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Journal of Clinical Microbiology, May 1998, p. 1371-1377, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genotypic and Phenotypic Analysis of
Mycoplasma fermentans Strains Isolated from Different
Host Tissues
Laura
Campo,1
Patrick
Larocque,1
Tiziana
La
Malfa,1
Warren D.
Blackburn,2 and
Harold
L.
Watson1,*
Department of Microbiology, University of
Alabama at Birmingham,1 and
Birmingham
Veterans Administration Medical Center,2
Birmingham, Alabama
Received 17 December 1997/Returned for modification 22 January
1998/Accepted 18 February 1998
 |
ABSTRACT |
A correlation was found between the expression of a specific
Mycoplasma fermentans surface antigen (Pra,
proteinase-resistant antigen) and the site of isolation of the organism
from the infected host. Strains which expressed Pra were most
frequently associated with cells of bone marrow origin, and strains
which lacked expression of Pra were most commonly isolated from the
respiratory tract, genital tract, and arthritic joints, i.e.,
epithelial cell surfaces. Pra was previously shown to be resistant to
degradation by proteinases and was hypothesized to play a protective
role at the organism surface and perhaps to influence which host tissue
site was colonized by the organism. The methods used for this
phenotyping scheme required isolation and growth of the mycoplasma in
quantities sufficient for immunoblot analysis using monoclonal
antibodies. We wanted to determine a more rapid and less cumbersome
technique to supplement this method for determining the Pra phenotype
directly in clinical specimens. Here we describe PCR studies to
investigate the movement of a previously identified M. fermentans insertion sequence (IS)-like element. These data
showed a correlation between a specific IS genotype and the
Pra+ phenotype. Production of a 160-bp product using a
single set of IS-based primers was associated with expression of Pra.
The genomic IS location resulting in the 160-bp product was determined by using Southern blot analysis and was found to be a stable insertion site characteristic of genotype I strains. Additional analyses of
sequences within and flanking the IS insertion sites revealed another
pair of PCR primer sites which resulted in the consistent production of
a 450-bp amplicon. The stability of this site was dependent on the
absence of the IS-like element between the primer sites. The production
of this 450-bp amplicon correlated with the Pra mutant phenotype and
was characteristic of genotype II strains. The data showed that the
sequence within the IS may be unstable and that reliable genotyping
sequences are more easily found in the stable genomic sites which flank
the IS element.
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INTRODUCTION |
First mistakenly identified as a
novel AIDS-associated virus (18), Mycoplasma
fermentans incognitus, during the ensuing years, was considered to
be a possible cofactor contributing to acceleration of the progression
of this immune disorder (8, 16, 20-22, 29). Immediately
following the first reports, several laboratories began probing into
this question, but to date, the hypothesis of a mycoplasma-AIDS
association remains unproved. However, these studies have added much to
our basic knowledge of mycoplasmas. It has been documented that
M. fermentans, as well as some other mycoplasmas, can occur
intracellularly, which was only an occasionally reported and unproved
observation prior to these studies. The ability of specific
subpopulations of these organisms to survive within host cells could
account, at least in part, for the characteristic chronicity of
mycoplasmal disease, as well as for the frequent difficulty of
isolation by culture. Additionally, an impressive volume of literature
is accumulating which describes the induction of various cytokines by
mycoplasma infection (3, 5, 15, 27, 28, 30, 37). The
potential to alternately stimulate or suppress the immune system would
impart a distinct advantage to any pathogen (or commensal organism)
attempting to survive in the hostile and changing environment of an
infected host.
Subsequent to the initial isolation of strain incognitus, M. fermentans was identified as the likely etiologic agent of an acute fatal disease in otherwise healthy adults (17). No
other infectious agents were found. A similar wasting syndrome leading to death was reported in silvered leaf monkeys after experimental infection with this same agent (19). Many years prior to
these recent studies, M. fermentans was isolated from bone
marrow of leukemic patients (24) and other reports
associated it with rheumatoid arthritis (2, 36). These
reports prompted further investigations, including some experimental
studies with animal models (9, 10, 26). None of these
studies resulted in data proving a cause-and-effect relationship
between M. fermentans infection and human disease. In fact,
early serologic studies provided evidence that antibodies to this
organism are common in adolescents and young adults (32).
Therefore, M. fermentans has been tentatively associated
with disease throughout its history but the precise etiologic role of
M. fermentans in disease remains unclear. This is, in part, due to the frequently unsuccessful attempts to isolate mycoplasmas in
general by routine culture methods (6) and to the presence of individuals harboring the organism without signs of disease. Even
though many cases have resulted in isolation of M. fermentans and each isolate has been assigned a new strain
designation, there has been no attempt to assign molecular or
functional characteristics to these strains which might assist in
determining if there is a characteristic or group of characteristics
which associate with specific diseases, or at least with sites of
isolation.
In the present study, we were interested in defining methods to
determine if specific strains exhibit characteristics which are more
frequently associated with particular tissue sites within an infected
host. We tested whether monoclonal antibodies (MAbs) developed against
M. fermentans antigens could distinguish between isolates of
M. fermentans to determine a possible correlation between
the expression of these factors and the site of isolation. We also
conducted the same correlative assessment for the chromosomal distribution of the M. fermentans insertion sequence
(IS)-like element, hypothesizing a role for this potentially mobile
element in the repression or activation of a specific gene expression.
| |
MATERIALS AND METHODS |
Sources of isolation.
The M. fermentans strains
evaluated in this study were isolated from various sources (see Table
1). Strains were obtained as follows: E10 (24) and K7
(25) were obtained from W. H. Murphy; 16700, 12406, and
DEPB were from the University of Alabama at Birmingham; AOU was from
Luc Montagnier (Pasteur Institute, Paris, France); Z62 was from P. Hannan (Beecham Labs) (24); incognitus was from Shyh Lo
(National Institute of Allergy and Infectious Diseases [NIAID])
(17, 18, 21); AMSO was from Ann Robinson (Laboratory of
Immune Genetics, NIAID); MT2 was from W. J. Leonard and N. F. Halden (National Institute of Child Health and Human Development)
(11); Elliman was from H. Elliman (University of Illinois,
Chicago); 48429 was from Andy Lewis (NIAID); M51, M39, M52, M64, M73,
and M70 were from R. Dular (Public Health Laboratory, Ottawa, Ontario,
Canada); KL4 and KL8 were from P. Hannan (Beecham Labs); and PG18 was
from Klieneberger-Nobel, Lister Institute, London, United Kingdom.
Organisms and growth conditions.
Cultures of M. fermentans were grown in SP-4 medium (mycoplasma broth base,
tryptone [Difco], peptone [Difco], arginine, phenol red [1%],
DNA, and antibiotics for SP-4, supplemented with 10% fetal bovine
serum, CMRL 1066, yeast extract, yeastolate, and glucose). The cultures
were incubated at 37°C. Samples were harvested and washed in
phosphate-buffered saline at pH 7.3. DNA was purified in accordance
with standard protocols (phenol-chloroform-isoamyl alcohol) and
concentrated by ethanol precipitation. DNA preparations were RNase A
treated (Sigma).
Southern blotting and DNA hybridization.
For Southern
blotting of the M. fermentans strains, 0.2 µg of genomic
DNA was digested with 5 U of HindIII (Promega, Madison, Wis.) for 2 h at 37°C. The samples were electrophoresed on 0.8% Tris-borate-EDTA agarose gels (50 V for 16 h) and transferred to
1× Hybond N+ nylon membranes. DNA was UV cross-linked to
the membrane in a UV Stratalinker 1800 (Stratagene). After
prehybridization, the membranes were hybridized with 5'-end
-32P-labeled oligonucleotide probe RW006 (5'-GCT GTG GCC
ATT CTC TTC TAC GTT-3'; see Fig. 3a) and probe ORF-1 (5'-GGA AAA CTC
TTA TTC AGC C-3'; see Fig. 3b), located within the insertion sequence transposase gene and open reading frame 1 (ORF-1). Hybridization was
performed at 42°C for 1 h in Rapid hyb (Amersham) hybridization buffer and followed by one washing in 5× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) for 20 min at room temperature and two washings in 0.5× SSC-0.1% SDS for 15 min each at 45°C. DNA hybrids were visualized by autoradiography
using Kodak XAR-5 film (Eastman Kodak Co., Rochester, N.Y.).
PCR.
After incubation and sufficient growth, the 21 strains
were processed with proteinase K combined with buffer A (1 M Tris-HCl [pH 8.0], 1 M KCl, 1 M MgCl2, Milli-Q distilled
H2O) and buffer B (1 M Tris-HCl [pH 8.0], 1 M
MgCl2, Triton X-100, Tween 20, Milli-Q distilled
H2O). One milliliter of culture was centrifuged for 20 min
at 4°C, the supernatant was discarded, and the pellet was resuspended
in proteinase K lysis buffer. The samples were incubated at 60°C for
1 h and then boiled for 10 min. Samples were incubated on ice for
10 min and then stored at 
70°C until ready for PCR.
Amplification of the M. fermentans strains was performed by
using four different primer pairs (see Fig. 2). Primers RS-47 and RS-49
and primers RW005 and RW004 were previously described by S.-C. Lo et
al. (34); primers MF-1 (5'-GGA AAA CTC TTA TTC AGC C-3') and
MF-2 (5'-GGA AAA CTC TTA TTC AGC ATG C-3') were synthesized by Keystone
Laboratories. Amplification of DNA was performed in a total volume of
50 µl. Basically, PCR was performed with 40 cycles of denaturation
(94°C, 25 to 30 s), annealing (60°C, 1 min), and extension
(72°C, 1 min). Another primer, MF-4 (5'-GCG GCA CCA TCA ATC ACA TAT
AC-3'), was used as the antisense primer along with the previously
described RS-47 sense primer. For this primer pair, an initial
denaturation at 94°C for 2 min was followed by 40 cycles as described
above. PCR products were resolved on 2% agarose gels and visualized by
ethidium bromide staining.
Immunoblotting.
SDS-polyacrylamide gel electrophoresis and
Western blot analysis were performed as described previously, by using
a 10% resolving gel and a 4% stacking gel, and then proteins were
separated and transferred to nitrocellulose (Bio-Rad) by the method of
Towbin et al. (33). Immunological reactions were visualized
with peroxidase-labeled conjugates (Sigma).
MAbs.
MAbs directed to M. fermentans incognitus
antigens were produced in conjunction with the Hybridoma Core Facility
of the Multipurpose Arthritis Center at the University of Alabama at
Birmingham. The basic procedure for the production and characterization
of MAbs has been described previously in detail (35).
| |
RESULTS AND DISCUSSION |
Phenotyping of M. fermentans strains and isolates.
The most frequently colonized sites in a mycoplasma-infected host are
epithelial cell surfaces (7, 12, 14, 23, 31). In the case of
M. fermentans, the second most frequent association is with
blood cells (17, 21). Are there characteristics that make
some species or some strains within a species uniquely qualified for
survival in one site as opposed to another? We previously identified an
M. fermentans surface antigen (Pra) that can be divided into
two distinct domains based on the immunoblot pattern obtained with
MAbs, i.e., a domain that is resistant to degradation by trypsin,
chymotrypsin, V-8 protease, and proteinase K and a second domain that
is sensitive to these same proteinases (40). Our preliminary
studies suggest that Pra is a complex surface network consisting of
acylated proteins, but the nature of the membrane anchor and the
noncovalent forces that mediate the interaction between the two domains
have not been fully characterized (39). Nonetheless, we
hypothesized that a correlation exists between the expression of the
proteinase-resistant domain, which may play a protective role at the
organism surface, and the association of the organism with particular
cell types. Results in Fig. 1 show the
variable expression of the Pra+ phenotype and the
distinctive, diffuse immunoblot pattern of the Pra+
phenotype which correlated with isolation of the organism associated with cells of bone marrow origin and frequently from immunocompromised patients (Table 1). This broad
distribution of the electrophoretic mobility of identical epitopes seen
for the Pra+ phenotype is not uncommon for mycoplasmal
antigens (38). The single exception to the above correlation
was isolate 16700, which was isolated from the urethra of a patient
with nongonococcal urethritis. Organisms lacking expression of the
proteinase-resistant phenotype were most commonly isolated from the
respiratory tract, from the genital tract, and from arthritic joints
(Fig. 1 and Table 1). These Pra mutants were presumably epithelial cell
associated. If the Pra+ phenotype does, in fact, provide
the organism with protection from proteolytic degradation, then the
above correlations support the possibility, although they certainly do
not prove, that the Pra+ phenotype resides in a
hydrolase-rich intracellular compartment. This niche may be represented
by the professional-phagocyte-rich cellular environment found in the
circulation.
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FIG. 1.
Pra phenotypes of representative M. fermentans strains. Organism proteins were separated by
SDS-polyacrylamide gel electrophoresis and then transferred to
nitrocellulose. Reactivity with MAb 1A2.6 was visualized by using
peroxidase conjugates. Strains incognitus and MT2 show the
characteristic Pra+ pattern, while the Pra
strains show no reaction with the MAb. Results for all strains are
summarized in Table 1.
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TABLE 1.
Relationships among the genotypes, phenotypes, and
sources of isolation of different M. fermentans strains
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Genomic distribution of the IS element.
Development of a
statistically sound proof of the association suggested above requires a
large number of isolates characterized with respect to Pra expression
and sites of isolation. Therefore, if a genetic marker also existed
(preferably associated with Pra in a nondissociable genotype-phenotype
relationship) which correlated with the site of isolation, then it
might be possible to develop a more sensitive and simple system for
discerning the Pra phenotype of new isolates. We initially looked for a
correlation between Pra expression and the genomic location of the
previously published M. fermentans IS-like element.
Disruption or activation of cryptic promoters is not an uncommon result
of IS movement (1, 4). Although it is unproved, M. fermentans may use this system to accommodate its surface
properties for survival in its current environment (13).
Figure 2 is a schematic showing the basic structure of the previously described IS-like element, as well as the
locations of the genotyping PCR primers and probes used in the current
study. Figure 3 shows the distribution of
IS-associated ORF-1 and ORF-2 (transposase gene) in
HindIII genomic digests of the different M. fermentans strains. These restriction patterns are consistent with
those previously shown for strain incognitus (13). Based on
the distribution of these two ORFs, the M. fermentans strains can be grouped into two basic genotypes (I and II). The published sequence of the incognitus strain IS-like element contains no
HindIII site (13). If the IS-like elements in
the other M. fermentans strains are sufficiently similar to
the IS-like element of strain incognitus and are intact, then ORF-1-
and ORF-2-specific probes should cohybridize to the same
HindIII restriction fragments. Examination of Fig. 3 by
using this criterion (i.e., identification of fragments common to Fig.
3a and b) indicates that genotype I has at least 10 copies of the
intact IS and genotype II has only 3. These are minimal estimates,
since a single fragment could contain multiple probe sites.
Identification of fragments that are not common to Fig. 3a and b
indicate that (i) all genotype I strains have one copy of ORF-1 which
is not associated with an IS (Fig. 3a, location 1); (ii) two of the
genotype I strains, incognitus and 16700, have one additional
non-IS-associated ORF-1 (Fig. 3a, location 2); (iii) 7 of the 10 genotype II strains have one non-IS-associated ORF-1 (Fig. 3a, location
3); and (iv) 6 of the latter 7 genotype II strains also have a
non-IS-associated ORF-2 (Fig. 3b, location 4). The apparent
non-IS-associated ORFs in these experiments may be the result of a
common mutational event producing a HindIII site between
ORF-1 and ORF-2. Strains KL4 and KL8 are unusual, since no IS elements
are detectable in Fig. 3. Even though the latter two strains may
actually constitute a third genotype, here we have placed them into
genotype II based on additional parameters to be discussed below.
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FIG. 2.
Schematic diagram of the M. fermentans
IS-like element and its flanking regions. The locations of all of the
primer pairs used in this study are indicated, as are the sizes, in
base pairs, of the respective amplicons. ORF-2 is the putative
transposase. ORF-1 and ORF-3 have no assigned putative functions.
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FIG. 3.
Southern blot analysis of the chromosomal distribution
of the M. fermentans IS-like element. DNAs isolated from all
of the strains were digested with HindIII, separated in
0.8% agarose, transferred to nylon membranes, and then probed. The
Southern blots were hybridized with an oligonucleotide specific for
ORF-1 (a), and the same strains were then hybridized with an
oligonucleotide specific for the transposase gene (b). Approximate
fragment sizes are indicated in kilobases. Differences between the two
basic genotypes (I and II) and the identification of fragments common
to the two panels are indicated by the numbered arrows (see text).
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Table
1 shows genotypes I and II in the context of Pra expression and
site of isolation. All strains in genotype I were Pra
+, and
all genotype II strains were Pra mutants.
Genotyping of M. fermentans strains by PCR.
The
pattern of distribution of the IS-like element genomic insertion sites
was a reliable reflection of Pra expression but required very
cumbersome methodology. The asymmetric hybridization of the ORF-1 and
ORF-2 probes seen in Fig. 3 indicated the presence of some differences
within the IS-like element which may provide useful sequence markers
for determining genotypes by using PCR. An M. fermentans-specific PCR primer pair has been previously described
(34). Amplification with this primer pair, located within
ORF-2 of the IS-like element (Fig. 2, RW005 and RW004), resulted in a
206-bp amplicon for the strains evaluated in that study. In the current
study, amplification with these same primers produced a 206-bp amplicon
for all of the strains listed in Table 1 (Fig.
4a). Although these primers performed as
predicted, they do not allow discrimination of the two genotypes.
Strains KL4 and KL8 showed no indication for the presence of either
ORF-1 or ORF-2 (Fig. 3), even though a typical 206-bp product was
obtained with the RW005-RW004 primer pair (Fig. 4a). This suggested
that even though the RW005 and RW004 primer sites were present, the intervening sequence was sufficiently different to disallow
hybridization of the RW006 probe. This may result in a dysfunctional
transposase, which could explain the absence of multiple insertion
sites in these two strains.
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FIG. 4.
PCR analysis of representative M. fermentans
strains from various sources (Table 1), using primers RW005 and RW004
(a) and primers RS47 and RS49 (b), which are located within the
transposase gene and upstream of the IS-like element, respectively
(Fig. 2). PCR amplicons were analyzed by electrophoresis in a 2%
agarose gel stained with ethidium bromide. Sizes of products are
indicated. Lanes: 1, M51; 2, KL8; 3, AMSO; 4, Elliman; 5, MT2; 6, DEPB;
7, M52; 8, AOU; 9, KL4; 10, M64; 11, E10; 12, 16700; 13, 12406; 14, Z62; 15, incognitus; 16, M39; 17, M73; 18, PG18; 19, K7; 20, 48429; 21, M70.
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In the same previous study as that described above, another primer pair
located immediately upstream of the IS-like element
(Fig.
2, RS47 and
RS49) produced a 160-bp product for only three
of the six strains
tested. The RS47-RS49 primer pair was not further
evaluated, since
those investigators were interested in defining
PCR primers for
detection of all strains of
M. fermentans. When
we evaluated
the strains listed in Table
1 with the RS47-RS49
primer pair, the most
common product was 160 bp (Fig.
4b). Other
products ranged from
80 bp
(M39 and M70) to over
860 bp (KL8,
KL4, and 12406). Some strains also
gave no distinct product (M51,
M52, M73, and PG18). These results are
recorded in Table
1, and
there is a complete correlation between
genotype I, Pra expression,
and the IS-like element location resulting
in a 160-bp product
with the RS primers. PCR amplification of all
genotype II strains
resulted in different-sized amplicons or no
amplicon. The lack
of an RS product for strains M51, M52, M73, and PG18
implies that
either one or both primer sites are missing (or lack
sufficient
homology) or that there is an insertion between the primer
sites
resulting in a template which was too large to amplify
efficiently.
Various PCR primer combinations (Fig.
2) were used to determine if
there were any differences in the sequence within the IS-like
element
which might allow the detection of a stable PCR product
that would be
representative of the genotype II strains. Amplification
with the
primers MF-1 and MF-2 suggested that ORF-1 was not significantly
different among the strains (Fig.
5a and
b). Strains KL4 and KL8
gave no product, and strain 16700 consistently
produced a weak
amplicon. Similarly, by using primers MF-1 and RW004,
we found
no differences in the linkage between ORF-1 and ORF-2 among
the
strains (Fig.
5c and d). Once again, strains KL4 and KL8 gave
no
product and strain 16700 was amplified poorly. The linkage
between the
RS genomic site and the ORF-1 IS site was evaluated
by using primers
RS-47 and MF-2 (Fig.
5e and f). Amplification
of all genotype I strains
resulted in a 546-bp amplicon, reaffirming
the stability of this
particular insertion site for the genotype
I strains. The genotype II
strains were inconsistently amplified
with this primer pair, indicating
the instability of this insertion
site in these strains.
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FIG. 5.
PCR analysis of M. fermentans strains.
Amplification of representative M. fermentans strains from
various sources (Table 1) by using primers MF-1 and MF-2, MF-1 and
RW004, and RS-47 and MF-2 (Fig. 2) is shown. PCR amplicons were
analyzed by electrophoresis in 2% agarose gels and stained with
ethidium bromide. Lanes in a, c, and e: 1, M51; 2, KL8; 3, AMSO; 4, Elliman; 5, MT2; 6, DEPB; 7, M52; 8, AOU; 9, KL4; 10, M64; 11, E10.
Lanes in b, d, and f: 12, 16700; 13, 12406; 14, Z62; 15, incognitus;
16, M39; 17, M73; 18, PG18; 19, K7; 20, 48429; 21, M70. Sizes of
products are indicated on the left.
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Since Fig.
4b,
5e, and
5f suggested that the genotype II strains do not
have an IS-like element consistently present near
the RS site, we used
primers RS-47 and MF-4, which flank the putative
insertion site, to
ascertain if the IS was consistently absent
from this site. Figure
6 shows a stable 450-bp product for all
genotype II strains, indicating that the genotype II strains have
a
stable linkage between the RS site and ORF-3 with no intervening
IS-like element.
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FIG. 6.
PCR analysis of the M. fermentans strains by
using primer sites which flank the IS-like element. Primer pair
RS-47-MF-4 (Fig. 2) amplicons were analyzed by electrophoresis in a
2% agarose gel and stained with ethidium bromide. Sizes of products
are indicated. Genotype II strains are represented by the 450-bp
product. Genotype I strains were not consistently amplified.
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These analyses indicate that the two
M. fermentans genotypes
consistently differed in the sites of insertion of the IS-like
element
but not in the sequence of the element itself. Also, due
to potential
sequence instability within the IS-like element,
the only reliable
genotyping markers reside outside the IS-like
element and in the
genomic sequences immediately upstream and
downstream of the IS
junctions. Therefore, the method of choice
for species detection
appears to be direct detection of specific
sequences within the IS-like
element. In contrast, for genotype
distinction, detection of stable
genomic insertion sites is required,
i.e., with primers RS47 and RS49
for genotype I and primers RS47
and MF-4 for genotype II. These primer
pairs should allow easy
and rapid genotyping of
M. fermentans in clinical samples and
thus obviate the need for the
frequently unsuccessful isolation
and culturing of this organism.
Aside from the immediate usefulness for diagnostic genotyping, these
data also will help to understand how this species may
adapt for
survival in a particular host population. At this time,
we have only a
suggestive link between the sites of insertion
of the IS-like element
and Pra expression, and as previously stated
by Lo et al.
(
13), there is no proof that this element is mobile.
Conclusive evidence will have to await a more direct connection
between
the
pra gene sequence and a specific insertion site. We
have
not analyzed a sufficient number of isolates to say that
proteinase
resistance is always associated with isolation from
cells of a
blood-related compartment or from immunocompromised
patients, but
completion of these analyses will determine if
M. fermentans
uses the mobility of this IS-like element coupled to
Pra expression as
a primary means of maintaining a specific niche
in its host.
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ACKNOWLEDGMENTS |
This research was supported by grant DAMD17-97-1-7001 from the
Department of the Army and grant 5R01 AI33197 from the National Institutes of Health. MAbs were produced in conjunction with the Hybridoma Core Facility of the Multipurpose Arthritis Center at the
University of Alabama at Birmingham, which is supported by grant 2P60
AR0614-20 from the National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Present address: Infectious
Diseases Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0438. Phone: (317) 277-0131. Fax: (317) 276-1743. E-mail: Watson_Harold_L{at}Lilly.com.
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Abstract
Introduction
