Molecular Basis for Hypervirulence of GAS • JID 2010:201 (15 March) • 855
M A J O R A R T I C L E
Dissection of the Molecular Basis for Hypervirulence
of an In Vivo–Selected Phenotype of the Widely
Disseminated M1T1 Strain of Group A Streptococcus
Bacteria
Rita G. Kansal,1,2,a Vivekanand Datta,3 Ramy K. Aziz,1,4 Nourtan F. Abdeltawab,1,2,5 Sarah Rowe,1,2 and Malak Kotb1,2,5
1Department of Veterans Affairs Medical Center, Research Service, and Departments of 2Molecular Sciences and 3Pathology, University
of Tennessee Health Science Center, Memphis, Tennessee; 4Department of Microbiology and Immunology, Faculty of Pharmacy,
Cairo University, Cairo, Egypt; and 5Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine,
University of Cincinnati, Cincinnati, Ohio
(See the editorial commentary by Bessen and Tengra, on pages 800–802.)
Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection
and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone:
a virulent wild-type (WT) strain, characterized by a SpeB+/SpeA
/Sda1low phenotype, and a hypervirulent
animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB
/SpeA+/Sda1high phenotype. This
AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-
component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of
the AP strain, we deleted covS from WT bacteria (DCovS) and were able to simulate the hypervirulence and
gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria
reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS
virulence.
The pathogenesis of group A Streptococcus (GAS) in-
fections reflects the complex interplay between bacterial
and host factors. GAS diseases vary from uncomplicated
pharyngitis to life-threatening streptococcal toxic shock
syndrome and necrotizing fasciitis. Multiple virulence
factors play important roles in distinct host niches and
different stages of GAS infections [1, 2]. Depending on
Received 15 July 2009; accepted 22 October 2009; electronically published 12
February 2010.
Potential conflicts of interest: none reported.
Financial support: American Heart Association (grant 0160234B to R.G.K.),
National Institutes of Health (grant AI40198-06 to M.K.), US Army Medical
Research Acquisition Activity (grant W81XWH-05-1-0227 to M.K.), and Research
and Development Office, Medical Research Service, Department of Veterans Affairs
(Merit Award to M.K.).
a Present affiliation: Department of Molecular Genetics, Biochemistry, and
Microbiology, University of Cincinnati, OH (kansalra@uc.edu).
Reprints or correspondence: Dr Rita G Kansal, University of Tennessee Health
Science Center and VA Medical Center, Research Service (151), 1030 Jefferson
Ave, Memphis, TN 38104 (rkansal@uthsc.edu).
The Journal of Infectious Diseases 2010; 201:855–865
 2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20106-0008$15.00
DOI: 10.1086/651019
the site of infection and host context, GAS may engage
different sets of virulence programs. For example, the
hyaluronic acid capsule, surface-bound M and M-like
proteins, fibronectin-, collagen-, and plasminogen-
binding proteins, SpeB, immunoglobulin-binding, de-
grading, and inactivating proteins, C5a-peptidase, and
a2-macroglobulin-binding protein are all used in the
primary stages of infection, where they promote bac-
terial adherence, colonization, resistance to phagocy-
tosis, invasion of host cells, and evasion of host defenses
[2–4]. Secreted proteins, including streptococcal su-
perantigens, cytotoxins (streptolysin O and streptolysin
S), and cell wall–associated peptidoglycans and lipo-
teichoic acid, elicit inflammatory responses that divert
host defenses, allowing bacterial colonization of specific
host niches [1, 5–7]. Furthermore, secreted deoxyri-
bonucleases (hereafter, DNases) allow the bacteria to
escape neutrophil killing [8, 9].
Under host pressure, the composition of the bacterial
community can drastically change, revealing pheno-
types that differ from the original inoculum [9–11].
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856 • JID 2010:201 (15 March) • Kansal et al
Figure 1. SpeB
/SpeA+/Sda1high AP bacteria are more virulent than SpeB+/SpeA
/Sda1low WT bacteria in CBA mice. Kaplan-Meier Survival curves
of CBA/J mice infected intravenously with CFU of the WT bacteria or the in vivo–derived AP bacteria of 2 M1T1 group A Streptococcus62
10
(GAS) isolates, 5448 (A) and 6050 (B). Mouse survival was recorded on daily basis. Statistical significance in survival curves was determined by the
log-rank test. (C) Bacterial loads in blood following infection: At 24 h after infection, 20 mL blood was drawn aseptically from the tail vein of mice
infected with the WT or AP bacteria of 5448 or 6050 GAS, and the bacterial loads were determined by plating a 100-fold dilution of the blood on
the blood agar plates. The plates were incubated at 37C for 24 h, and b-hemolytic colonies were counted. Differences between bacterial counts in
blood were analyzed by 2-tailed Student t test. (D) Loss in body weights following infection. Infected mice were weighed daily for the entire observation
period or until they died as a result of the infection. The weight loss is presented as the percentage of mice initial weight. Differences between
weight loss were analyzed with 2-tailed Student t test. The results presented are representative of 2 or 3 experiments.
Such phenotypic switching has been documented for the glob-
ally disseminated M1T1 strain and may be a part of the bacterial
strategy to evade host defense mechanisms [12]. Analysis of the
in vivo–manifested phenotypes uncovered molecular events as-
sociated with the emergence of a virulent M1T1 clonal strain
that differs markedly from its ancestral M1T1 SF370 strain in
prevalence, dissemination, and virulence [12–17]. This clonal
M1T1 strain emerged in the 1980s, coinciding with the resur-
gence of streptococcal toxic shock syndrome and necrotizing
fasciitis [12]. Its virulence and prevalence have been partially
attributed to the acquisition of 2 prophages, which introduced
the speA and sda1 genes, encoding the superantigen SpeA and
the highly potent DNase Sda1, respectively [14, 15].
In analyzing large numbers of clinical isolates, we identified
2 phenotypic variants of the emergent M1T1 strain that differed
significantly in their expression of the virulence factors SpeB,
SpeA, and Sda1. Using our chamber model of localized GAS
infection [11], we separated these 2 phenotypes. The wild-type
(WT) phenotype is characterized by high expression of SpeB
but low expression of SpeA and Sda1 (SpeB+/SpeA
/Sda1low),
and the animal-passaged (AP) phenotype expresses no SpeB
but high levels of SpeA and Sda1 (SpeB
/SpeA+/Sda1high) [10,
11]. These variants differ considerably in their invasive poten-
tial, virulence, and ability to thrive in vivo [9, 18, 19]. Inter-
estingly, all bacteria exhibiting the AP phenotype have muta-
tions in covS [9, 12, 19], the sensor part of a key 2-component
regulatory system CovR/S that regulates expression of many
streptococcal genes [20–23].
We undertook this study to determine whether the hyper-
virulent phenotype of the AP M1T1 bacteria is directly related
to the covS mutation, or whether other factors contribute more
significantly to its invasive properties and hypervirulence.
METHODS
Generation of in-frame covS allelic exchange knockout and
reverse-complemented strains. We used the parental WT and
AP phenotypic variants of 2 representative M1T1 GAS clinical
isolates 5448 and 6050 [6]. We amplified a 2253 base pair (bp)
GAS chromosomal DNA fragment containing covS, along with
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