Regulation of polysaccharide utilization contributes to the persistence of group a streptococcus in the oropharynx.

Abstract

Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its DeltamalR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the DeltamalR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the DeltamalR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of DeltamalR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.

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INFECTION AND IMMUNITY, June 2007, p. 2981–2990
0019-9567/07/$08.00?0 doi:10.1128/IAI.00081-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 6
Regulation of Polysaccharide Utilization Contributes to the Persistence
of Group A Streptococcus in the Oropharynx?
Samuel A. Shelburne III,1Nnaja Okorafor,1Izabela Sitkiewicz,2Paul Sumby,2David Keith,1
Payal Patel,2Celest Austin,3Edward A. Graviss,3and James M. Musser2*
Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, Houston, Texas 770301; Center for
Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute,
Houston, Texas 770302; and Department of Pathology, Baylor College of Medicine, Houston, Texas 770303
Received 13 January 2007/Returned for modification 13 March 2007/Accepted 21 March 2007
Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization
show growth phase-dependent expression in human saliva. We sought to determine whether the putative
polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR
helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR
is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and
the levels increased significantly during growth in human saliva compared to the levels during growth in
glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide
utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization
proteins in the parental serotype M1 strain MGAS5005 and its ?malR isogenic mutant derivative. The
transcript levels of all eight genes were significantly increased in the ?malR strain compared to the parental
strain, especially during growth in human saliva. Following experimental infection, the ?malR strain persis-
tently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers
of ?malR strain CFU recovered were significantly lower than the numbers of the parental strain CFU
recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in
polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.
Genetic studies have shown that some of the microbial genes
that are most widely and differentially expressed are those that
encode proteins putatively involved in carbohydrate transport
and metabolism (1, 3, 32, 37, 44). Changes in the transcript
levels of these genes are often accompanied by alterations in
genes encoding classical virulence factors, such as secreted
toxins, proteases, immune effector molecules, and capsule syn-
thesis proteins (17, 25, 28, 35, 46). Thus, genome-wide inves-
tigations of major bacterial pathogens have suggested that
there is a close link between basic metabolic processes and
pathogenesis (25, 32, 35). These findings in turn have led to
recent investigations attempting to more clearly elucidate the
relationship between bacterial nutrition and infection (7, 17,
34, 41).
In humans, group A Streptococcus (GAS) causes diverse
infections ranging from innocuous (e.g., simple colonization
and uncomplicated pharyngeal and skin infections) to life
threatening (e.g., necrotizing fasciitis and toxic shock syn-
drome) (10). This diversity implies that GAS infection involves
complex regulatory networks that are different in different
environments (21). When faced with new surroundings, GAS
not only alters the transcription of genes involved in meeting
basic metabolic demands but also alters the transcription of
genes encoding major virulence factors (15, 25, 35, 38). There-
fore, gene regulation in GAS forms a link between basic nu-
trition and pathogenesis.
The major site of GAS infection and colonization in humans
is the oropharynx (29). A key mediator of acquired and innate
immunity in the human oropharynx is saliva, and we have
recently been exploring molecular interactions between GAS
and human saliva (33). Using expression microarrays, we an-
alyzed GAS transcription during the logarithmic and stationary
growth phases of GAS organisms in human saliva (35). Of the
nearly 1,700 genes in the GAS genome, we found that 14 of the
15 genes that had the largest growth phase-dependent changes
in transcript levels encode proteins that are thought to be
involved in carbohydrate transport and metabolism (35).
Moreover, 8 of these 14 genes encode proteins that are either
putatively or known to be involved in the uptake and process-
ing of polysaccharides (35). In studies of GAS growth in a
nonhuman primate model of pharyngitis, in human blood ex
vivo, and in soft tissue infection in mice, we found that the
transcription of genes encoding polysaccharide utilization pro-
teins was highly dynamic (14, 15, 44). Taken together, these
data suggest that the regulation of genes involved in polysac-
charide degradation, transport, and utilization likely contrib-
utes to GAS host-pathogen interactions.
Recently, we demonstrated that the GAS putative maltodex-
trin binding protein MalE was important for colonization of
the oropharynx (34). The transcript level of malE was in-
creased nearly 100-fold during growth in human saliva com-
pared to growth in a standard laboratory medium (34). In this
work we sought to determine whether the transcript levels of
seven other genes encoding proteins putatively involved in
* Corresponding author. Mailing address: Center for Molecular and
Translational Human Infectious Diseases Research, The Methodist
Hospital Research Institute, Houston, TX 77030. Phone: (713) 441-
5890. Fax: (713) 441-3447. E-mail: jmmusser@tmh.tmc.edu.
?Published ahead of print on 2 April 2007.
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polysaccharide digestion, transport, and metabolism were ele-
vated in human saliva in a fashion similar to malE. We also
investigated whether the transcript levels of putative polysac-
charide utilization genes were influenced by MalR, a putative
transcriptional repressor not previously studied in GAS. We
discovered that MalR influences the expression of at least
eight genes residing in two distinct locations on the GAS
chromosome. Moreover, we found that MalR is essential for
high-level persistence of GAS in human saliva and in the
mouse oropharynx.
MATERIALS AND METHODS
Bacterial strains and culture media. The genome of serotype M1 strain
MGAS5005 has been completely sequenced (39). Strain MGAS5005 has been
studied extensively in animal models of GAS infection and in vitro, including
transcriptome analysis during growth in human saliva (34, 35, 40, 44). The other
bacterial strains used in this study are listed in Table 1. GAS was grown on
Trypticase soy agar containing 5% sheep blood (BSA) (Becton Dickinson) or in
Todd-Hewitt broth containing 0.2% (wt/vol) yeast extract (THY) (Difco) at 37°C
without shaking in 5% CO2. Spectinomycin (Sigma) was added to THY or THY
agar at a concentration of 150 ?g/ml when appropriate. Carbohydrates were
added at a concentration of 1.0% (wt/vol) to a carbohydrate-free preparation of
a commercially available chemically defined medium (CDM) (JR Biosciences)
(45). In this paper, we use the terms glucose-medium, maltose-medium, malto-
triose-medium, etc., to refer to the carbohydrate-free CDM with the carbohy-
drate indicated added at a concentration of 1.0% (wt/vol).
Growth of GAS in human saliva. GAS was grown in human saliva as previously
described (33). Briefly, saliva was collected, briefly centrifuged, and passed
through a 0.20-?m filter. GAS was grown in saliva without shaking at 37°C with
5% CO2. Healthy volunteers donated saliva according to a protocol for human
subjects approved by the Baylor College of Medicine Institutional Review Board
(33). Saliva pooled from at least four donors was used to minimize the effects of
donor variation on the study results. All comparative growth experiments were
done in duplicate on five separate occasions for a total of 10 replicates. To
determine whether any observed growth differences might be related to differ-
ences in clumping induced by saliva, Gram staining was performed on culture
aliquots. The average numbers of GAS in aggregates were determined by count-
ing 10 high-power fields for each aliquot.
DNA sequence analysis. Chromosomal DNA was isolated using a DNeasy kit
(QIAGEN). DNA sequencing primers were designed on the basis of the serotype
M1 strain MGAS5005 genome (39). The malR open reading frame M5005_Spy_
TABLE 1. Homology of MalR in various GAS strains
Strain
M
serotype
Year
isolated
Disease or
source
Nucleotide sequence
changes compared with
strain MGAS5005
% MalR amino acid
sequence identity
with strain
MGAS5005
SF370
MGAS2221
MGAS9506
MGAS10606
MGAS9490
MGAS10270
MGAS10599
MGAS315
SSI-1
MGAS10652
MGAS11787
MGAS9503
MGAS10750
MGAS9512
1
1
1
1
2
2
2
3
3
3
3
4
4
6
1985
1988
2001
2002
2001
2002
2002
1988
1994
2002
2003
2001
2002
2001
Wound infection
Scarlet fever
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Toxic shock
Toxic shock
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
None
None
None
None
G93A, A648G
G93A, A648G
G93A, A648G
G93A, C843T
G93A, C843T
G93A, C843T
G93A, C843T
G93A, A648G
G93A, A648G
G93A, A320T, C324T, T369C,
T513C, A648Ga
G93A, A320T, C324T, T369C,
T513C, A648Ga
G93A, A320T, C324T, T369C,
T513C, A648Ga
G93A, A320T, C324T, T369C,
T513C, A648Ga
G93A, A320T, C324T, T369C,
T513C, A648Ga
T168C, A648G
T168C, A648G
T168C, A648G
T168C, A648G
T168C, A648G
G93A, T624G, A648G
G93A, T624G, A648G
G93A, A648G
G93A, A648G
G93A, A648G
G93A, A648G
G93A, T624G, A648G
G93A, A648G
G93A, A648G
G93A, T624G, A648G
G93A, A648G
G93A, A648G
100
100
100
100
100
100
100
100
100
100
100
100
100
99.7
MGAS103946 2001 Pharyngeal99.7
MGAS11802 62003Pharyngeal
99.7
MGAS117916 2003Pharyngeal99.7
MGAS11847112003Pharyngeal99.7
MGAS2096
MGAS9429
MGAS9511
MGAS10604
MGAS11785
MGAS8232
MGAS11789
MGAS10603
MGAS6180
MGAS9513
MGAS10602
MGAS10600
MGAS9516
MGAS10601
MGAS9502
MGAS11790
MGAS11861
12
12
12
12
12
18
18
22
28
28
28
75
77
77
89
102
124
1980
1998
2001
2002
2003
1987
2003
2002
1998
2001
2002
2002
2001
2002
2001
2003
2003
APSGNb
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Blood
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
Pharyngeal
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
aThe A320T nucleotide substitution in serotype M6 and M11 strains results in an Asp107Val replacement at the amino acid level.
bAPSGN, acute poststreptococcal glomerulonephritis.
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1057 corresponds to open reading frame SPy1293 in serotype M1 strain SF370
(13). Sequence data obtained from both DNA strands with an Applied Biosys-
tems 3730XL DNA analyzer were assembled with Sequencher 4.5. The inferred
amino acid sequences were aligned and compared using the software program
CLUSTALW. All sequences that differed from the strain MGAS5005 sequence
were confirmed by repeating the entire DNA sequence analysis.
RNA isolation and transcript level analysis. Serotype M1 strain MGAS5005
was grown to the mid- and late-logarithmic phases. RNA was isolated with a Fast
Prep Blue kit (Q/BioGene) and was purified using an RNeasy kit (QIAGEN)
(35). The quality and the concentration of RNA were assessed with an Agilent
2100 Bioanalyzer and by analysis of the A260/A280ratio. cDNAs were created
from the RNA using Superscript III (Invitrogen) by following the manufacturer’s
instructions. For comparison of gene transcript levels during growth in various
media, TaqMan real-time quantitative reverse transcription-PCR (QRT-PCR)
was performed in quadruplicate with an ABI Thermocycler 7700 (Applied Bio-
systems) using the ?CTmethod of analysis (8). To compare gene transcript levels
of the parental and ?malR and ?malRv2 isogenic mutant strains, the ??CT
method was employed (user bulletin no. 2, ABI PRISM 7700 sequence detection
system; Applied Biosystems). TaqMan primers and probes for genes of interest
and the internal control gene proS are listed in Table 2. All experiments were
performed in quadruplicate on two separate occasions.
Creation of ?malR isogenic mutant strain. The ?malR isogenic mutant strain
was created by nonpolar insertional mutagenesis from parental serotype M1
strain MGAS5005 using the PCR-mediated method described by Kuwayama et
al. (23). Primers were designed to amplify the 5? and 3? ends of the malR gene
region along with nucleotide sequences that were complementary to the 5? or 3?
portion of the spectinomycin (spc) resistance cassette (Table 2). A third set of
primers containing nucleotide sequences complementary to the 5? and 3? ends of
the malR gene region was used to amplify the spc cassette from plasmid pSL60
(24). Fusion PCR was then used to link the 5? and 3? malR gene region PCR
products to the spc cassette via the overlapping nucleotide sequence regions (11,
23). This resulted in a PCR product in which the spc cassette, flanked by the 5?
and 3? malR gene regions, was inserted in place of nearly the entire malR open
reading frame. The gene disruption PCR construct was used to transform com-
petent GAS cells, which were then selected via spectinomycin resistance. The
?malR isogenic mutant strain was analyzed by Southern hybridization and DNA
sequencing to confirm that the proper genetic construct was obtained (data not
shown). Extensive efforts to complement the ?malR strain in trans were unsuc-
cessful. Therefore, we addressed the possibility that the effects observed in the
?malR strain were due to unrelated spontaneous mutations acquired during
construction of the mutant by creating a second, independent ?malR isogenic
mutant strain. Southern hybridization and DNA sequencing were used to con-
firm that the proper genetic construct was obtained (data not shown).
Carbohydrate uptake assays. Strain MGAS5005 and its ?malR isogenic mu-
tant derivative were grown to the mid-exponential phase in CDM in the presence
of the carbohydrate of interest added at a concentration of 1.0%. The bacteria
were collected by centrifugation, washed, and suspended to an optical density at
600 nm (OD600) of 0.5 in 150 ?l of carbohydrate-free CDM. [14C]maltose (600
TABLE 2. Primers and probes used in this study
PrimerSequence (5?33?) Target
malR-A
malR-B
GTT AGC TTG TTT AAA GGT ACC ACC
GTT ATA GTT ATT ATA ACA TGT ATT TGC CTT TTT GAG CGA
CAT CTT
CTA TTT AAA TAA CAG ATT AAA AAA ATT ATA AGA AAC AAA GAG
AAA GCG TCC GT
AAC TTC TAC ACT GTC CGC AGA AAC
AAG ATG TCG CTC AAA AGG CAA ATA CAT GTT ATA ATA ACT ATA AC
5? primer for 5? region of malR
3? primer for 5? region of malR with
spc sequence overlap
5? primer for 3? region of malR with
spc sequence overlap
3? primer for 3? region of malR
5? primer for spc along with malR
overlap sequence
3? primer for spc along with malR
overlap sequence
5? primer for Southern blotting
3? primer for Southern blotting
5? malR primer for QRT-PCR
3? malR primer for QRT-PCR
malR TaqMan probe
5? malP primer for QRT-PCR
3? malP primer for QRT-PCR
malP TaqMan probe
5? malQ primer for QRT-PCR
3? malQ primer for QRT-PCR
malQ TaqMan probe
5? malE primer for QRT-PCR
3? malE primer for QRT-PCR
malE TaqMan probe
5? malF primer for QRT-PCR
3? malF primer for QRT-PCR
malF TaqMan probe
5? malG primer for QRT-PCR
3? malG primer for QRT-PCR
malG TaqMan probe
5? pulA primer for QRT-PCR
3? pulA primer for QRT-PCR
pulA TaqMan probe
5? dexB primer for QRT-PCR
3? dexB primer for QRT-PCR
dexB TaqMan probe
5? msmK primer for QRT-PCR
3? msmK primer for QRT-PCR
msmK TaqMan probe
5? proS primer for QRT-PCR
3? proS primer for QRT-PCR
proS TaqMan probe for QRT-PCR
malR-C
malR-D
malR-spcF
malR-spcR GGA CGC TTT CTC TTT GTT TTA AAC CTT ATA ATT TTT TTT AAT CTG
TTA TTT AAA TAG
CGT CGT TGG CTA GAA TTT GCT
AGA ATC CAG CTT TTG CGA TG
AAC CCT TCG ACG GTG AGT AG
AAT CTG TGC TGC GAC ATT TG
TCC AAA TCT GCC ATC GCT TTT C
TGA TGA AGC GGT AGC TGT TG
GCA GTT GGC CAT TTT TCA AG
TGG TTG GTT ACA CTA ACC ACA CTA TTC TTG CA
CTC AGC CCC AGG TAT AGC AT
GG TTT TCT GCG ATG ATA GGG
TCC TAA GGC CTC ACG AAC CGC
TGA AAT CAT GGC AAA AAG TTA TCG
GAT CCA CAT CCC ACT AAC AAG GTA CT
GCA AGT GTC AAA CTT GCT CCG CCG
GAT TGG GTG GGA CTT GCT AA
CAG CCC AAA TCA AAG TCC AT
TTC CTGCCA TAC GGC CGC TT
TCA ATG CTT CAG CCA AGA TAC C
AGT GAT TGG GAC GGC AAT TA
ATG GCC TTC ACT GCT GGT TCT GTC
GGA CTA GCC CGC GAT GAA
CCT TGC TAG ACG ATT GAA GAC CAT
CTC AAC AAG GAG ATG GCA ATG CTA AAT TCT GG
AGC GTC AGC TTG GAG AAG AG
CCA CAT CTG TAT TGG CGA TG
TGC CAC AGA CTT AGC AGG TGC ACA
GTT AAA GAT GGC CGC ATT GT
ACC AGC AGC TTC AAG CAT TT
TGA CAT TGC TAT TCC AGA AGG ACA GCA
TGA GTT TAT TAT GAA AGA GGC TAT AGT TTC
AAT AGC TTC GTA AGC TTG ACG ATA AT
TCG TAG GTC ACA TCT AAT CTT CAT AGT TG
malR-SouthF
malR-SouthR
malR-F
malR-R
malR-P
malP-F
malP-R
malP-P
malQ-F
malQ-R
malQ-P
malE-F
malE-R
malE-P
malF-F
malF-R
malF-P
malG-F
malG-R
malG-P
pulA-F
pulA-R
pulA-P
dexB-F
dexB-R
dexB-P
msmK-F
msmK-R
msmK-P
proS-F
proS-R
proS-P
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mCi/mmol; 200 ?Ci/ml) and [14C]maltotriose (900 mCi/mmol; 100 ?Ci/ml) were
purchased from American Radiolabeled Chemicals, St. Louis, MO. [14C]maltose
was judged to be ?98% pure by thin-layer chromatography, whereas [14C]malto-
triose was found to be ?90% pure (American Radiolabeled Chemicals, personal
communication). Forty microliters of the14C-labeled sugars was added to GAS
cells at 37°C to obtain a final volume of 200 ?l and a final concentration of
radiolabeled carbohydrate of 40 ?M. At the times indicated below, 40-?l samples
were removed and filtered through a 0.45-?m nitrocellulose membrane (Milli-
pore), which was then rinsed twice with carbohydrate-free CDM. The radioac-
tivity retained on each filter was determined by liquid scintillation counting
(Beckman model LS7500). Samples were taken every 30 s for the first 120 s, a
period during which the carbohydrate transport rates were found to be linear.
The uptake rates were adjusted to the amounts of protein in the cultures as
determined by the Bradford assay (Bio-Rad). All experiments were performed in
quadruplicate on two separate occasions.
Mouse colonization experiments. All experiments with mice were performed
according to a protocol approved by the Baylor College of Medicine Institutional
Animal Care and Use Committee. Mouse throat colonization studies were
conducted with adult (18- to 20-g) female outbred CD-1 Swiss mice (Harlan
Sprague-Dawley Inc.) (24, 34). The MGAS5005 and ?malR strains were grown
in THY and harvested at an OD600of approximately 0.5. The cells were washed
once with sterile phosphate-buffered saline and suspended in phosphate-buffered
saline at a concentration of 1 ? 108CFU/ml. Each nostril of each mouse was
inoculated with 50 ?l of the GAS suspension (total inoculum, 1 ? 107CFU), and
there were 35 mice in each group. In previous experiments with MGAS5005 this
dose resulted in colonization of approximately 65% of the inoculated animals at
72 h (24). The throat of each mouse was swabbed before inoculation and then
daily thereafter. The throat swabs were streaked onto BSA and grown overnight
to obtain beta-hemolytic colonies. These colonies were then tested for the pres-
ence of GAS carbohydrate antigen via latex agglutination (BD Biosciences).
Blood collected by cardiac puncture from dead mice was cultured on BSA
overnight.
Statistical analysis. Growth and RNA transcript levels were compared using
Student’s two-sided t test. Radiolabeled carbohydrate uptake rates were com-
pared using linear regression. The ?2test was used to assess statistical differences
in throat colonization rates between the mice infected with strain MGAS5005
and the mice infected its ?malR isogenic mutant derivative. Student’s two-sided
t test was used to test for differences in the number of CFU recovered from
animals infected by the two strains. Statistical significance was assigned a two-
sided P value of 0.05 using Bonferroni’s adjustment for multiple comparisons
when appropriate. Statistical calculations were performed using the NCSS soft-
ware, version 2004.
RESULTS
GAS genome contains maltodextrin and pullulanase gene
regions. Maltodextrin is the term given to molecules composed
of two to seven linked glucose monomers. Polysaccharide is the
term for molecules containing more than seven glucose mono-
mers. In gram-positive bacteria, genes encoding proteins pu-
tatively involved in the binding, transport, and utilization of
maltodextrins and polysaccharides may be either contiguous or
located in separate parts of the chromosome (22, 42). In all 12
GAS strains sequenced to date, including strain MGAS5005,
these genes are found in two regions (2, 4, 5, 13, 16, 26, 36, 39).
Putative maltodextrin binding, transport, and utilization genes
are located in the gene region from M5005_Spy_1055 to
M5005_Spy_1060 (Fig. 1). This region includes M5005_Spy_
1058 (malE), a gene encoding a maltodextrin binding cell sur-
face lipoprotein recently shown to be necessary for GAS to
colonize the oropharynx (34). Genes encoding proteins puta-
tively involved in the production of maltodextrin from poly-
saccharides, maltodextrin degradation, and ATP binding are
located in a separate part of the chromosome, from M5005_
Spy_1680 to M5005_Spy_1682 (Fig. 2). This region includes
the gene encoding M5005_SPy_1680 (PulA), an LPXTG cell
wall-anchored pullulanase thought to be involved in GAS vir-
ulence (20, 30, 31).
In this paper, we refer to the region comprising the genes
M5005_Spy_1055 to M5005_Spy_1060 as the maltodextrin gene
region and the region comprising the genes M5005_Spy_1680 to
FIG. 1. Schematic diagram of the maltodextrin and pullulanase gene regions in the chromosome of GAS strain MGAS5005. The M5005_spy
number is the gene number in serotype M1 strain MGAS5005. The same gene arrangement is present in all 12 GAS strains sequenced to date (2,
4, 5, 13, 16, 26, 36, 39).
FIG. 2. Transcript levels of maltodextrin (A) and pullulanase
(B) region genes are increased during growth in a maltotriose-medium
and human saliva. Strain MGAS5005 was grown to the mid-exponen-
tial or late-exponential phase in either standard laboratory medium
(THY), CDM containing 1% maltotriose, or human saliva. TaqMan
real-time QRT-PCR was performed using probes and primers listed in
Table 2. The transcript levels of target genes were normalized to those
of proS, a gene that is expressed constitutively throughout the GAS cell
cycle and whose transcript levels are similar when the organism is
grown in THY and when the organism is grown in saliva (35, 43). For
each gene, the order of bars from left to right is as follows: mid- and
late-exponential growth phases in THY, maltotriose-medium, and hu-
man saliva. The transcript levels are the means ? standard deviations
of quadruplicate measurements obtained on two separate occasions.
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M5005_Spy_1682 as the pullulanase gene region. The maltodex-
trin gene region contains three of the four genes that putatively
encode an ATP binding cassette (ABC) transport system, a
common mechanism used by bacteria to import low-molecular-
weight substrates (18). The fourth gene, M5005_Spy_1682
(msmK), which encodes the putative ATP-binding portion of
the maltodextrin ABC transport system, is located in the pul-
lulanase gene region. The gene encoding the putative tran-
scriptional regulator MalR (M5005_SPy_1057) is located in
the maltodextrin gene region and is transcribed divergently
from malE. The pullulanase gene region does not contain a
putative transcriptional regulator. The lack of a gene encoding
a transcriptional regulator in the pullulanase gene region sug-
gests that expression of genes in this region is influenced by
proteins encoded in other regions of the GAS genome.
Transcript levels of genes in the maltodextrin and pullula-
nase gene regions are increased in human saliva and a malto-
triose-medium compared to a nutrient-rich medium. Before
the present study, we had demonstrated that malE transcript
levels were significantly higher during growth in human saliva
and in a maltodextrin-medium than during growth in a nutri-
ent-rich medium (THY) (34). Now, we asked whether the
same was true for putative polysaccharide utilization genes
other than malE. As assessed by real-time QRT-PCR, the
transcript levels of malP, malQ, malF, and malG during the
mid-exponential phase of growth in human saliva and in a
maltotriose-medium were increased an average of 75-fold
compared to the levels in THY (P ? 0.001 for both human
saliva and in the maltotriose-medium) (Fig. 2A). Moreover,
the transcript level for each of the maltodextrin region genes in
a maltotriose-medium and in saliva was higher during the mid-
exponential phase than during the late-exponential phase (Fig.
2A) (34). Similar to the findings for the maltodextrin region
genes, the transcript levels of each of the three genes in the
pullulanase gene region were significantly higher during
growth in a maltotriose-medium and human saliva than during
growth in THY (Fig. 2B). The pulA transcript levels followed
a pattern nearly identical to the pattern for the maltodextrin
utilization genes; they were higher during the mid-exponential
growth phase than during the late exponential phase in a malto-
triose-medium and human saliva (Fig. 2B). The transcript lev-
els of dexB and msmK were less growth phase dependent.
Taken together, these data suggest that gene transcript levels
in the maltodextrin and pullulanase gene regions were in-
creased during growth in a maltotriose medium and human
saliva and that similar regulatory mechanisms influenced the
two gene regions.
Putative maltodextrin and pullulanase transcriptional reg-
ulator MalR is highly conserved among diverse GAS strains.
As the marked elevation of the transcript levels for the malto-
dextrin and pullulanase region genes in a maltotriose-medium
and human saliva suggested that the two gene regions share
some aspects of transcriptional regulation, we next focused on
MalR, the only putative transcriptional regulator encoded in
the two gene regions. Some transcriptional regulatory proteins
are highly variable, whereas others are well conserved among
GAS strains (6). Moreover, single nucleotide changes or inser-
tions have been shown to affect the function of GAS transcrip-
tional regulators (4, 40). To determine whether MalR is con-
served among genetically diverse GAS strains, we sequenced
the malR gene from 35 strains representing 18 different M
serotypes (Table 1). All strains tested had the malR gene. Nine
nucleotide changes were identified, and the mean frequency of
these changes was 2.46 nucleotide substitutions in the 1,017-bp
open reading frame compared to the reference allele in sero-
type M1 strains. Eight of the nucleotide changes were synon-
ymous substitutions resulting in less than one amino acid re-
placement per 339 amino acid sites (Table 1). There were no
insertions, deletions, or nucleotide changes that resulted in
truncated proteins. Thus, MalR is very highly conserved among
genetically diverse GAS strains.
malR transcript levels are significantly higher during
growth in human saliva and a maltotriose-medium than dur-
ing growth in a nutrient-rich medium. Data on how MalR
functions in gram-positive organisms other than GAS are con-
flicting. For example, the homologue of MalR acts as a tran-
scriptional repressor in Streptococcus pneumoniae, whereas in
Staphylococcus xylosus and Enterococcus faecalis it acts as a
gene activator (12, 19, 27). To begin to investigate the function
of MalR in GAS, we determined the transcript levels of malR
in various growth conditions. Similar to the levels of other
genes in the maltodextrin gene region, malR transcript levels
were significantly higher during the mid-exponential growth
phase in a maltotriose-medium and in saliva than in THY (P ?
0.01 for each comparison) (Fig. 3). Coupled with the increase
in maltodextrin and pullulanase region gene transcript levels
observed in a maltotriose-medium and human saliva, the pat-
tern of malR transcript levels suggested that MalR may posi-
tively influence gene expression in GAS.
Uptake of maltodextrins is similar in parental and ?malR
isogenic mutant strains. To gain further insight into the role of
MalR, we replaced the malR open reading frame in the sero-
type M1 strain MGAS5005 with a spectinomycin resistance
cassette in a nonpolar fashion (24). Four genes in the malto-
dextrin and pullulanase gene regions (i.e., malE, malF, malG,
and msmK) encode proteins that putatively form an ABC
FIG. 3. malR transcript levels are elevated during growth of GAS
in a maltotriose-medium and human saliva. Strain MGAS5005 was
grown to the mid-exponential phase and late-exponential phase in
THY, a maltotriose-medium, and human saliva. TaqMan real-time
QRT-PCR was performed using primers and probes listed in Table 2.
The transcript levels of target genes were normalized to those of proS,
a gene that is expressed constitutively throughout the GAS cell cycle
and whose transcript levels are similar when the organism is grown in
THY and when the organism is grown in saliva (35, 43). The transcript
levels are the means ? standard deviations of quadruplicate measure-
ments obtained on two separate occasions.
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transport system for maltodextrins. To test the hypothesis that
MalR activates maltodextrin transport, we studied the uptake
of14C-radiolabeled maltose and maltotriose by parental and
?malR GAS strains and found that it was similar for both
strains (P ? 0.84 for maltose and P ? 0.57 for maltotriose)
(Fig. 4). The finding that MalR was not needed for optimum
transport of maltose or maltotriose suggests that MalR is not
required for the activation of maltodextrin transport genes
in GAS.
?malR mutant strain persists at a lower number of CFU
than the parental strain in human saliva. In light of the ra-
dioactive uptake data, we next tested the hypothesis that MalR
is not necessary for the growth of GAS in media containing
maltodextrin. Consistent with this hypothesis, there was no
significant difference between the parental and ?malR strains
in growth in THY, a maltose-medium, or a maltotriose-me-
dium (Fig. 5A to C) (P ? 0.791 for THY, P ? 0.187 for
maltose-medium, and P ? 0.115 for maltotriose-medium).
Given the elevated malR transcript levels that we observed
during growth in saliva, we hypothesized that MalR is impor-
tant for optimal growth of GAS in human saliva. To test this
hypothesis, we grew GAS in saliva and analyzed the resulting
growth using CFU analysis. Numbers of CFU were used rather
than density readings because the normal tendency of GAS to
aggregate in saliva interferes with optical density readings (9,
33). In human saliva, strain MGAS5005 grew to a density of
approximately 2.5 log10CFU/ml from the baseline and per-
sisted at a density of approximately 1 ? 107CFU/ml, whereas
the ?malR isogenic mutant strain grew to a density of only
approximately 1.9 log10CFU/ml and persisted at a density of
approximately 5.5 ? 105CFU/ml (Fig. 5D) (P ? 0.03 for
growth and P ? 0.004 for persistence). Repeated Gram stain
analysis of the saliva showed no difference between the aggre-
gation of strain MGAS5005 and the aggregation of the ?malR
strain, indicating that differences in cell clumping were unlikely
to account for the growth disparity. Further analysis of the data
revealed that the growth of the parental strain and the growth
of the ?malR strain were initially identical, but the viability of
the ?malR strain decreased rapidly during the shift from rapid
growth to high-level persistence during the stationary phase
(Fig. 5D). These data suggest that in human saliva, mainte-
nance GAS from the rapid growth phase to the stationary
phase requires MalR.
Despite extensive efforts, we were unable to genetically com-
plement the ?malR strain. Therefore, to show that the changes
observed in the ?malR strain were not due to spurious muta-
tions that occurred during creation of the mutant strain, we
created a second, independent ?malR strain (see Materials
and Methods). The growth curves of the second ?malR strain
in THY, maltose-medium, maltotriose-medium, and human
saliva were identical to those of the original ?malR strain (data
not shown).
MalR negatively influences the transcription of maltodex-
trin and pullulanase region genes. The radioactive uptake and
growth data suggested that MalR does not activate maltodex-
trin utilization genes in GAS. To test the hypothesis that MalR
negatively influences the transcription of genes in the malto-
dextrin and pullulanase gene regions, we assayed gene tran-
script levels in the MGAS5005 and ?malR strains. The tran-
script level of each of the eight genes assayed increased in the
?malR strain during growth in THY and saliva and to a lesser
degree during growth in a maltotriose-medium compared to
the transcript level in strain MGAS5005 (Fig. 6A). For all five
genes in the maltodextrin gene region, the differences between
the transcript levels in the mutant and parental strains were
greater during the late-exponential phase of growth in human
saliva than during the mid-exponential phase. This observation
correlates with the finding that the significant difference in
growth between the parental and ?malR strains in human
saliva occurred during the transition from the late exponential
phase to the stationary phase (Fig. 5D). The pattern of pulA
transcript levels in the parental and ?malR strains was quite
FIG. 4. Uptake of maltodextrins in GAS is not affected by deletion
of malR. Serotype M1 strain MGAS5005 and its isogenic ?malR de-
rivative were grown in a maltose- or maltotriose-medium to the mid-
exponential phase. Cells were harvested by centrifugation, washed with
carbohydrate-free CDM, and suspended to an OD600of 0.5. [14C]maltose
or [14C]maltotriose was added to a final concentration of 40 ?M.
Samples were removed every 30 s for 120 s and passed through a
0.45-?m filter. The filters were washed twice with carbohydrate-free
CDM, and the radioactivity retained was determined using a liquid
scintillation counter. The data are the means ? standard deviations for
four replicates done on two separate occasions.
FIG. 5. Growth curves of parental and ?malR strains in various
media. OD600readings were taken at different times to measure
growth in a nutrient-rich medium (THY) (A) and CDM with either 1%
maltose (B) or 1% maltotriose (C). CFU were quantified and analyzed
as a measure of growth in human saliva (D) as previously described
(33). The data are the means ? standard deviations of duplicate
measurements for five independent experiments.
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similar to the pattern observed for genes in the maltodextrin
gene region, and the largest difference among the conditions
tested occurred during the late-exponential phase in saliva
(Fig. 6A). Similar to the results of the growth experiments, we
found that the transcript levels of the maltodextrin and pullu-
lanase region genes in the second ?malR strain were essen-
tially identical to those in the original ?malR strain (data not
shown).
We next sought to determine whether there was an increase
in the maltodextrin and pullulanase region gene transcript
levels during growth in a maltotriose-medium or human saliva
in the ?malR strain. Unlike the results obtained for strain
MGAS5005, for the ?malR strain there were no significant
differences in the transcript levels for the maltodextrin and
pullulanase region genes between growth in THY and growth
in a maltotriose-medium (Fig. 6B). The lack of a difference
between the two growth media was due to an increase in gene
transcript levels in THY. However, an increase in maltodextrin
and pullulanase region gene transcript levels was observed in
the ?malR strain during growth in human saliva compared to
growth in either THY or a maltotriose-medium (Fig. 6B).
Taken together, these data suggest that MalR either directly or
indirectly negatively influences the expression of all genes in
the maltodextrin and pullulanase gene regions and that the
influence of MalR is maximal during growth in human saliva.
?malR mutant strain persists at a significantly lower level
than the parental strain in the mouse oropharynx. In light of
our finding that the ?malR strain was unable to persist at a
high level in human saliva, we next tested the hypothesis that
optimal GAS persistence in the oropharynx requires MalR. In
the first 3 days after inoculation of adult outbred CD-1 mice
with either the parental or ?malR GAS strain, there was no
significant difference between the two groups of mice in terms
of either the percent colonized or the average number of CFU
per mouse recovered from the oropharynx (Fig. 7A). However,
starting at day 4 and continuing through the remainder of the
experiment, significantly fewer mice were colonized by the
?malR strain than by the parental strain. Moreover, starting at
day 4 and continuing through the remainder of the experiment,
the average numbers of GAS CFU were significantly greater in
mice inoculated with the parental strain than in mice inocu-
lated with the ?malR strain (Fig. 7B). Together, these data
show that MalR is required for the optimal persistence of GAS
in the oropharynx.
malR is expressed during human pharyngitis. Inasmuch as
malR contributes to the persistence of GAS in human saliva ex
vivo and in the mouse oropharynx, we hypothesized that malR
is transcribed and expressed in humans with GAS pharyngitis.
We tested this hypothesis by using TaqMan real-time PCR to
assay malR gene transcript levels in RNA isolated from throat
swabs of six patients with GAS pharyngitis (43). Transcripts of
malR were present in all six specimens at levels that were
1.5-fold higher than the levels of transcripts of the internal
control gene proS (Fig. 8). These observations demonstrate
that malR is actively transcribed during GAS pharyngitis in
humans and further implicate MalR as a protein that partici-
pates in the GAS host-pathogen interaction.
DISCUSSION
Although GAS has long been known to be the leading cause
of bacterial pharyngitis in humans, our understanding of the
molecular mechanisms by which it colonizes and infects the
human upper respiratory tract has remained relatively limited.
FIG. 6. MalR negatively influences the transcription of genes in the maltodextrin and pullulanase gene regions. Strain MGAS5005 and its
isogenic ?malR derivative were grown to the mid-exponential or late-exponential phase in standard laboratory medium (THY), a maltotriose-
medium, or human saliva. TaqMan real-time QRT-PCR was performed using probe and primers listed in Table 2. The transcript levels of target
genes were normalized to those of proS, a gene that is expressed constitutively throughout the GAS cell cycle and whose transcript levels are similar
during growth in THY and during growth in saliva (35, 43). (A) Gene transcript levels are expressed as the log2fold difference between the ?malR
mutant and the wild-type strain (??CTmethod); therefore, positive values indicate higher transcript levels in the ?malR strain. (B) Gene transcript
levels in the ?malR strain relative to those of proS (?CTmethod). For each gene, the order of bars from left to right is as follows: mid- and
late-exponential growth phases in THY, maltotriose-medium, and human saliva. The data are the means ? standard deviations of quadruplicate
measurements obtained on two separate occasions.
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To this end, we have been utilizing the interaction of GAS with
human saliva as a model for investigation of previously unstud-
ied portions of the GAS genome (33–35). The high transcript
levels of putative polysaccharide utilization genes during GAS
host-pathogen interactions suggest a role for polysaccharide
utilization proteins in GAS pathogenesis (14, 15, 35, 44). In-
deed, a recent investigation showed that MalE, a cell surface
maltodextrin binding protein, was critical for initial coloniza-
tion of the oropharynx by GAS (34).
The finding that interaction with human saliva markedly
increased the transcript levels of malE (34) led us to investigate
whether other genes encoding proteins putatively involved in
polysaccharide utilization were similarly affected by growth in
human saliva. Examination of the 12 available GAS genomes
showed that genes encoding proteins putatively involved in
polysaccharide utilization are located in two separate chromo-
somal regions (Fig. 1). All of the genes in these two locales,
which we termed the maltodextrin and pullulanase gene re-
gions, had marked increases in their transcript levels during
growth in human saliva compared to the levels during growth
in a standard laboratory medium (Fig. 2). These findings led us
to focus on MalR, the only putative transcriptional regulator
encoded in the maltodextrin and pullulanase gene regions.
The fact that the levels of malR transcripts were increased in
saliva in a fashion similar to the fashion observed for the
polysaccharide utilization genes studied suggested that MalR
might function as a transcriptional activator in GAS (Fig. 3).
However, as shown by the markedly elevated transcription of
genes in the maltodextrin and pullulanase gene regions in the
?malR strain during growth in THY and saliva (Fig. 6), we
surmised that MalR negatively influences gene expression.
Whether MalR directly influences gene transcription, acts via a
secondary transcriptional regulator, or affects gene expression
through some other mechanism remains unclear. If MalR does
function as a direct repressor of polysaccharide utilization
genes, then one must ask why the transcript levels of MalR and
polysaccharide genes both increase under the same conditions.
Investigation of the mechanism by which MalR influences gene
transcription is ongoing.
One critical finding of our transcriptional analyses was that
the gene transcript levels of parental and ?malR strains dif-
fered more significantly during the late exponential growth
phase in human saliva than during the rapid growth phase. This
observation correlated well with our finding that the ?malR
strain not only failed to make an effective transition from
growth to high-level persistence in human saliva but also failed
to persist in the mouse oropharynx. As experimental analysis of
GAS pharyngitis in nonhuman primates has shown, malR tran-
script levels are highest during the persistence phase (44).
These data have led us to conclude that MalR plays its most
important role during the prolonged colonization phase of
GAS in the oropharynx.
In an earlier investigation, the maltodextrin binding protein
MalE was needed for optimal colonization of the oropharynx
by strain MGAS5005 (34). If MalR represses expression of
malE, our present finding that MalR contributes to the persis-
tence of MGAS5005 in the oropharynx appears at first glance
to contradict the MalE results. However, recent GAS infection
experiments in nonhuman primates have demonstrated that
gene expression by GAS varies significantly during the initial
colonization, acute infection, and persistence phases of phar-
yngitis (44). Therefore, we propose that MalE and MalR dif-
ferentially function in two distinct phases of GAS pathogene-
sis. The maltodextrin binding protein MalE appears to be most
FIG. 7. Failure of the ?malR isogenic mutant strain to persist in
mouse orophyarnx. Adult outbred CD-1 mice (35 mice per group)
were inoculated with ?1.0 ? 107CFU of either MGAS5005 or its
?malR derivative. Mice oropharynges were swabbed daily, and the
swabs were plated on BSA. The plates were incubated for 24 h, and
beta-hemolytic colonies were counted and tested for GAS carbohy-
drate antigen using latex agglutination. (A) Percentages of mice with
GAS isolated on different days. (B) Average numbers of CFU isolated on
different days. The data are the means ? standard errors of the means. P
values were determined by a ?2test (A) or Student’s t test (B).
FIG. 8. Presence of malR transcripts in vivo during pharyngitis in
humans. malR transcript levels for six patients with GAS pharyngitis
were determined by TaqMan real-time PCR. The M serotypes of the
infecting GAS strains are indicated in the circles, and the numbers in
parentheses are the median fold increases in transcript levels relative
to the levels of GAS proS gene transcripts (43). The error bars indicate
the standard deviations of quadruplicate measurements obtained on
two occasions.
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important during the initial colonization of the oropharynx by
GAS, when rapid growth of the organism requires nutrient
acquisition (34). Then, once the organism reaches maximal
density, it must persist. In this situation, a protein that nega-
tively influences gene expression, such as MalR, may become
critical for fine-tuning the balance between energy utilization
and protein production.
In addition to influencing the production of maltodextrin
utilization genes, MalR also appears to affect the expression of
genes in the distant pullulanase gene region. Our finding that
MalR influences gene expression in two distinct chromosomal
gene regions makes physiologic sense when the putative roles
of the encoded proteins are considered. First, PulA is an
LPXTG cell wall-anchored protein that degrades starch and
other polysaccharides into maltodextrin and other transport-
able sugars (20, 31). Second, MsmK is the putative ATP-bind-
ing portion of the ABC transporter that mediates the uptake of
maltodextrins. Third, the other three proteins that make up the
ABC transporter are putatively encoded in the maltodextrin
gene region. Finally, putative MalR binding sites are present in
the maltodextrin and pullulanase regions (unpublished data).
Thus, we hypothesize that MalR coordinates a system that is
capable of breaking down starch into maltodextrins, incorpo-
rating maltodextrins into the cell, and converting maltodextrins
into usable energy sources.
In conclusion, we used the results of multiple genome-wide
expression microarray analyses to concentrate our investiga-
tions on the putative maltose/maltodextrin transcriptional reg-
ulator MalR. Our most important findings are that MalR in-
fluences the expression of at least eight GAS genes in two
distinct chromosomal regions and that MalR is required for
GAS to persist at high levels in human saliva and in the mouse
oropharynx. These findings expand our insight into how GAS
nutrient utilization contributes to pathogenesis. Comparative
genomic analyses have shown that many mucosal human
pathogens have conserved their mechanisms for the acquisition
and selective utilization of nutrients. Therefore, further inves-
tigation of the links between nutrition and pathogenesis may
yield broadly applicable insights into microbial host-pathogen
interactions.
ACKNOWLEDGMENTS
We thank Richard Hull for his advice on carbohydrate uptake.
This work was supported by American Heart Association grant
0565133Y (S.A.S.) and National Institutes of Health grant K08
RR17665-04 (S.A.S.).
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Editor: D. L. Burns
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