Download Spontaneous Mutations in the CsrRS Two

1043
Spontaneous Mutations in the CsrRS Two-Component Regulatory System
of Streptococcus pyogenes Result in Enhanced Virulence in a Murine Model
of Skin and Soft Tissue Infection
N. Cary Engleberg,1,2 Andrew Heath,1 Alita Miller,2,3
Clarise Rivera,2 and Victor J. DiRita2,3
Departments of 1Internal Medicine and 2Microbiology
and Immunology and 3Unit for Laboratory Animal Medicine,
University of Michigan Medical School, Ann Arbor
CsrS/CsrR is a 2-component system in Streptococcus pyogenes that negatively regulates
hyaluronic capsule and several exotoxins. To detect spontaneous mutations in csrRS, mucoid
and large colony variants of M1 strain MGAS166 were isolated from experimental murine
skin infections. By use of complementation with a csrRS+ plasmid, relevant mutations were
also detected in 7 of 12 human clinical isolates. The presence of spontaneous mutants in mouse
infection was associated with larger, more necrotic lesions. Most spontaneous changes in CsrR
resulted from single amino acid substitutions, whereas most csrS mutations were frameshift
or nonsense mutations. In 2 instances, IS1548 insertions were found in csrS. Experimental
inoculation of mixtures of wild-type (wt) and csrRS2 bacteria yielded larger, more necrotic
lesions than did either strain at twice the inoculum, which suggests that these variants may
exhibit pathogenic synergy. Spontaneous emergence of csrRS2 mutants in vivo enhances the
virulence of wt bacteria and increases severity of murine skin infection.
Skin and soft tissue infections with Streptococcus pyogenes
may be life-threatening or relatively benign [1, 2]. Differences
in the aggressiveness of these infections may be partly explained
by host factors, strain characteristics, or portal of entry. However, none of these explanations fully accounts for the variation
in outcome among infected persons. Although the very young
and the very old have higher rates of invasive streptococcal
disease, most cases occur among older children and adults !65
years old [3, 4]. Various states of immunocompromise and underlying diseases increase the risk of severe infection (i.e., necrotizing fasciitis); however, aggressively invasive cases also are
common among healthy persons with normal immunity. Similarly, certain clones of S. pyogenes have been associated with
worldwide resurgence of invasive streptococcal disease, but
such strains do not account for all cases, nor do they always
cause severe disease [5–10]. One might speculate that the site
and depth of inoculation would influence the type and progress
Received 16 October 2000; revised 28 December 2000; electronically published 1 March 2001.
Presented in part: American Society for Microbiology General Meeting,
Chicago, June 1999 (session 187); 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, September 1999 (abstract 141B).
Financial support: National Institutes of Health (grants RO1-141682 to
N.C.E, GM-20420 to A.M., and RR-00042 for technical assistance). C.R.
is supported by a Rackham minority fellowship.
Reprints or correspondence: Dr. N. Cary Engleberg, Div. of Infectious
Diseases, Dept. of Internal Medicine, University of Michigan Hospitals,
3116B Taubman Center, Box 0378, Ann Arbor, MI 48109 (cengleb@
umich.edu).
The Journal of Infectious Diseases 2001; 183:1043–54
q 2001 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2001/18307-0008$02.00
of skin infection; however, blunt trauma is associated with necrotizing fasciitis as often as penetrating trauma [11]. Therefore,
these factors account for some of the variation in severity and
outcome of streptococcal skin and soft tissue infection, but they
do not explain it fully.
Control of several streptococcal virulence factors is mediated
by a 2-component regulatory system designated CsrR/CsrS
(capsule synthesis regulation) [12–15]. This system consists of
a membrane-spanning sensor kinase (CsrS) and a DNA-binding cytoplasmic protein (CsrR). When it is phosphorylated,
CsrR binds upstream of certain genes and represses transcription [13]. The CsrR-repressed genes include those that encode
the synthesis of hyaluronic acid capsule, streptolysin S (SLS),
pyrogenic exotoxin B (SpeB), and streptokinase [14, 15]. Because CsrR is a transcriptional repressor, mutants that affect
the expression or integrity of this protein result in enhanced
expression of the regulated genes. Thus, csrR mutants are highly
mucoid and produce increased SLS activity and SpeB protein.
In a mouse model of skin and soft tissue infection, such strains
are significantly more likely than the CsrR1 parental strains
from which they are derived to produce rapidly enlarging necrotic skin lesions and lethality [12, 15]. Similarly, a large, sitedirected deletion of csrS in 1 clinical isolate resulted in a phenotype of large colony (LC) size in response to 5% CO2
atmosphere [16]. Colonies grown under these conditions also
appear to be more mucoid but do not have increased amounts
of hyaluronic acid to account for this glossy appearance (authors’ unpublished data). Although the biologic basis for this
phenotype is not known, skin inoculation with this LC strain
resulted in rapidly expanding ischemic lesions that were also
associated with enhanced mortality.
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Engleberg et al.
The above observations were made with directed mutations
and transposon insertions in clinical strains. We reasoned that
a similar enhancement of streptococcal virulence might also
occur as a result of spontaneous mutations in csrR or csrS.
Here we report the results of a study in a mouse model.
Materials and Methods
Bacterial strains. Streptococcal strains used for this study were
derived from wild-type (wt) strain MGAS166 (table 1). Strains were
grown in Todd-Hewitt broth supplemented with 0.2% yeast extract
(THY broth; Difco) or on Todd-Hewitt yeast extract agar (THYA)
plates (Difco). In certain experiments, antibiotic-resistant streptococci were grown on selective agar media containing 100 mg/mL
spectinomycin or streptomycin (Sigma).
Strain MGAS166 is a streptomycin-resistant type M1 isolate
from a case of invasive streptococcal disease that has a matte colonial appearance when grown on THYA [5]. Strain SBmuc7 is a
derivative of MGAS166 that has an insertion of Tn916 immediately
upstream of csrR [15]. Strain UMAA2392 is a site-directed mutant
of MGAS166 that has both a large in-frame deletion of csrR and
a substitution of ACG for ATG as the start codon in the csrS gene
[15]. Strain UMAA2690 is a site-directed mutant that has a large
deletion of the csrS gene and an intact copy of csrR [16]. SBmuc7
and UMAA2392 both are constitutively mucoid and produce rapidly enlarging dermonecrotic lesions in the mouse model described
below. UMAA2690 has the LC phenotype when grown on THYA
in 5% CO2 and produces rapidly enlarging dermal ischemia in the
mouse model.
To investigate expression of capsular synthesis genes, we constructed a reporter plasmid, phas-phoZ, which carries the first 200
Table 1.
JID 2001;183 (1 April)
bp of the hasA gene fused to alkaline phosphatase. This plasmid
was generated from vector pABG-5 [17] by inserting the hasA promoter upstream of a truncated copy of the phoZ gene that lacks
its promoter and signal sequence. The hasA gene fragment includes
the 235 and 210 sequences that are reportedly protected by CsrR
in DNA footprinting experiments [13]. As a negative control, we
used a derivative of pABG-5 that contains a rofA-phoZ transcriptional fusion [17] (pABG-5 and the rofA-phoZ derivative were provided by M. Caparon, Washington University, St. Louis). Capsuledeficient strains used in these experiments included UMAA2497,
a derivative of MGAS166 that has an in-frame deletion between
the capsular synthesis genes, and hasA, hasB, and UMAA2526, a
nonencapsulated derivative of CsrRS mutant UMAA2392 containing the same hasAB deletion as UMAA2497 [15].
Assays for bacterial products in culture. Uronic acid levels in
culture supernates were quantified by using the stains-all method,
as described elsewhere [15]. Phosphatase activity in 200 mL of supernate samples was measured by mixing with 800 mL of 1 M Tris
(pH 9.5) and 200 mL of p-nitrophenyl phosphate (10 mg/mL). The
assay mixture was incubated at room temperature until a yellow
color developed. The reactions were stopped by the addition of
200 mL of KH2PO4, and absorbance was read at OD420.
Isolation and analysis of spontaneous CsrRS mutants. Animal
isolates were screened for mutations in csrR or csrS by growth on
THYA in air and 5% CO2. Isolates that produced larger or more
mucoid colonies were selected for further study. Some isolates were
subjected to DNA sequencing of the entire csrRS locus. This was
accomplished by amplification of 2 overlapping segments (1.4 kb
and 1.9 kb) of the 2.3-kb locus by polymerase chain reaction (PCR)
with Taq polymerase. Sequencing was done by the University of
Michigan Molecular Biology Core Laboratory.
A larger number of variant strains was screened for csrRS mu-
Bacterial strains used in this study.
Designation
Description
Source
MGAS166
SBmuc7
UMAA2392
Type M1 clinical isolate
Tn916::csrR
MGAS166, DcsrR, csrS2 (spontaneous mutation
of the initiation codon from ATG to ACG)
MGAS166, DcsrS
MGAS166, DhasAB
UMAA2392, DhasAB
a
Clinical isolate; blood
a
Clinical isolate; surgical wound
a
Clinical isolate; necrotic skin lesion
Clinical isolate; blood
Clinical isolate; blood
Clinical isolate; tissue source unknown
a
Clinical isolate; wound
a
Clinical isolate; tissue source unknown
a
Clinical isolate; wound
Clinical isolate; blood
Clinical isolate; blood
Clinical isolate; subdural empyema
Clinical isolate; blood
Clinical isolate; necrotic skin lesion
a
Clinical isolate; wound
Clinical isolate; tissue source unknown
[5]
[15]
[15]
UMAA2990
UMAA2497
UMAA2526
UMAA2254
UMAA2259
UMAA2712
UMAA2713
UMAA2714
UMAA2715
UMAA2716
UMAA2717
UMAA2718
UMAA2719
UMAA2720
UMAA2721
UMAA2722
UMAA2723
UMAA2724
UMAA2725
a
b
[16]
[15]
[15]
b
Toronto, Canada
b
Toronto, Canada
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Ann Arbor, MI
Colony size or mucoidy decreased after transformation with pASH2477.
Kindly provided by Don Low (University of Toronto).
JID 2001;183 (1 April)
Spontaneous CsrR/CsrS Mutants of S. pyogenes
tations by complementation analysis with pASH2477. This plasmid
is a derivative of spectinomycin-resistant broad host-range vector
pJRS525 that contains the entire csrRS locus, including its promoter,
inserted into the multiple cloning site (figure 1A) [15, 18]. For electroporation with either plasmid, bacteria were grown for ∼2 h in 30
mL of THY broth with 0.2 M glycine and hyaluronidase (0.1 mg/
mL to an OD600 of 0.1). The bacteria were recovered by centrifugation
at 47C, were washed twice in 5 mL of 15% glycerol, and were resuspended in 0.5 mL of 15% glycerol. We added 0.2 mL of this
bacterial suspension to 5–10 mg of DNA, which was electroporated
at 1.75 kV (capacitance, 25 mF; resistance, 400 ohms) in a gene pulser
with pulse controller (Bio-Rad). The mixture then was diluted in 5
mL of THY broth and was incubated on ice for 45 min and at 307C
for 2 h. Bacteria were pelleted, were resuspended in 0.75 mL, and
were plated on THYA with spectinomycin.
Spectinomycin-resistant transformants that reverted to a small
matte colonial morphology were cured by passage on nonselective
media to confirm a reversion to the original phenotype after loss
of the plasmid. Isolates obtained from invasive streptococcal infection of humans also were screened by plasmid complementation
and curing, to determine whether they had a csrRS-repressible phenotype. All strains were compared on media incubated in air and
in 5% CO2.
We estimated the approximate copy number of pASH2477 in S.
pyogenes by PCR analysis. A series of 2-fold dilutions of total DNA
extracted from UMAA2392 (pASH2477) was amplified with a
primer that binds 416 bp upstream of the csrR initiation codon (50CTTGCTATTCCGCTACAGG-30) and with one that binds 308
bp downstream of the csrR termination codon (50-CCTAATGACTCGACTGCCCTTTCT-30). This primer pair yields 1405-bp amplicons from the intact csrR gene on the plasmid and 966-bp amplicons from the DcsrR gene on the chromosome of UMAA2392.
Comparison of the intensity of ethidium bromide staining of these
2 bands in the dilutional series of DNA allowed for estimation of
the plasmid copy number.
Mouse model of skin and soft tissue infection. Streptococci were
harvested at midlog phase (OD600 p 0.6–0.7) and were diluted in
THY broth to produce various inocula in a 100-mL bacterial suspension. The inoculum was injected subcutaneously into the right
flank of hairless 4-week-old male crl:SKH1(hrhr) Br mice (Charles
River), as described elsewhere [19]. Where noted, Cytodex beads
(Sigma-Aldrich) were mixed with the inoculum immediately before
injection. This manipulation enhances the virulence of the wt strain
[19].
Mice were weighed before inoculation and every 24 h thereafter.
Total lesions (induration, abscess, ulcer, and erythema) and any
regions of necrosis were measured daily, and the areas were calculated by using the equation area p p(L 3 W )/4 , where L is the
long axis and W is the short axis of the lesion. Edema was noted
in some animals but was not measured separately. Mice that were
immobilized by large lesions were killed and were counted as lethal
outcomes of infection.
In experiments that required quantitation of streptococci from
wounds, the mice were killed, and a wide area surrounding the
lesion was excised and was homogenized in PBS (pH 7.4) by using
a 7-mL tissue grinder (Tenbroek Pyrex). Aliquots were plated at
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Figure 1. Complementation of csrRS by pASH2477. A, Map of
pASH2477. White- and black-filled lines, vector sequences derived from
pJRS525; heavy solid lines and arrows, inserted csrRS locus. For reference, relative position of DcsrR positioned on chromosome of strain
UMAA2392 is indicated by dotted brackets adjacent to intact csrR
gene on plasmid map. P1 and P2 indicate where primers used for
polymerase chain reaction (PCR) analysis hybridize. B, PCR products
of 2-fold dilutions of total DNA from UMAA2392 (pASH2477). Amplicons from intact plasmid csrR gene (∼1.4-kb band); ∼1-kb band
contains amplicons from chromosomal DcsrR gene. Intensity of each
1-kb band is comparable to that of 1.4-kb band at next dilution, which
suggests that there are ∼2–4 copies of the plasmid gene for each chromosomal copy (after correction for ∼50% increase in band intensity
because of amplicon length). C, Growth curves of indicated streptococcal strains in Todd-Hewitt yeast broth in air. Samples taken at 6.7
h (f) were analyzed for bacterial cell-associated uronic acid (inset).
various dilutions on THYA with streptomycin, and streptococcal
colonies were counted.
Results
Complementation of mutations in csrRS with pASH2477.
Bacteria suspected of having a significant mutation in the csrRS
locus were electroporated with pASH2477 (csrRS1) and were
examined for suppression of a mucoid or LC phenotype. To
demonstrate that plasmid complementation suppresses csrR
mutations in a physiologic manner, we estimated the plasmid
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Engleberg et al.
copy number and measured uronic acid levels at the end of the
exponential phase. By comparing the intensity of ethidium bromide–stained PCR amplicons derived from the chromosomal
and plasmid copies of csrR, we were able to estimate copy
number of pASH2477 as 2–4 copies per bacterial cell (figure
1B). To confirm that the suppression of the mucoid phenotype
by pASH2477 is physiologic, we compared total uronic acid
levels from MGAS166 and UMAA2392 (pASH2477). At the
end of the exponential phase, production of uronic acid by the
complemented mutant was near (but not less than) the wt level
(figure 1C, inset). Insertion of pASH2477 into MGAS166 produced no change in the colony appearance or size, compared
with MGAS166 that carry the vector plasmid (figure 2). Taken
together, these findings confirm that pASH2477 does not suppress capsule synthesis to a greater degree than does a single
intact copy of csrRS on the chromosome.
Emergence of spontaneous csrRS mutations. Our initial hypothesis that csrRS mutations might occur spontaneously was
JID 2001;183 (1 April)
supported by finding rare mucoid streptococcal colonies in cultures from mice infected with strain MGAS166. Eight such
isolates were saved: 6 were from blood or spleen tissue of mice
that had been infected with MGAS166 and Cytodex, one was
a mucoid variant from a persistent streptococcal abscess, and
one was a rare mucoid colony that appeared on routine passage
of strain MGAS166 on THYA. DNA sequencing of the entire
csrRS locus in these 8 strains showed that all had alterations
in either csrR or csrS (the first 8 strains listed in table 2). In
each strain, the variant phenotype (constitutive mucoid or LC)
corresponded with the alterations in csrR or csrS, respectively.
In 2 of the 4 csrS mutants, the deletion of thymidine residues
occurred within a string of thymidines (i.e., TTTTTTTTT at nt
75–83 of the open-reading frame). A third mutation involved the
spontaneous deletion of a thymidine at nt 492 from the sequence
TATA(T)TCTAAT. The fourth csrS mutation occurred as a result of an insertion of IS1548 at nt 345. All these mutations led
to early termination of CsrS translation. All 4 mutants were
Figure 2. Complementation of spontaneous csrRS mutants with pASH2477 (csrRS1). Upper panels, Portions of nonselective Todd-Hewitt
yeast extract agar plates streaked with representative variant strains. Spontaneous constitutive mucoid variant UMAA2904 (left) and spontaneous
large colony variant UMAA2899 (right) were grown in 5% CO2 with pASH2477-transformed derivative and plasmid-cured spectinomycin-sensitive
derivative of transformant. Note smaller size and matte appearance of pASH2477 transformant of both strains. As controls, MGAS166 (wild
type) and UMAA2392 (CsrRS2) are shown with their respective pASH2477-transformed and pJRS525-transformed derivatives (lower panels).
Neither plasmid affected the appearance of MGAS166. Although pASH2477 complemented the mucoidy of UMAA2392, the vector pJRS525
had no effect on the appearance of this constitutive mucoid strain. These controls confirm that the presence of the csrRS locus suppresses the
variant phenotype in the spontaneous mutants.
JID 2001;183 (1 April)
Table 2.
Spontaneous CsrR/CsrS Mutants of S. pyogenes
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Alterations in DNA sequence of csrRS among variant isolates of MGAS166.
a
Isolate no.
Gene
Type of mutation
Consequence of mutation
UMAA2207
UMAA2209
UMAA2210
UMAA2211
UMAA2057
UMAA2058
UMAA2088
UMAA2345
csrS
csrS
csrS
csrS
csrR
csrR
csrR
csrR
Frameshift (DT between nt 76 and 83)
Frameshift (DT at nt 492)
IS1548 insertion at nt 342
Frameshift (DT between nt 76 and 83)
Missense
Missense
Missense
Deletion of 30 nt bases
UMAA2749
UMAA2752
UMAA2759
UMAA2760
UMAA2762
UMAA2763
UMAA2764
UMAA2765
UMAA2767
UMAA2880
UMAA2882
UMAA2887
UMAA2875, UMAA2877, UMAA2878,
UMAA2881, and UMAA2884
csrR
csrS
csrS
csrS
csrS
csrR
csrS
csrR
csrS
csrS
csrS
csrS
csrS
Nonsense
Missense
Frameshift (DAA at nt 321)
IS1548 insertion (detected by PCR)
Frameshift (DGAAAA at nt 218)
Missense
None (sequence incomplete)
Nonsense
Nonsense
Missense
Frameshift (insertion of GC at nt 1136)
Missense
Frameshift (insertion of T at nt 1105)
Early termination after aa residue 35
Early termination after aa residue 181
Insertional inactivation
Early termination after aa residue 35
R203S
R94C
M86V
Deletion of aa 211–220 (W1 region of
DNA binding domain)
E159ochre
M260T
Early termination
Early termination
Early termination
G204D
E159ochre
E159ochre
S254F
Early termination
R241S
Early termination
Complemented
by pASH2477
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ND
Yes
Yes
Yes
ND
ND
ND
ND
ND
Yes
Yes
ND
No
ND
NOTE. aa, Amino acid; ND, not done; nt, nucleotide; PCR, polymerase chain reaction.
a
Nucleotide residues in csrS are numbered beginning at first base of start codon.
restored to the wt, nonencapsulated (matte colony) phenotype
by introduction of pASH2477 (csrRS1) by electroporation.
To assess the virulence potential of these mutants, 3 spontaneous variants were selected for mouse inoculation experiments.
These 3 strains were compared with the wt MGAS166 and a
defined CsrRS mutant. The results of these experiments confirmed that the spontaneous mutants produced enhanced dermonecrosis (table 3). The spontaneous csrR mutants produced
highly destructive lesions identical to those induced by the sitedirected CsrRS mutant. The appearance of the lesions produced
by the csrS mutant (thymidine deletion) was equivalent to that
previously observed with another strain carrying a large sitedirected deletion in csrS. Infection with these mutants causes a
distinctive spreading dermal ischemia with late necrosis and
sloughing of the skin, but the ultimate frequency of dermonecrosis is similar to that observed with csrR mutants [16].
To determine how frequently csrR or csrS mutants emerge
during infection, we conducted a prospective study of 10 mice
after infection with 3 3 10 6 cfu of MGAS166 (table 4). Five
mice were killed on day 2 because of advanced lesions; the
other 5 were killed on day 5. Table 4 shows the sizes of the
lesions that developed in these animals, the frequency of bacteremic isolates, and the occurrence of mucoid strains. In this
experiment, only those mice that developed necrotic lesions
were bacteremic. Larger lesions occurred among animals with
mucoid isolates from either the wound or blood. The largest
lesion was seen in the animal with the highest frequency of
variant isolates from the lesion. Finally, 5 of the 6 animals that
eventually developed necrosis had mucoid isolates taken from
either the lesion or blood. None of the 4 animals that did not
develop necrosis had mucoid isolates taken from any site
(P p .05, Fisher’s exact test, 2-tailed). In all 5 mice with mucoid
isolates, there were isolates with the LC phenotype, which is
consistent with csrS mutation, and 3 of these mice also had
isolates with constitutive mucoidy, which is consistent with csrR
mutation. We demonstrated an IS1548 insertion in csrS in 1
LC isolate (table 2).
In a subsequent experiment, LC isolates were obtained from
all 8 of 8 necrotic wounds that were cultured 72 h after inoculation. Three animals with very large necrotic wounds were
killed before 72 h, and cultures were not done. Five of these
wounds had isolates with the identical frameshift mutation csrS
(an insertion of thymidine at nt 1106), which suggests that this
mutation arose in broth culture before inoculation. Three other
unique csrS mutations were observed in isolates from 3 other
animals—frameshift due to insertion of GC at nt 1137, missense
resulting in an S254F substitution, and missense resulting in
an R241S substitution (table 2). Electroporation of pASH2477
suppressed the LC phenotype of the S254F mutant strain but
not the R241S mutant strain; consequently, the significance of
the latter mutation remains uncertain. The strain may carry an
additional mutation affecting the phenotype.
In a final experiment, mucoid or LC variants of MGAS166
were obtained from 16 mice with necrotic lesions. Five mice
had only LC variants, 6 had constitutive mucoidy, and 5 had
isolates with both phenotypes. Three of the constitutive and 3
of the LC strains were transformed with pASH2477; in all but
1 (an LC isolate), the variant phenotype was suppressed by the
presence of the plasmid and was returned after curing. Figure
2 shows representative isolates.
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Engleberg et al.
Table 3. Lesion type and lethality in dermal infections of mice with
MGAS166 and spontaneous mutants.
Strain
MGAS166 (wt)
UMAA2392 (DcsrRS2)
UMAA2057
UMAA2058
UMAA2211
Colony
phenotype
Total
no.
Matte
Mucoid
Mucoid
Mucoid
Large colony
5
5
5
5
5
No. with
No. with
No.
lesions skin necrosis died
3
5
5
5
5
0
5
5
5
4
0
4
4
2
1
Spontaneous csrR mutation in a human clinical isolate. Since
the csrRS sequences of nonisogenic streptococcal strains may
have silent or conservative substitutions, we examined clinical
isolates by complementation with pASH2477, to determine
whether they possess a functional csrRS locus. A panel of 16
isolates from cases of invasive human infections was transformed with the plasmid and was compared with respect to
colony size and appearance after growth in CO2. After transformation, 7 of these isolates yielded smaller colonies; all 7
yielded larger colonies again when the plasmid was cured. These
7 strains are indicated in table 1. One strain, UMAA2716, had
a dramatic loss of its highly mucoid appearance when the plasmid was present, and curing of the plasmid restored abundant
capsule production (figure 3). DNA sequence analysis of this
isolate showed that the csrRS locus differs from that of
MGAS166 by substitutions of 4 nt. Two substitutions in csrS
and 1 in csrR do not alter the amino acid sequence. One substitution in csrR (CrT) predicts a substitution of cysteine for
arginine at position 144 in CsrR (i.e., R144C). These findings
suggest that nearly half the clinical isolates lack CsrR or CsrS
activity and that 1 of the 12 carries a variant csrR associated
with abundant mucoidy.
Enhancement of virulence by coincident populations of csrR
or csrS mutants. The previous experiments demonstrate that
mouse skin infection enriches for spontaneous mutations in
both csrR and csrS and that similar strains can be found in
invasive human infections as well. The results in table 3 suggest
that the emergence of spontaneous mucoid and LC variant
strains (probably csrR or csrS mutants) are associated with
more dermonecrosis and a higher frequency of bacteremia.
However, these isolates usually comprise a relatively small proportion of the total streptococcal population in the infected
animals. To explain the severity of the lesions, we hypothesized
that the mutant population may facilitate the growth of the wt
strain in vivo and that enhanced growth of the wt strain may
facilitate the formation of lesions.
All the currently recognized CsrRS-regulated gene products
are related to the expression of secreted substances (i.e., capsule
and exoproteins). With respect to pathogenesis, these bacterial
products presumably exert their primary action in the extracellular space in vivo. Therefore, we considered the possibility that
enhanced production of these secreted products by a population
of csrR or csrS mutants might substitute for production that
JID 2001;183 (1 April)
remains repressed by CsrRS during infection with the wt strain.
We hypothesized that a mixed infection with equivalent numbers
of wt and mutant bacteria would enhance the survival and growth
of the wt strain in vivo. Groups of animals were injected subcutaneously with either 106 cfu of MGAS166 or a mixture of 106
cfu of wt and 106 cfu of strain SBmuc7 (csrR::Tn916). The
transposon in strain SBmuc7 allows the 2 coinoculated strains
to be distinguished on antibiotic-containing media. At 48 h, lesions from the coinoculated animals and a comparable area of
skin from the animals that received only the wt strain were excised, were homogenized in a tissue grinder, and were cultured
quantitatively on media with streptomycin or with streptomycin
and tetracycline. We calculated the log-fold increase in bacteria
by using 106 as the starting quantity of each population. The
results of the experiment, shown in figure 4, demonstrate a 3.5log increase in the growth of the wt strain in vivo when coinoculated with a csrR mutant. When inoculated alone, the recovery
of MGAS166 from areas of minimal erythema at the site of
inoculation showed a 1 log decrease in colony-forming units.
To determine whether the csrR mutant exerts a systemic effect
in the mouse that permits growth of the wt, we inoculated
MGAS166 (wt) and strain SBmuc7 separately into opposite
flanks of the same mouse (n p 6) and together in the same site
in another set of 6 mice. Lesions developed in all 6 mice that
received the mixed inoculum and at the site of SBmuc7 inoculation in those mice in which inocula were separated. No lesions developed at sites where MGAS166 was inoculated alone.
This experiment demonstrates that the growth enhancement
effect is purely a local phenomenon and not a systemic effect
of the mutant.
Given this local growth enhancement, we were interested in
Table 4.
Spontaneous variant isolates of Streptococcus pyogenes
MGAS166 obtained from infected mice inoculated with 3 3 106 cfu.
Area, mm2
Lesion,
isolate no.
Necrosis
UMAA2749
UMAA2752
Necrosis
Not viable
Abscess
Necrosis
Necrosis
UMAA2759
UMAA2760
Necrosis
UMAA2762
UMAA2763
Abscess
UMAA2764
None
Abscess
Necrosis
UMAA2765
NOTE.
Cultures
Total
Necrosis
Lesion
Blood
232
153
20% Mucoid
Matte
Spleen
10% LC
170
126
151
31
—
28
3% Mucoid
Matte
Matte
Matte
Neg
Matte
141
141
Matte
LC
Neg
Neg
Matte
LC
113
3
50% LC
Rare mucoid
Neg
Neg
66
—
88
—
—
—
Rare LC
Neg
Matte
Neg
Neg
Neg
Neg
Neg
Neg
170
25
Mucoid
NT
NT
LC, large colony; Neg, negative; NT, not tested.
JID 2001;183 (1 April)
Spontaneous CsrR/CsrS Mutants of S. pyogenes
1049
Figure 3. Complementation of mucoid human clinical isolates with pASH2477 (csrRS1). Isolates were transformed with either pASH2477 or
vector plasmid, pJRS525, and were grown on adjacent sectors of Todd-Hewitt yeast extract agar plates containing spectinomycin in 5% CO2.
Strain UMAA2716 was a highly mucoid isolate that became matte when pASH2477 was introduced (upper left); strain appearance was unchanged
by transformation with pJRS525. Clinical isolate UMAA2723 did not change colonial morphology with either plasmid (upper right). Both strains
also were plated on nonselective media with respective pASH2477-transformed derivative and a spectinomycin-sensitive cured derivative (lower
panels). This experiment confirms that the matte appearance of UMAA2716 is dependent on the persistence of pASH2477. Some spontaneous
curing of pASH2477 occurred on nonselective media and produced occasional mucoid colonies among a lawn of matte colonies (left sector, lower
left panel).
determining whether the presence of a subpopulation of a csrR
or csrS mutant in an infection with predominantly wt bacteria
would produce a synergistic increase in virulence. To address
this question, we designed a checkerboard synergy experiment.
From preliminary experiments, we knew that the wt strain begins to yield necrotic lesions with inocula between 106 and 107
cfu, whereas the csrR mutant strain SBmuc7 produces necrosis
at inocula of 105–106 cfu. Consequently, these 2 strains were
grown to midexponential phase, and suspensions were prepared
containing 4 dilutions of each strain (i.e., 8 3 10 6, 4 3 10 6,
2 3 10 6, and 1 3 10 6 cfu per inoculum of MGAS166 and
8 3 10 5, 4 3 10 5, 2 3 10 5, and 1 3 10 5 cfu per inoculum of
SBmuc7) and mixtures of these inocula in various combinations. Each inoculum preparation then was injected subcutaneously into groups of 5 mice. We examined the mice at 24 and
48 h to measure the total lesion size and the presence and size
of any dermonecrosis. Results are shown in figure 5.
Certain combinations of strain MGAS166 and a csrR mutant
produce more necrotic lesions than twice the inoculum of either
strain alone. This was particularly so at the lowest inoculum
in which the strains were given in a 10:1 ratio (wt:mutant).
The total size of lesions (figure 5) and the area of necrosis (data
not shown) were also greater in certain coinfections than in
larger inocula of either strain alone. Mice with no lesions or
no necrosis were included in the calculation of the mean as
zeros. This experiment was repeated with similar results. In
brief, inoculation of MGAS166 and SBmuc7 at a 10:1 ratio
yielded more necrotic lesions at 24 h (3 of 5) than did a 2-fold
larger inoculum of either MGAS166 alone (0 of 5) or SBmuc7
alone (2 of 5).
In a similar experiment, we compared a DcsrS mutant,
UMAA2690, with the wt in a similar array of combinations.
No differences were noted when the strains were combined.
However, as in the experiment depicted in table 4, we discovered
that most animals inoculated with MGAS166 alone that developed necrotic lesions had LC variants isolated from their
wounds, which is consistent with spontaneous csrS mutants
(table 1). Several spontaneous mutants were complemented by
1050
Engleberg et al.
Figure 4. Recovery of MGAS166 (wild type) from murine skin lesions after inoculation alone or with csrR mutant. V, Recovery of
MGAS166 from lesions after inoculation alone; v, recovery of
MGAS166 from lesions after mixed inoculation (1:1) with SBmuc7;
m, recovery of SBmuc7 from same mixed infections.
transformation with pASH2477 and lost the LC phenotype,
which suggests that they are spontaneous csrR or csrS mutants.
Signaling between CsrR mutant and wt bacteria. To investigate mechanisms to explain the enhanced growth and apparent
pathogenicity of wt bacteria during coinfection with CsrRS mutants, we focused on the genes that encode capsular synthesis. A
reporter plasmid expressing the streptococcal gene for alkaline
phosphatase under the control of the hasA promoter (phas-phoZ)
was introduced into the wt strain MGAS166 and its DhasAB
derivative, UMAA2497. Overnight cultures of these indicator
strains were mixed at a 10:1 ratio (OD600 p 0.05) with overnight
cultures of various reporterless strains (OD600 p 0.005 ) and were
grown in THY broth at 377C for 1–2 h (to OD600 p 0.4–0.5).
Culture supernates were analyzed for PhoZ activity and were
normalized to activity measured from cultures of wt only. The
results show that cocultures of wt harboring phas-phoZ and a
csrRS mutant at a 10:1 ratio (wt:mutant) have enhanced transcription of hasA (figure 6). When the reporter strain is a DhasA
mutant, the transcriptional enhancement is enhanced further.
Conversely, the effect is completely eliminated when either reporter strain is cocultivated with UMAA2526, a DcsrRS, DhasAB
mutant. However, enhancement does not require the presence of
an intact capsule, since the transcriptional enhancement is unaffected by the addition of hyaluronidase 0.1 mg/mL to the coculture. The specificity of hasA induction is confirmed by the
failure of any of these conditions to enhance expression from the
rofA promoter [20].
To determine whether encapsulated strains produce a soluble
substance that induces wt expression of hasA, we next collected
and tested supernates from cultures of various strains. Supernates were obtained from cultures sampled at early log phase
(OD600 p 0.4), midlog phase (OD600 p 0.8), and early station-
JID 2001;183 (1 April)
ary phase (OD600 p 1.2) and were filtered through a 0.45-mm
Millipore membrane. Aliquots from a log-phase culture
(OD600 p 0.4) of reporter strain UMAA2497 (DhasAB) carrying the phas-phoZ were centrifuged, and the pellets were resuspended in the filtered supernates at 377C for 1–2 h. Culture
supernates then were assayed for alkaline phosphatase and were
normalized to values obtained by incubating supernates from
the DhasAB mutant (UMAA2497) with the reporter strain. The
results show that midlog-phase supernates from mucoid strain
UMAA2392 (DcsrRS2) induced nearly a tripling of alkaline
phosphatase production (figure 7). The inducer was not present
at the earlier time point and dissipated dramatically in stationary-phase cultures. These observations are consistent with
the hypothesis that capsule expression generates the inducer,
since the most abundant inducing activity coincides with the
period of abundant capsule synthesis in UMAA2392. In this
experiment, the addition of hyaluronidase reduced the peak
induction of hasA transcription (figure 7). As expected, supernates from the DcsrRS, DhasAB mutant possessed no inducing
activity at any time point (figure 7).
Discussion
For positively regulated virulence genes, mutation of the regulator or regulatory system results in the loss of function and
pathogens with reduced capacity to cause infection or disease.
The CsrRS system of group A streptococcus is unusual in that
all the known virulence factors that it controls are regulated
negatively [14, 15]. Consequently, mutation of the genes encoding this system result in enhanced expression of capsule and
certain toxins and in increased virulence in a mouse skin infection model. This report demonstrates that such mutations
occur in vivo. This phenomenon raises several interesting questions about the role of spontaneous mutants and the regulatory
system itself.
Spontaneous mutants were usually apparent by inspection
of colonies growing on agar medium. We obtained confirmation
that these strains have mutations in the csrRS locus by plasmid
complementation analysis. Although this method provides 2–
4 copies of the csrRS locus in trans, we found no evidence that
the increased copy number results in oversuppression of the
CsrR-regulated genes. Sequencing of the csrRS locus of complemented strains invariably showed DNA alterations in the
suspected gene.
We observed that most spontaneous mucoid variants that
arise during mouse infections have mutations in the csrR gene.
Similarly, spontaneous LC variants were associated with mutations in csrS. For both variant phenotypes, the introduction
of an intact csrRS locus of a plasmid returned the bacteria to
their original wt colonial appearance, and curing of the plasmid
restored the variant mucoid or LC phenotype, respectively. Although the plasmid used for these complementation experiments provides 2–4 copies of the csrRS locus in trans, we found
JID 2001;183 (1 April)
Spontaneous CsrR/CsrS Mutants of S. pyogenes
1051
Figure 5. Skin infection of mice with MGAS166 (white squares and bars), SBmuc7, a csrR mutant (dark gray squares and bars), or mixtures
of the 2 strains (light gray squares and bars). Inocula for each data point on checkerboard are indicated by intersection of X-axis (2-fold dilutions
of SBmuc7; 13, 105 cfu) and Y-axis (2-fold dilutions of MGAS166; 13, 106 cfu). Bars in upper portion of graph indicate mean total lesion areas
in mm2 (erythema plus induration or abscess plus necrosis, Y-axis) at 24 and 48 h. Below the 3-dimensional bar graph, a similar checkerboard
plot shows the no. of mice in each group of 5 that developed dermonecrotic lesions.
no evidence that the increased copy number results in more
suppression of CsrR-regulated genes than occurs in the wt
strain with a single chromosomal copy of csrRS.
Both the mucoid and LC variants also produce enhanced
mouse virulence, as measured by lesion size and progression to
necrosis. We suspect that the LC variants are identical to those
described by Raeder et al. [21] and that csrS null mutation is
the usual mechanism by which these variants arise. Both the
variants described by this group and our LC mutants have
decreased expression of SpeB [16].
The spontaneous csrR mutations described in this report consist mostly of single amino acid substitutions in or near critical
domains required for the repressor function of this protein. In
contrast, mutations in csrS occurred more frequently and were
more often frameshift or nonsense mutations. Initially, mutations in a string of thymidines in csrS led us to speculate that
a mechanism of phase variation of this gene might result from
slipped-strand mispairing during replication, as described for
Bordetella pertussis by Stibitz et al. [22]. Subsequently, we encountered frameshift mutations in numerous other locations
within the gene and concluded that this T-rich segment is not
the site of most mutations. Nevertheless, one might predict that
reversion to wt csrS may occur at high frequency in a subset
of our spontaneous mutants.
Examination of LC variant mutants also showed insertions
of IS1548 into the csrS reading frame. This IS element was
found in all group A streptococci examined. Insertion of a
mobile element is of particular interest as a modifier of virulence, since this element also inserts into the hyaluronidase gene
of group B streptococcus and abrogates its function [23]. A
review of 4 insertion sites of IS1548 in other locations on the
group A streptococcal chromosome suggests that the sequence
TGTCTA is found close by. This sequence is present once in
the csrR open-reading frame and 3 times in csrS. One copy of
this sequence is adjacent to the csrS insertion that we sequenced.
The presence of this possible IS element “hot spot” suggests
that csrS may have evolved to generate ablative mutations at
a high frequency. A hypermutable csrRS locus might be adaptive if the loss of CsrRS regulation actually offers the organism
some survival advantage.
The experiment depicted in figure 4 shows that a CsrRS2
mutant coinoculated with the wt strain dramatically enhances
growth of the wt bacteria in vivo. The simplest explanation for
this observation is that the mutant creates a microenvironment
that is permissive for growth of the coinoculated wt strain. This
is consistent with the observation that the known CsrRS-regulated virulence factors are extracellular. Since the wt bacteria
do not have the burden of synthesizing large amounts of capsule
1052
Engleberg et al.
JID 2001;183 (1 April)
well-established example of this phenomenon is the emergence
of mucoid Pseudomonas aeruginosa among patients with cystic
fibrosis (CF). As in streptococci, pseudomonal mucoidy arises
by mutations affecting regulators. In the case of P. aeruginosa,
these regulators are S factors that control alginate synthesis. One
of these regulators, j54, serves as a negative regulator, and spontaneous mutations in the rpoN gene, which encodes j54, leads
to mucoidy [24]. In addition, the mucoid isolates from CF patients arise in naturally hypermutable strains. This mutator phenotype was not found in P. aeruginosa isolated from patients
without CF [25]. It is not known whether our wt strain,
MGAS166, possesses a hypermutable phenotype or whether such
a phenotype would predict more virulent streptococcal infection.
In our mouse experiments, we observed enhanced virulence
even when the spontaneous mutants accounted for a relatively
small proportion of the total infecting bacterial population. We
therefore wanted to determine what mixtures of mutant and
Figure 6. Transcription of hasA in wild-type (wt) bacteria during
coculture with encapsulated strains. MGAS166 (wt) and hasAB deletion mutant were used as reporter strains after electroporation with
phas-phoZ or prof-phoZ. Reporter strains were cocultured with various
other strains in 10:1 ratio, as indicated under diagram: MGAS166, a
DcsrRS mutant (with or without addition of hyaluronidase 1 mg/mL),
and DcsrRS, DhasAB mutant. After 1–2 h of coculture at 377C, supernates were assayed for alkaline phosphatase, and activities were
normalized to MGAS166 coculture. A 1:10 proportion of encapsulated
DcsrRS strain induced hasA transcription in wt strain and to higher
levels in unencapsulated DhasAB reporter strain. In contrast, a nonencapsulated DcsrRS, DhasAB mutant failed to induce hasA above baseline. Reporter strain carrying a rofA-phoZ fusion was not induced by
any of these coculture conditions.
and toxins, they may actually outgrow the mutant strains in
vivo. Similarly, the emergence of spontaneous mutants in vivo
in experimentally infected mice was associated with more severe
outcomes of infection, including larger lesion size and necrosis
(table 4). It was therefore of interest to determine whether such
strains emerge in human disease as well.
We analyzed a small panel of human clinical isolates derived
from patients with invasive streptococcal disease by complementation with a plasmid carrying the csrRS locus; several had
significant phenotypic changes associated with the presence of
the plasmid. A striking example is isolate UMAA2716 (shown
in figure 3). DNA sequencing of this isolate identified a substitution in csrR that most likely accounts for the loss of function and the hypermucoid appearance of this strain. We speculate that the emergence of such mutants in humans may also
affect the outcome of infection.
The notion that a pathogen may mutate to a more virulent
state in vivo is novel but not unprecendented. One striking and
Figure 7. Enhanced transcription of hasA in MGAS166 carrying a
DhasAB mutation by addition of supernates from cultures of encapsulated bacteria. Filtered supernates were obtained from streptococcal
strains grown to various densities (as indicated) in liquid culture and
were added to a midlog-phase culture of reporter strain UMAA2497
(DhasAB) with phas-phoZ for 1–2 h. Supernates were derived from cultures of DcsrRS2 (M), DcsrRS2 plus hyaluronidase 1 mg/mL (m), and
DcsrRS, DhasAB (V). After incubation with reporter strain, supernates
were assayed for alkaline phosphatase activity and were normalized to
results obtained with supernate from DhasAB strain UMAA2497 (m).
hasA transcription was increased by incubation with filtered supernate
from late log-phase culture of encapsulated (DcsrRS) strain but not an
unencapsulated (DcsrRS, DhasAB) strain. Addition of hyaluronidase
reduced peak induction by encapsulated filtrate by ∼50%.
JID 2001;183 (1 April)
Spontaneous CsrR/CsrS Mutants of S. pyogenes
wt bacteria would produce enhanced virulence. Figure 5 shows
that wt infections containing a subpopulation of CsrRS2 bacteria cause more aggressive disease than did the wt strain inoculated alone at comparable levels. Although less dramatic,
there is also a suggestion that the mutant strain may be more
aggressive when a wt population is present in the lesion. For
example, a mixture of 106 cfu of the wt strain (“13” in figure
5) and 105 cfu of the CsrRS mutant (also labeled “13”) produces larger lesions with more necrosis than does “23” of either
strain inoculated alone. This pathogenic synergy suggests that
each of the bacterial strains in the mixture may profit from the
presence of the other in the lesion.
The mechanisms responsible for this enhancement are not
known, but we generated preliminary evidence that some signaling may occur between the mutant and wt bacteria. Specifically, we found that encapsulated strains produce a nonfilterable substance that induces transcription of hasA in wt bacteria.
The modification of the response by addition of hyaluronidase
and the absence of the effect when the DcsrRS, DhasAB mutant
is mixed with reporter strains suggest that the inducer may be
an oligomer of hyaluronic acid or some other soluble degradation product derived from the capsule. We conclude that
group A streptococci possess a positive amplification system
for capsule production and that a small proportion of heavily
encapsulated bacteria may stimulate a neighboring population
to produce capsule as well. This may partly explain the enhanced survival and growth of wt bacteria in mixed infections
with CsrRS mutants and the observed pathogenic synergy in
mouse skin infections.
Of note, we demonstrated synergy with csrRS mutants, even
though csrR and csrS mutations may have arisen spontaneously
from the wt bacteria in the inocula. This potential problem did
not obscure the demonstration of synergy with a csrR mutant;
however, it confounded our attempt to demonstrate synergy
with a csrS mutant. Since csrS mutants are less aggressively
virulent than csrR mutants, the experiment may not have been
sufficiently robust to detect any differences. In future experiments, it will be necessary to generate wt strains for comparison
that cannot easily undergo csrRS mutation (e.g., a csrRS merodiploid strain). Nevertheless, it is possible that csrS mutants
do not synergize with wt bacteria, since they do not produce
the excess capsule that is characteristic of csrR mutants and
therefore should not signal the wt strain to produce capsule.
Reliable induction of mouse skin infections with the wt strain
requires large inocula unless Cytodex beads are mixed with the
bacteria [19]. In our synergy experiments, Cytodex was not used,
and we observed that increasing inocula did not always result in
larger lesions. Because of the relative insensitivity of mice to wt
infection, the range between the minimal infectious dose and the
dose that produces the maximal measured response (e.g., a plateau in lesion size) may be narrow. Some very large inocula produced measurably smaller lesions. The reason for this paradoxical
1053
finding is not obvious, but we speculate that bacteria injected at
very high density may grow more slowly and spread less rapidly
in tissue [26]. In addition, we observed that very large, severe
lesions sometimes are measurably smaller on subsequent measurements, possibly as a result of dehydration. Both factors may
limit the usefulness of lesion size as an indicator of virulence at
the severe end of the disease spectrum.
The large inocula required to generate lesions in the mouse
model may not be typical of the number of organisms that initiate
naturally occurring human disease. Since some of our experiments yielded identical mutants from individual mice, we can
deduce that csrRS mutants already were present in the culture
from which the initial inocula were prepared. In humans, we do
not know whether csrRS mutants are acquired as such or emerge
spontaneously in vivo. In the first case, we would expect de novo
infections with csrRS mutants to be relatively more virulent.
Assuming that all evolutionary changes are adaptive, it is likely
that complete derepression of the CsrRS regulon must also have
some counteradaptive features; otherwise, this control mechanism would not exist or all streptococci would be csrRS mutants.
Counteradaptive features of the variants might include poor
transmissibility or adherence, or perhaps the enhanced virulence
of these variants per se is ultimately counterselective and is unlikely to be maintained as a consistent feature of group A
streptococci.
The second case, that csrRS mutations emerge in vivo, presents several interesting possibilities. For example, a spontaneous mucoid mutant in a superficial colonizing population
may be more likely to survive after invasion of mucosal barriers
than is the less consistently encapsulated strain from which it
is derived. Alternatively, a csrRS mutant arising from a population that has already occupied a sterile body site might synergize with its parental strain to produce more aggressive disease. In this latter situation, infections that develop from
smaller inocula of CsrRS1 bacteria would be less likely to give
rise to csrRS mutants. The rare mutational event occurring
early in the course of infection would lead to the largest proportion of variant bacteria and the most severe clinical manifestations. One might speculate that the severity of a streptococcal infection may be stochastically determined by the timing
of the emergence of the variant population.
The notion that variant populations influence the outcome
of human streptococcal infection is unproven, but it is consistent with our data. Our findings demonstrate that human infections may be associated with CsrR2 or CsrS2 strains, but
we do not know whether these strains were acquired with these
mutations or whether they arose in vivo. Nor do we know what
proportion of the infecting population they represent, since only
single isolates were obtained from each patient. If disease outcome is influenced by >1 virulence-enhancing subpopulations
coexisting with the primary infecting strain, then characterization of a single isolate from an infected person may not pro-
1054
Engleberg et al.
vide an accurate picture of the pathogenic process in that person
or the potential virulence of the infecting strain.
Acknowledgments
We gratefully acknowledge the assistance of Michael Caparon
(Washington University, St. Louis), Neil Barg (Internal Medicine Association of Yakima, Yakima, Washington), Don Low (University of
Toronto, Toronto), June R. Scott (Emory University, Atlanta), and
Kevin McIver (University of Texas–Southwestern, Dallas) for providing helpful advice or bacterial strains.
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