Similar carrying capacities can be assumed from the organism burdens
for each form shown in Table
1 and Fig.
3, and was set at
10
10 organisms, a figure based on previous enumeration
studies in
our laboratory. For all of the calculated scenarios, forms 1 and
2 remained above the limits of detection by CHEF analysis,
suggesting
that extrinsic factors were influencing the observed
population
decreases.
A more compelling argument for an active suppression of growth rather
than disparate growth rates can be found in the organism
burdens in
rats receiving form 1 first and then form 2 after 20
days. If there was
simply a difference in growth rate, one would
have anticipated an
organism burden similar to that of the control
rats that received form
1 alone. However, a dramatic decrease
in organism burden was observed
in these rats, suggesting a more
complicated mechanism. This dramatic
reduction in organism burden
was not observed in coinoculations of
forms 1 and 6, further suggesting
that this mechanism may be specific
for the form pairs
inoculated.
If, therefore, the organism burden and identity of the infecting
population were not due to a difference in growth rate and
appeared to
be specific to the organism forms inoculated, an alternative
explanation might be the active suppression of one population
by
another. Such interactions are commonly reported in other fungi,
including
Neurospora crassa and other ascomycetes (
4,
17)
and in some myxomycetes (
5). In those cases,
the fungal population
which has established growth actively inhibits
coinfection by
another strain of the same fungus. We posited that a
similar mechanism
may have been involved in the exclusion of incoming
populations
of
P. carinii attempting to colonize the same
rat lung. We tested
this hypothesis by exposing the form 1 and form 2 populations
to one other in an in vitro setting, but we found no
detrimental
effects on viability, as determined by ATP levels. Rather,
an
additive effect in which both populations contributed to the ATP
levels was observed. These results suggested that a direct detrimental
effect of one population on the other was not a likely explanation
for
the predominance of the first population over the
second.
Potential role for the host.
Lack of evidence supporting a
direct effect of one population on another, protective effects of
autoclaved organism inoculations, and an apparently specific response
with successive inoculations of live forms 1 and 2 or forms 1 and 1 with 20-day gaps strongly suggests a role for the host immune response
in these findings. A simple explanation for the majority of these
observations could be framed in the context of a changing immune
response in the chronically immunosuppressed rat model of infection.
The first inoculation of a P. carinii f. sp.
carinii form occurred after 2 weeks of corticosteroid
treatment in a P. carinii f. sp. carinii-naive host. The host is sufficiently immunosuppressed to permit replication of the organisms and subsequent colonization of the lung alveoli by
attachment to the type I pneumocytes. In nonimmunocompromised antigen-naive rats, an immune response usually takes 10 days to 3 weeks
after antigen exposure to develop (P. Mirley [Charles River
Laboratories, Wilmington, Mass.], personal communication). By
extrapolation to our model, one would predict that the second inoculation 10 days after the first would provide organisms easily accessible to the cellular mediators of a developing, yet truncated immune response (due to the effects of the corticosteroids), thus permitting eradication. The first population may withstand this response by its privileged position in the alveolar hypophase and tight
adherence to the type I pneumocytes or because of the immature immune
response. After 20 days, an immune response to P. carinii f.
sp. carinii would have had time to develop, even in the
immunosuppressed host (36), and inoculation of the second organism population could then result in a more dramatic immune response not only resulting in elimination of the incoming organisms but also of some of the resident organisms if the two populations were
sufficiently similar in antigenic composition.
This scenario is supported by an understanding of the immunological
changes in the corticosteroid-treated rat model of
P. carinii pneumonia and by recent reports implicating
CD8
+ cells as mediators of lung injury. Walzer et al.
(
33) tracked
the generalized depletion of lymphocytes and
inversion of the
CD4
+/CD8
+ lymphocyte ratios in
the chronic corticosteroid-treated rat model
of
P. carinii
pneumonia. After 3 weeks of immunosuppression, the
lymphocyte numbers
are <1,000/mm
3 and the CD4
+/CD8
+
ratio has fallen from 2.4 in the nonimmunosuppressed state to
1.0. At 4 weeks of immunosuppression, this ratio drops to 0.6.
In the present
study, the second inoculation of the 10-day gap
coincides with 3 weeks
of immunosuppression (2 weeks prior and
1 week after the first
inoculation), while the 20-day gap period
coincides with 4 weeks of
immunosuppression. When the second
P. carinii f. sp.
carinii population was introduced 10 days after
the first,
there was likely a sufficient immune response that
could eliminate
these organisms prior to their initiating colonization
of the alveolar
lining cells. However, after a gap of 20 days,
the CD8
+
cell population was the predominant cell type. Rats receiving
inoculations of form 1 and 2 combinations may have had organism
burdens
reduced by a specific inflammatory response exacerbated
by the
CD8
+ cells. A recent report by Wright et al.
(
35) implicated the
CD8
+ cell as a major
factor in immune-mediated lung injury in CD4
+-depleted mice
infected with
P. carinii f. sp.
mus. These
effects
were more pronounced in later infection than during an earlier
phase of infection, a result similar to our own findings. Although
the
experimental design used by Wright et al. was not identical
to ours,
the role of the host immune response in mediating the
infective process
in immune-deficient rodents is
supported.
Harmsen et al. reported a somewhat similar observation of a reduction
in organism burden mediated by two inoculations of
P. carinii f. sp.
mus in a mouse model of pneumocystosis
(
18).
These investigators showed that intratracheal
inoculation of mice
with live
P. carinii f. sp.
mus could reduce the infection that
followed when the same
mice were depleted of CD4
+ cells and reinoculated with
P. carinii f. sp.
mus.
Results from inoculation of paired form 1 and form 2 live and
autoclaved organisms support the role of the immune response
in the
reduction of organism burdens and in the elimination of
secondary
populations. Not only were these autoclaved organisms
easily accessible
to the immune system, they likely evoked a more
vigorous response due
to exposure of additional antigens as a
result of the autoclaving
process.
An unique aspect of the present study was the use of characterized
organisms which were found to play a significant role in
the outcome of
the infections. The reduction in burden severity
was most clear upon
coinoculations of live forms 1 and 2 with
a gap of 20 days between
inoculations. Organism burdens were not
significantly reduced by paired
inoculations of forms 1 and 6,
but a clear dominance in karyotype was
observed by the population
that was first inoculated. These data would
be consistent with
the hypothesis described above such that the second
inoculation
was easily accessible to the immune system but, in this
case,
did not initiate a specific immune response as seen for forms
1 and 2. The role for a specific immune response in burden reduction
is
supported additionally by results in rats given live form 1
20 days
after the initial form 1
inoculation.
Failure to detect form 2 organisms by PCR suggests elimination
rather than growth inhibition.
The absence of a P. carinii f. sp. carinii form on a PFGE gel does not
necessarily reflect the absence of organisms, since the level of
sensitivity on these gels is estimated to be 5 million organisms (M. Cushion, unpublished data). PCR was used to evaluate the presence of
organisms in rat lungs which were not visible on PFGE gels. Form 2 amplicons could be detected in preparations in which the form 2 karyotype was visible, e.g., inoculations of equal numbers of forms 1 and 2 administered at the same time or a singular inoculation of form
2. However, no amplicons resulted from preparations in which the form 2 population could not be visualized by PFGE. These results show that the
form 2 organisms not only failed to grow but were also present in far
fewer numbers than when inoculated, suggesting the elimination of this population.
Coinfections of forms 2 and 1 were not due to physical separation
within the lung.
The ability of the form 1 population to exist as
a coinfection when inoculated 10 days after form 2 was puzzling in
light of the results from all other coinoculations. The same result was
reproduced in a second study using different preparations of form 1 and
2 organisms and thus was not a spurious finding. We postulated that
perhaps the two forms colonized different lobes of the rat lungs and
might coexist as physically isolated colonies. However, studies to
assess the extent of colonization of the lobes of the lungs by forms 1 and 2 showed that both populations were able to migrate throughout the
entire lung by the termination of the study when they were inoculated
as admixtures or when form 1 followed form 2. Thus, it appeared these
two populations under this specific inoculation regimen were able to
establish infection in the rat lungs and could coexist within the same
lobes of the lung. Further studies will be required to understand the
conditions permitting form 1 and form 2 coexistence. However, such
findings contribute to our growing appreciation of the diversity of
members of this family of fungal pathogens, their interactions within the host, and their ability to cause infection. This result also illustrates the differences in biological properties that can now be
associated with karyotype profiles. Only forms 2 and 1 were able to
produce this coinfection. Forms 1 and 6 did not.
Factors permitting coinfection in the rat have relevance to human
infection.
The P. carinii f. sp. hominis
populations infecting human beings exhibit levels of polymorphisms in
the large-subunit mitochondrial rRNA (24) and in the
intergenic regions of the nuclear rRNA locus (25, 26)
similar to those differences observed between the P. carinii
f. sp. carinii karyotype forms used in the present study.
Whether the organism strains coinfecting human beings also differ in
karyotypic profiles is not known, since most samples (usually
bronchoalveolar lavage fluids) from humans do not supply the necessary
numbers of organisms needed for visualization of the chromosomes by PFGE.
Our data suggest that establishment of successful coinfections is
dependent upon the genotype of organism populations and
the timing of
their introduction into the lung, with some mediation
of outcome by the
host. Reports of the prevalence of infections
in humans containing more
than one strain range from 10 to 69%
(
3,
19), depending
on the method used for detection. Furthermore,
Beard et al. reported
the presence of multiple genotypes associated
with primary infection
but not recurrent
P. carinii f. sp.
hominis pneumonia (
3). The studies presented here experimentally
reproduced
coinfections similar to those occurring in humans and
extended
our understanding of the factors leading to infection with
more
than a single
P. carinii population. Previous
colonization with
one
P. carinii f. sp.
carinii
prevented the growth of a second
population in all but one case. Based
on our studies, it would
be reasonable to hypothesize that the lack of
multiple genotypes
in recurrent human infection may be mediated by the
host response
to organisms of the primary infection. Coinfections could
be established
when
P. carinii f. sp.
carinii
populations were inoculated at
the same time, suggesting that primary
human infections may arise
from the acquisition of multiple genotypes
within a relatively
short time frame or even as a transmission of
multiple genotypes
from a single
source.
In the present study, we have shown that the establishment of
coinfections in immunosuppressed rats was dependent on the identity
of
the populations used for the coinoculations and on the time
between
those inoculations. The inhibition of growth of some karyotype
forms by
others suggests that an active process by the host, but
not by the
organisms themselves, may be involved. Such findings
have implications
for attempts to inoculate organisms into rats
previously colonized with
another population and should be considered
in the experimental design
of a study, especially those measuring
the immune response. These
studies also revealed clear biological
differences among
P. carinii f. sp.
carinii defined by karyotype
profiles,
thus associating phenotype with genotype. Inoculations
of defined
P. carinii f. sp.
carinii karyotype forms should
facilitate
experimental approaches for defining the transmission and
acquisition
of
P. carinii f. sp.
hominis in
humans.
| |
ACKNOWLEDGMENTS |
This work was supported by grants RO1 AI29839 (M.T.C.) and RO1
AI36701 (J.R.S.) from the National Institutes of Health.
We thank Jonathan Arnold, University of Georgia, for his invaluable
assistance in calculating the logistical growth curves of forms 1 and 2.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH
45267-0560. Phone: (513) 861-3100, ext. 4417. Fax: (513) 475-6415. E-mail: Melanie.Cushion{at}Uc.Edu.
Editor:
W. A. Petri Jr.
| |
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Infection and Immunity, January 2001, p. 97-107, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.97-107.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
