Menzies School of Health Research,1 Cooperative Research Centre for Aboriginal and Tropical Health, Darwin,2 Queensland Institute of Medical Research, Brisbane, Australia3
Received 20 March 2003/ Returned for modification 1 May 2003/ Accepted 6 May 2003
Epidemiologically unrelated
Streptococcus pyogenes strains isolated
from blood, throat, and skin were assayed for adherence to HEp2
and HaCaT cells. Invasive isolates showed significantly higher
avidity for these cell lines than isolates from skin and throat.
In general,
S. pyogenes showed greater binding to HaCaT cells
than to HEp2 cells.
Streptococcus pyogenes (group A streptococcus ) is an etiological
agent for diverse human diseases, including pharyngitis, pyoderma,
and severe invasive diseases. In addition, the pathogen is associated
with potentially life-threatening sequelae such as poststreptococcal
glomerulonephritis and acute rheumatic fever. In the Northern
Territory (NT) of Australia the incidence of acute rheumatic
fever is very high among the indigenous population (
3), despite
a low throat isolation rate of GAS. Furthermore, pyoderma from
GAS infection is extremely common and poststreptococcal glomerulonephritis
is endemic in many remote Aboriginal communities (
4,
7). While
asymptomatic throat carriage is often the reported reservoir
for strains associated with invasive disease (
5), in populations
where impetigo is endemic, such as in Aboriginal communities
in the NT, the primary reservoir is the skin. Irrespective of
which tissue is the primary site of infection, the first event
the pathogen needs to achieve is adherence to host cells. The
S. pyogenes genome encodes numerous genes that could be regarded
as encoding adhesins. These genes are highly regulated, and
individual strains do not have the genetic potential to encode
all of these proteins. The adhesins include M protein (an antiphagocytic
molecule), capsule, and fibronectin binding proteins. There
are many different fibronectin binding proteins, such as SfbI
(
8,
12), PrtF2 (
9,
10), Fbp54 (
2), and SfbII (
11). The adherence
capacity of an individual strain could vary depending on the
repertoire of genes for the adhesins that the strain possesses
and their level of expression. This in turn may reflect the
differences in the ability to colonize and persistently infect
different tissue sites. A corollary of this is that isolates
from different tissue sites may exhibit differences in adherence
capacity. To test this, we have determined the extent of binding
of GAS isolates from skin, throat, and blood to HEp2 and HaCaT
cell lines, representing human laryngeal epithelial cells and
keratinocytes, respectively.
GAS isolates from the NT were collected between 1990 and 2002. The 72 strains analyzed in this study were isolated from blood (n = 26), skin (n = 22), and throat (n = 24) (Table 1). Blood isolates were from severe disease, and the remaining strains were from uncomplicated infections. The isolates were Vir typed as described previously (6). Vir typing involves restriction fragment length polymorphism of the mga regulon, which includes the gene for highly variable M protein. To ensure inclusion of epidemiologically unrelated strains, one representative isolate from each Vir type was included. Cultures were grown overnight at 37°C in an orbital shaker to stationary phase in Todd-Hewitt broth (Oxoid, Basingstoke, United Kingdom) supplemented with 1% yeast extract. To prepare the GAS inoculum for adherence assays, overnight cultures were centrifuged, and the pellets were washed in phosphate-buffered saline (PBS; Life Technologies Gibco BRL, New York, N.Y.) and resuspended in serum-free and antibiotic-free RPMI 1640 medium (Life Technologies) to an optical density at 600 nm of 0.05. This represents approximately 1 x 107 to 1.5 x 107 bacteria per ml.
| fig.ommitted |
TABLE 1. Vir type, binding, and source of isolation of GAS isolates used in this study
| |
Human laryngeal epithelial (HEp2) cells were maintained in RPMI
1640 medium supplemented with 10% fetal calf serum (Life Technologies),
1% Fungizone (Life Technologies), 20 µg of vancomycin
HCl (David Bull Laboratories, Sydney, Australia) per ml, and
100 µg of streptomycin sulfate (Sigma, St. Louis, Mo.)
per ml. Human adult skin keratinocytes (HaCaT cells) were maintained
in Dulbecco's modified Eagle medium (Life Technologies) supplemented
with 10% heat-inactivated fetal calf serum. For adherence assays,
10
5 cells/ml were seeded onto 12-mm-diameter glass coverslips
in the bottoms of 24-well tissue culture plates (Nunc, Roskilde,
Denmark). After overnight growth at 37°C in 5% CO
2 atmosphere,
the cells were washed with PBS (pH 7.4) and inoculated with
500 µl of the GAS inoculum. After 2 h of incubation at
37°C, the coverslips were washed five times by adding 1
ml of PBS to each well, and after gentle mixing, the wash solution
was removed by aspiration. After removal of the nonadherent
bacteria, the host cells and adherent bacteria were fixed with
95% methanol and air-dried. After heat fixing, the coverslips
were placed on slides and Gram stained for viewing under oil
immersion. In each experiment, cells in several random fields
were analyzed, and attachment was expressed as the average number
of GAS chains per cell. All assays were performed in duplicate,
and the mean binding was determined for each strain. All statistical
analyses were performed with Stata Statistics/Data Analysis
program version 7.0 (Stata Corporation, College Station, Tex.).
Data were analyzed with
t tests.
GAS strains adhered to both cell types, and the degree of binding varied from strain to strain (Table 1). There was good correlation between independent experiments with 20 isolates repeated at two time intervals (data not shown), suggesting the avidity of binding is reproducible and strain specific. Overall, GAS binding to HaCaT cells is greater than to HEp2 cells (P < 0.05). When the data in Table 1 were separated based on the tissue site of isolation, an average of 270 chains of GAS strains from blood bound to 50 HaCaT cells (Fig. 1). In contrast, skin and throat isolates bound on average only 169 and 178 chains per 50 HaCaT cells, respectively. These differences are statistically significant (P = 0.0044 and 0.0063, respectively). Interestingly, for HEp2 cells the differences are less pronounced and not statistically significant. However, when the data were reanalyzed based on invasive versus uncomplicated infections by combining data for the skin and throat isolates, significant differences between the two categories were found in both cell lines (P = 0.0011 for HaCaT; P = 0.0238 for HEp2).
| fig.ommitted |
FIG. 1. Comparison of binding of GAS to HaCaT and HEp2 cell lines. Statistically significant results are indicated by asterisks. Error bars represent standard errors.
| |
Earlier work from this laboratory showed that many commonly
circulating strains of
S. pyogenes could cause invasive disease
with skin as the primary site of infection (
1). These observations
are consistent with the present findings of higher avidity of
the NT GAS strains for HaCaT than HEp2 cell lines and blood
isolates being able to bind in greater numbers than the isolates
from uncomplicated infections. Possible explanations for the
high adherence propensity of
S. pyogenes blood isolates include
both genotypic and phenotypic differences between isolates from
invasive and noninvasive disease sources. Further studies are
required to define the nature of this binding avidity and to
determine whether it is consistent within clonal populations.
We are indebted to D. Gordon and E. Giannakis for advice and
for providing HaCaT cells.
Funds from Cooperative Research Centre for Aboriginal and Tropical Health supported this work.
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作者：
Mandy L Edwards Peter K. Fagan Heidi Smith-Vaugh 2007-5-10