Department of Laboratory Medicine, Lund University, Lund, Sweden1
Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, CT, USA2
Author for correspondence: Debra E. Bessen. Tel: +1 203 432 7890. Fax: +1 203 432 6533. e-mail: debra.bessen{at}yale.edu
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ABSTRACT |
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Keywords: streptococci, proteolysis, plasmin, inflammation
Abbreviations: GAS, group A streptococci; HA, hyaluronic acid; Plg, plasminogen; PMN, polymorphonuclear leukocyte; URT, upper respiratory tract
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INTRODUCTION |
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Decades of epidemiological findings support the existence of GAS strains that have strong preferences for either the throat or skin, as well as other strains whose tissue site preference is less clear cut (Anthony et al., 1976 ; Bisno & Stevens, 2000
; Maxted, 1980
; Wannamaker, 1970
). Thus, there should exist tissue-specific genotypes among GAS strains. The emm pattern is a genetic marker that distinguishes many throat- and skin-tropic strains of GAS (Bessen et al., 1996
, 2000
; Dicuonzo et al., 2001
); this pattern is defined by the chromosomal arrangement of emm subfamily genes. The emm genes, in turn, encode M surface proteins, which display extensive heterogeneity in their structure and function (Cunningham, 2000
; Fischetti, 2000
). Plasminogen (Plg)-binding group A streptococcal M protein (PAM) is uniquely associated with emm pattern D strains (Svensson et al., 1999
), which are usually recovered from impetigo lesions and rarely from cases of pharyngitis. Despite their niche separation, there is an ample flow of neutral housekeeping genes between throat and skin strains (Kalia et al., 2002
), and high rates of genetic recombination within GAS as a whole (Feil et al., 2001
). In instances where neutral housekeeping alleles are randomly distributed with respect to ecologically distinct populations (Kalia et al., 2002
), genetic variation that is strongly associated with the different populations may be directly responsible for adaptation to the ecological niche, i.e. emm gene products (or closely linked genes) may have a direct role in tissue tropism. Thus, based on the population genetics findings, a reasonable argument can be made for PAM as a strong candidate for having a tissue-specific role in the development of streptococcal impetigo.
Human Plg can be bound by at least three distinct GAS surface proteins, whereupon it can be converted to its active form (plasmin), through the action of streptokinase, a secreted product of GAS that functions as a Plg activator (Lottenberg et al., 1992 ; Pancholi & Fischetti, 1992
, 1998
). Plasmin acts as a broad-spectrum proteinase. It has been proposed that the acquisition of unregulated surface-associated plasmin by GAS and other bacterial pathogens promotes its spread into deep host tissue (Boyle & Lottenberg, 1997
; Coleman et al., 1997
; Rasmussen & Bjorck, 2002
; Sodeinde et al., 1992
). However, PAM is distinguished from other GAS Plg-binding proteins in that it has a high affinity for Plg (affinity constant, 8x10-8 M-1) (Berge & Sjöbring, 1993
). The concentration of human Plg in tissue is highest in the circulation and interstitial fluids (12x10-6 M), suggesting that PAM is adapted for functioning in a micro-environment where the Plg concentration is low. This is consistent with the idea that the interaction of PAM with streptokinase promotes adaptation at a superficial tissue site (i.e. the epidermis).
To test the contribution of specific GAS products to skin infection, an in vivo model was developed that closely mimics impetigo in humans, based on histopathology (Scaramuzzino et al., 2000 ). Human neonatal foreskins are grafted onto the backs of SCID mice, which fail to reject the xenotransplants. In an effort to mimic natural infection in humans, skin grafts are slightly damaged and GAS are topically applied. The hu-skin-SCID mouse model for impetigo exhibits a high degree of sensitivity, as evidenced by low inoculating doses for virulent strains, and high specificity, whereby emm pattern D strains display greater overall levels of virulence than typical throat strains (emm pattern AC). Using the hu-skin-SCID mouse model in conjunction with isogenic mutants of GAS, it can be shown that the secreted cysteine proteinase SpeB plays a key role in virulence (Svensson et al., 2000
). Furthermore, levels of secreted cysteine proteinase activity are highest for emm pattern D strains and lowest for emm pattern AC strains. Thus, experimental findings combined with epidemiological data can provide compelling evidence for the role of a streptococcal product as a determinant of tissue tropism.
In this study, mutants of emm pattern D strain ALAB49 were constructed, whereby the pam gene or the ska gene underwent allelic replacement. The isogenic mutants of ALAB49 were compared to the parent strain for virulence in vivo, using the hu-skin-SCID mouse model for impetigo.
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METHODS |
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Mutant construction.
Directed mutagenesis, via allelic replacement and insertion of the Km-2 interposon, using the temperature-sensitive shuttle vector pJRS233 harbouring an erythromycin-resistance (EmR) gene, was used to generate
pam,
ska and
speB mutants of ALAB49 (Caparon, 2000
; Kotarsky et al., 2000
; Perez-Casal et al., 1993
; Ringdahl et al., 1998
; Svensson et al., 2000
). Plasmid vectors used for GAS gene inactivation were constructed in Escherichia coli. Following electroporation of GAS with purified plasmid DNA, EmR transformants were selected by growth on medium containing 15 µg erythromycin ml-1. Colony picks of EmR transformants were grown to high density in THY broth at 30 °C and shifted to the non-permissive temperature (in the presence of 200 µg kanamycin ml-1), to select for chromosomal integration. Transformants arising from double crossovers were identified by screening for erythromycin sensitivity by replica plating. Construction of the ALAB49
speB mutant has been described in detail already (Svensson et al., 2000
). For inactivation of the central emm gene of ALAB49, encoding PAM (emm53 or pam), two PCR-amplified fragments were prepared, corresponding to the 3' portion of mrp53 (upstream) through the 5' portion of emm53 and to the 3' portion of emm53 through the 5' portion of enn53 (downstream). The two PCR fragments were ligated into pJRS233, with
Km-2 introduced between the two PCR fragments, and directed mutagenesis was performed as indicated above. The ALAB49
ska mutant was constructed using the replacement vector described for strain AP53 (Ringdahl et al., 1998
). This vector was constructed from pJRS233 via ligation of two PCR-amplified fragments corresponding to the 5' and 3' portions of the ska gene, which were flanked by the
Km-2 interposon. All mutations were confirmed by PCR-based mapping using the general approach described by Svensson et al. (2000)
.
In vivo model for impetigo.
The hu-skin-SCID mouse model for streptococcal impetigo was used to measure the virulence of ALAB49 wt and its isogenic mutants (Scaramuzzino et al., 2000 ; Svensson et al., 2000
). Briefly, human neonatal foreskins were grafted onto SCID mice. Healed skin grafts were subsequently damaged by a series of gentle, superficial cuts made with a scalpel blade and topically inoculated with 50 µl THY broth containing the appropriate concentration of bacteria that had been freshly grown for 24 h at 37 °C in THY broth. A wide range of inoculation doses was tested, based on the dilution of the broth cultures. To determine the actual dose administered, duplicate 10-fold dilutions were plated onto agar; duplicate c.f.u. counts were highly concordant with each other and with inoculums used in other experiments. The ratios of c.f.u. to optical density (OD) were consistent for ALAB49 wt and isogenic mutants, suggesting low variation in the mean chain length. At all steps, broth cultures were vigorously vortexed, to break up any aggregates.
Infected human skin grafts were occluded with a bandage. At 7 days post-inoculation, the bandages were removed and the human skin grafts were scored for gross pathology and the animals killed. Following biopsy, each graft was weighed and then split in (approximately) half through the centre with a blade. Half of the graft was used for histopathology and the other half of the graft was used to determine the c.f.u. count. The portion of the graft used for determining the c.f.u. count was reweighed, and bacteria were released from the tissue by vigorous vortexing; final calculations were made by taking a measured portion of the supernatant containing released bacteria, determining the c.f.u. counts following 10-fold serial dilutions (averaged duplicates) on THY/blood agar plates (containing kanamycin for mutant strains) and multiplying the c.f.u. mg-1 by the weight of total tissue biopsied prior to splitting. Spleens were recovered and cultured for the growth of GAS; all mice infected with ALAB49 wt, ALAB49pam and ALAB49
ska were negative for septicaemia, with the exception of one spleen which yielded a single colony of GAS. Haematoxylin- and eosin-stained tissue sections were scored, in a blinded fashion, for inflammation and epidermal destruction on a number scale of 03, reflecting the severity of the alterations (3 being the most severe). Although gross and microscopic pathology scoring is subjective, it is highly reproducible, even when done in a blinded fashion (Scaramuzzino et al., 2000
).
In vitro assays
Mutants were compared to the ALAB49 wild-type strain for several phenotypic traits that are of relevance to the directed mutagenesis and/or other known virulence properties of GAS.
(i) Bactericidal assay.
The Lancefield basic bactericidal test was used to measure antiphagocytic activity, but with bacterial inoculating doses ranging from 150 to 780 c.f.u. ml-1, which more closely reflects the Minneapolis method (Johnson et al., 1996 ). The ability of GAS to survive and multiply in heparinized human blood following a 3 h rotation at 37 °C was assessed by c.f.u. counts. Since this assay measures exponential growth, variance can be large; therefore, to minimize the effects of data skewing, bacteria were tested in four separate experiments, rather than tested as replicates in a single experiment. GAS yielding a 32-fold increase in c.f.u. values (5 population doublings) are regarded as having a completely intact antiphagocytic capacity (Johnson et al., 1996
).
(ii) Binding of Plg to bacteria.
The binding of radiolabelled Plg, from both human and mouse sources, to whole bacteria was measured, using a standard assay (Ringdahl et al., 1998 ; Svensson et al., 1999
). Heat-killed ALAB49 wt and isogenic mutants were tested over a range of concentrations, generated by serial fivefold dilutions of bacteria. Prior to heat-killing, GAS were grown to an OD620 value of 0·8 in THY broth, which is equivalent to the late-exponential phase.
(iii) Plasmin acquisition in plasma.
Plasmin acquisition by viable ALAB49 wt and the isogenic mutants (1010 c.f.u.), which were grown overnight and washed with PBS, was assessed by measuring bacterial-bound plasmin following their inoculation into fresh THY broth containing 30% freshly prepared human plasma, for the times indicated, as described by Ringdahl et al. (1998) . Bacterial-bound Plg was eluted with glycine (pH 2·0) and monitored by Western immunoblot using a rabbit antibody specific for human Plg (Dako A/S).
(iv) Streptokinase production.
ALAB49 wt and isogenic mutants were grown in THY broth overnight, washed in fresh THY broth and then grown to several OD values reflecting different stages of growth. Bacteria were pelleted by centrifugation and 1 ml of the supernatant was precipitated in 10% trichloroacetic acid (Svensson et al., 2000 ). The suspension was incubated on ice for 1 h and the precipitated proteins were collected by centrifugation. The final pellet was resuspended in SDS sample buffer and analysed by SDS-PAGE and Western blot. The immunoblots were incubated with polyclonal rabbit antibodies raised against purified streptokinase derived from the group C streptococcal strain H46A, generously provided by Dr Horst Malke (Friedrich Schiller University, Jena, Germany). Streptase (highly purified streptokinase from group C streptococci) was purchased from Hoechst Marion Roussel AB.
(v) Capsule content.
The ALAB49 wild-type strain and isogenic mutants were grown to exponential phase, and their hyaluronic acid (HA) capsule contents were measured using Stains-All (Sigma) as described by Ashbaugh & Wessels (2001) . Overnight THY broth cultures of bacteria were diluted 1:100 in fresh THY broth and grown to an OD600 value of between 0·2 and 0·3 (mid-exponential phase), prior to HA extraction. C.f.u. counts were determined in duplicate and data were expressed as c.f.u. (fg HA)-1, based on a standard curve generated with known quantities of bovine HA.
Statistical analyses.
Statistical tests were performed using STATISTICA 5.5 (StatSoft). Tests included the t-test for independent samples (unpaired, two-tailed), the non-parametric MannWhitney U-test, Fishers exact test for independence (two-tailed) and Pearsons correlation coefficient.
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RESULTS |
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The level of streptokinase production by the mutant and wild-type organisms was monitored by Western immunoblot, using a polyclonal antiserum directed against streptokinase (Fig. 2). Following growth to late-exponential phase, culture broth supernatants derived from both ALAB49 wt and the ALAB49
pam mutant revealed a strongly immunoreactive band at 44 kDa (Fig. 2
, lanes 1 and 2), which corresponded to the expected size for streptokinase. In contrast, the ALAB49
ska mutant lacked a 44 kDa band, but instead displayed a strongly immunoreactive band of 27 kDa (Fig. 2
, lane 3), consistent with the expected size for the truncated gene product.
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Growth curves in THY broth, measured at OD620, were nearly identical for ALAB49 wt and the ska and
pam mutants (data not shown). In addition, the wild-type and the mutant constructs produced equivalent amounts of the mature 28 kDa form of SpeB in late-stationary phase culture supernatants, and displayed complete conversion of SpeB from the 40 kDa zymogen form (data not shown). Despite the strongly polar nature of
Km-2, evidence for decreased expression of downstream genes in our mutants is lacking. The ska transcript is monocistronic (Malke, 2000
; Malke et al., 2000
) and, based on the SF370 (M1) genome (Ferretti et al., 2001
), the nearest downstream gene is 4·5 kb away (SPy1984, encoding a putative ribonucleotide reductase, NrdI); intervening ORFs are transcribed on the opposite strand, and rel shares a bidirectional transcriptional terminator with ska (Malke, 2000
; Malke et al., 2000
). Using gene-specific primers, the relative chromosomal arrangements of ska (SPy1979), rel (SPy1981) and nrdI (SPy1984) were found to be similar for ALAB49 and SF370 (data not shown), unlike the gene order observed for some strains of Streptococcus dysgalactiae subsp. equisimilis (Geyer & Schmidt, 2000
). emm genes are also transcribed monocistronically in several GAS strains that have multiple emm genes (Bessen & Fischetti, 1992
; Podbielski et al., 1995
; Yung & Hollingshead, 1996
).
In summary, phenotypic analysis of the ska and
pam mutants constructed in ALAB49 revealed that each mutant exhibits the changes that were expected, but neither mutant has measurable alterations in several unrelated properties that may be key for virulence. As expected, the ALAB49
ska mutant is unchanged in its Plg binding, resistance to non-immune phagocytosis, HA capsule and SpeB contents and growth in THY broth; however, it fails to secrete streptokinase and fails to convert surface-bound Plg to plasmin. The ALAB49
pam mutant is unchanged in its streptokinase and SpeB production, HA capsule content and growth curve but, as expected, shows loss in binding Plg and loss in the antiphagocytic effect, properties that are both attributed to the M53 protein (i.e. PAM).
In vivo effects of ska and pam inactivation
The ALAB49ska and
pam mutants were tested for virulence in the hu-skin-SCID mouse model for impetigo, over a wide range of inoculating doses. In this model, virulence can be defined as a function of the increase in c.f.u. counts following topical inoculation of GAS onto slightly damaged (scratched) human skin grafts. The 7 day time point chosen for skin biopsy is based on empirical evidence showing a large net growth of virulent strains and histopathological alterations that closely mimic a late stage of the human disease (Scaramuzzino et al., 2000
; Svensson et al., 2000
). The hu-skin-SCID mouse model is technically challenging, and there is a limit to the number of graft biopsies that can be processed at any one time. Furthermore, since the inoculum doses, expressed as c.f.u. values, are only known in retrospect, it is not possible to test two different constructs at precisely the same dose using the highly accurate c.f.u. measure. Given these constraints, we chose an experimental design whereby each graft receives a different inoculating dose, spanning a wide range of doses, rather than multiple grafts receiving only one or two identical doses. This approach further ensures against overlooking a critical threshold dose, which may differ for the wild-type and each mutant. In natural infections, inoculating doses are likely to fall within a wide range and be dependent on the degree of direct contact and various risk factors, such as skin integrity.
For skin graft biopsies taken at 7 days post-inoculation (Table 2), each mutant showed a partial attenuation in virulence, relative to ALAB49 wt. There were no significant differences for the ranges of inoculating doses tested for ALAB49 wt versus each mutant (Table 2
). The difference between ALAB49 wt and each of the mutants in terms of the two-log change in c.f.u. counts (i.e. doubling or halving of the bacterial population) was statistically significant (t<0·05; unpaired, two-tailed). Although the
ska mutant showed greater attenuation than the
pam mutant (t=0·0067 and 0·0393, respectively), the
ska versus
pam mutants failed to show significant differences relative to one another. Using a non-parametric statistical test (MannWhitney U-test), loss of virulence for the
ska mutant was highly significant (P<0·001), whereas the
pam mutant showed no significant difference relative to ALAB49 wt. Thus, it appears that the
ska mutant is highly attenuated for virulence, whereas the
pam mutant is attenuated to a lesser degree. However, the
pam mutant exhibited net reproductive growth for only 44% of the inoculated skin grafts, in contrast to 100% of the grafts infected with ALAB49 wt (P=0·029, Fishers exact test, two-tailed). If only half of the infections caused by PAM-deficient strains are productive, there may be a significant impact on transmission dynamics (Anderson, 1998
). Although statistical analysis strongly supports partial attenuation in virulence resulting from the ska and pam mutations, the loss in virulence does not follow a strict dose-response for either mutant (Table 2
); this may be partly due to microscopic graft-to-graft variation in the depth of the skin surface scratches, a procedure that is difficult to control with precision and which can on occasion extend beyond the epidermis into the dermal layer (Scaramuzzino et al., 2000
).
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When considering all grafts inoculated with GAS in this study (n=25), the correlation between gross pathology score and the sum of the scores for inflammation and epidermal destruction is statistically significant (r=0·801; Pearsons correlation coefficient). Grafts showing a net increase in c.f.u. counts at 7 days post-inoculation (Table 2), as compared to grafts showing a net decrease in c.f.u. counts, reveal differences in gross pathology scores that are highly significant (t=0·00027). Grafts showing a net increase or decrease in c.f.u. counts at 7 days post-inoculation also showed a strong correspondence with histopathological scores for epidermal destruction and inflammation (t=0·0036 and 0·0008, respectively). The latter finding is consistent with the idea that bacterial reproduction in tissue depends on a source of nutrients provided by extravasated inflammatory cells and that the inflammatory response and/or replicating bacteria contribute to destruction of the epidermal layer.
The hu-skin-SCID mouse model measures the c.f.u. values for bacteria released from the skin graft surface. However, in severely infected tissue, dermal ulcers packed with bacteria can arise; this is characteristic of ecthyma observed in human disease (Scaramuzzino et al., 2000 ). Therefore, the number of c.f.u. recovered from heavily infected tissue in the hu-skin-SCID mouse model may be an underestimate of the total bacteria associated with the skin graft. Since the wild-type strain yields the most extensive tissue damage (Table 3
), it is possible that the net increase in bacterial growth within the total skin graft is higher than reported for vortex-released bacteria (Table 2
). Nonetheless, in natural infections in humans, bacteria present on the superficial skin surface are the most likely to undergo transmission to a new host.
Interactions of streptokinase and PAM with SpeB
In a previous study, it was shown that the secreted cysteine proteinase SpeB is critical for GAS infection in the hu-skin-SCID mouse model for impetigo (Svensson et al., 2000 ). Other investigators have reported that the M1 protein facilitates the conversion of the zymogen form of SpeB to the mature form (Collin & Olsen, 2000
). In contrast, the M53 protein (i.e. PAM) of the ALAB49 strain has no noticeable effect on SpeB conversion. Using culture broth conditions used to prepare inoculums for hu-skin-SCID mice (24 h growth in THY broth), there was complete conversion of SpeB to the mature form, and near-equivalent levels of SpeB for the
pam mutant and ALAB49 wt (data not shown). Also, there were no obvious differences in SpeB production by the ALAB49
ska mutant.
Cysteine proteinase activity due to SpeB can result in the degradation of M proteins (Berge & Björck, 1995 ; Raeder et al., 1998
). Studies show that SpeB activity is maximally expressed during the stationary phase of growth (Collin & Olsen, 2000
; Lyon et al., 1998
), whereas Mga-regulated emm transcription peaks during the exponential phase (McIver & Scott, 1997
). Control experiments show that the ALAB49
speB mutant (Svensson et al., 2000
) binds human Plg at levels equivalent to those measured for ALAB49 wt and ALAB49
ska, following growth in THY broth to late-exponential phase (Fig. 1a
; data not shown). The ALAB49
speB mutant shows no change in antiphagocytic activity in the bactericidal assay (Svensson et al., 2000
), consistent with the presence of functionally intact PAM in ALAB49 wt. Thus, under the set of in vitro conditions tested, there is no evidence for modulation of PAM by SpeB in ALAB49 wt nor is there any indication that the loss of PAM influences SpeB activity. Nonetheless, we cannot rule out the possibility that SpeB has an effect on PAM under other experimental conditions (such as stationary phase) or that SpeB modulates the low-affinity Plg-binding surface proteins of ALAB49.
The production of streptokinase by ALAB49 wt and the ALAB49pam mutant was monitored by Western immunoblot analysis of culture broth supernatants (Fig. 2
). Streptokinase was evident in late-exponential phase cultures as a strong 44 kDa band (Fig. 2
, lanes 1 and 2), but was significantly degraded in overnight cultures (Fig. 2
, lanes 4 and 5). However, addition of the cysteine proteinase inhibitor E64 led to an accumulation of streptokinase in stationary phase cultures (Fig. 2
, lanes 6 and 7). That SpeB degrades streptokinase is further supported by findings with overnight culture supernatants of the ALAB49
speB mutant grown in the absence of E64, which yielded ample quantities of non-degraded streptokinase (Fig. 2
, lane 8). The finding of degradation of Ska by SpeB is consistent with an earlier report (Johnston & Zabriskie, 1986
).
Mid-exponential phase cultures of ALAB49 wt and ALAB49pam, grown to OD620 values of 0·4, each reveal an immunoreactive band that is somewhat less intense than that observed for late-exponential cultures (data not shown). Streptokinase production was unaffected by the presence of the E64 inhibitor during growth. This finding is consistent with the extracellular production of streptokinase beginning at or before the mid-exponential phase, and streptokinase accumulating throughout the later stages of exponential growth.
In summary, there is no evidence that the inactivation of pam or ska results in alterations in the production of the mature form of SpeB. However, the accumulation of SpeB in stationary phase broth cultures leads to the degradation of extracellular streptokinase in the ALAB49 wild-type strain.
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DISCUSSION |
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In the hu-skin-SCID mouse model for impetigo, ALAB49 wt and its mutants exhibit a strong correlation between gross pathological alterations and histopathological findings on the infiltration of inflammatory cells and epidermal erosion. Furthermore, the findings of this study show that the severity of both gross and microscopic alterations correlates with a net increase in bacterial growth at the skin. Previous studies have shown that at an early stage of infection with virulent GAS (48 h post-inoculation) the stratum corneum is lost over large sections of skin, and single cocci or short chains are observed in close association with the newly exposed apical surface of the viable keratinocytes; furthermore, the number of tissue-associated bacteria is low and few PMNs are present (Scaramuzzino et al., 2000 ). We propose that at this early stage of infection, the bacteria are in a nutrient-poor environment (Fig. 4
). A nutrient-poor environment is consistent with a stationary phase of growth and maximal expression of cysteine protease activity due to SpeB (Lyon et al., 1998
; Podbielski et al., 1999
; Svensson et al., 2000
). Inactivation of speB leads to complete attenuation of virulence in the experimental model for impetigo (Svensson et al., 2000
), suggesting that SpeB plays a critical role during early stages of infection, prior to extensive bacterial growth.
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Extracellular streptokinase is produced during exponential growth (Fig. 2); therefore, streptokinase appears to be essential at some time during the nutrient-rich phase of infection (Fig. 4
). Host Plg is (presumably) present in the local micro-environment (i.e. purulent exudate) and its activation by streptokinase appears to be critical for ensuing bacterial reproduction, although incomplete attenuation of the ALAB49
ska mutant suggests that limited bacterial growth (or survival) can occur in the absence of streptokinase. If streptokinase is critical during the earliest stage of the nutrient-rich phase of infection, it may act, directly or indirectly, to further amplify the inflammatory response, leading to the recruitment of additional PMNs and host plasma. Bacterial-generated plasmin activity can lead to fibrinolysis, which, in turn, may prevent the skin lesion from drying out and healing. If streptokinase plays a role during the late stages of the nutrient-rich phase of infection, it may act to promote invasion of GAS into the host tissue. Unlike PAM, streptokinase does not appear to directly contribute to resistance to phagocytosis, based on findings with the in vitro bactericidal assay. By 7 days post-inoculation, bacterial reproduction has increased even further and dermal pockets of Gram-positive cocci are readily observed (Scaramuzzino et al., 2000
).
It is proposed that PAM exerts its virulence properties during the nutrient-rich phase of infection (Fig. 4), since emm gene transcription is up-regulated during the exponential phase (McIver & Scott, 1997
). It is not known whether the slight attenuation observed for ALAB49
pam in vivo is related to the partial loss in its antiphagocytic property or to the decreased bacterial-bound plasmin activity in vitro. The ALAB49
ska mutant exhibits a greater loss in virulence than the ALAB49
pam mutant, suggesting that PAM function can be partly compensated by low-affinity Plg-binding proteins present on the bacterial surface (Lottenberg et al., 1992
; Pancholi & Fischetti, 1992
, 1998
) and/or by fluid-phase plasmin activity. Since the affinity of PAM for mouse Plg is 10- to 20-fold lower than its human counterpart, the contribution of PAM to virulence may be delayed until additional plasma is dragged to the epidermal surface by extravasating PMNs. A possible consequence for the hu-skin-SCID mouse model is that the minimal infective dose is somewhat higher than for natural infection in humans.
As ALAB49 re-enters another round of stationary growth, the increased expression of cysteine protease activity due to SpeB results in the degradation of extracellular streptokinase. It is not yet known whether streptokinase is deleterious to GAS transmission and/or the early stages of a newly acquired infection. Conceivably, bacterial-generated plasmin might act to degrade streptococcal adhesins or host keratinocyte receptors that are critical for the initial hostpathogen interaction. Alternatively, SpeB degradation of streptokinase might simply be incidental.
Although the ALAB49ska and
pam mutants are only partially attenuated in the experimental model for impetigo, it is quite possible that in the natural world comparable mutants would become completely lost. In a human host population, GAS infectious doses are probably at the lower end of our experimental range. In order for a micro-organism to survive, its basic reproductive rate (R0) must be greater or equal to one (Anderson & May, 1991
; Anderson, 1998
), which essentially means that for each infected host that recovers, the organism must be successfully transmitted to at least one new host. If bacterial doses are naturally low and the mutated variant can induce infection in only a fraction of newly exposed hosts, the organism will quickly disappear.
From the standpoint of evolutionary success, changes in c.f.u. counts are highly relevant measures. Fitness is defined in the context of the environmental niche, and the organism with the highest fitness is that which yields the greatest number of progeny in that niche (i.e. it is the most highly adapted). An increase in the number of (transmissible) progeny increases the frequency of adaptive alleles in the gene pool of that subpopulation. The finding that the PAM phenotype and high levels of SpeB activity display statistically significant associations with emm pattern D strains and impetigo isolates (Svensson et al., 1999 , 2000
) is consistent with the idea that they play a direct role in promoting skin infection. In addition to emm pattern D strains, emm pattern E strains of GAS are often associated with impetigo, although they are prevalent at the URT as well (Bessen et al., 1996
, 2000
; Dicuonzo et al., 2001
). To date, pattern E strains have not been carefully investigated in the hu-skin-SCID mouse model. Since emm pattern E strains lack PAM (Svensson et al., 1999
), it is possible that they cause impetigo by using an entirely different molecular strategy than pattern D strains.
Following mutagenesis of any gene, unintended polar effects can potentially arise, not only through the introduction of strong transcriptional terminators within the Km-2 element (as is the case for the ALAB49
ska and
pam mutants) but also through indels generated by allelic replacement and ensuing shifts in codon usage and translational activity. In many respects, an ideal control for the ALAB49
ska and
pam mutants is the reintroduction of the wild-type gene by transcomplementation, although there is a downside lying in variable gene dosage effects. Transcomplementation was achieved for the ALAB49
pam mutant, and the binding activity of human Plg to whole bacteria, when tested in vitro, was fully restored (data not shown). However, when this construct was tested in the hu-skin-SCID mouse model for impetigo, a confusing picture emerged whereby gross and microscopic pathological alterations were evident in the absence of net bacterial growth at the skin. These findings are probably an artefact of plasmid instability resulting from shifts in selective pressures that occur during the course of infection, i.e. uneven diffusion of the selectable antibiotic to the superficial tissue site and changing needs for PAM. Once the plasmid is lost due to a transient lapse in positive selection, the bacteria can be eliminated following subsequent exposure to antibiotics or PMNs. Despite the lack of this particular control, the numerous phenotypic tests performed in vitro provide strong support that the ALAB49
ska and
pam mutants did not undergo unintended alterations in several phenotypes that are known to affect virulence. Although the
Km-2 element contains strong transcriptional terminators, both ska and emm genes are expressed as monocistronic transcripts, and the ORF transcribed on the strand opposite to ska shares a bidirectional transcriptional terminator positioned downstream of ska (Bessen & Fischetti, 1992
; Malke, 2000
; Malke et al., 2000
; Podbielski et al., 1995
; Yung & Hollingshead, 1996
); therefore, expression of adjacent genes should be unaffected. In summary, there is no evidence that the ALAB49
ska and
pam mutants have alterations in other genes.
It has been proposed that bacterial-generated plasmin acts to modulate the micro-environment of the bacterium and its movement through host tissue (Boyle & Lottenberg, 1997 ; Coleman et al., 1997
; Rasmussen & Bjorck, 2002
; Sodeinde et al., 1992
). However, the ability to spread into deep tissue and cause invasive disease does not appear to be a long-term evolutionary strategy for GAS. The URT and epidermis of the skin are the primary tissue reservoirs for GAS. Because infection at the throat and skin is typically mild and superficial, the infected host is fairly mobile and has a high potential for exposing numerous individuals, via respiratory droplets or direct contact. Since GAS undergo expansive reproductive growth at the superficial epithelium and transmission to new hosts is launched from the superficial epithelium as well, it is at the URT and epidermis where the evolution of GAS is most profoundly shaped by the forces of natural selection. Thus, it seems most likely that the primary function of bacterial-generated plasmin activity is to enhance survival at a superficial epithelial site, as opposed to deep tissue.
The epidemiological findings, in combination with the experimental data, make a strong case for a role for both extracellular plasmin (this study) and cysteine proteinase activities (Svensson et al., 2000 ) as key determinants of tissue tropism at the skin. An interesting aspect of this finding is that one usually envisions determinants of host or tissue range as attachment factors that recognize a specific host-cell receptor, yet, here, extracellular enzymes appear to play that role. By modulating their local micro-environment through proteolytic pathways, GAS have been highly successful in exploiting this ecological niche.
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ACKNOWLEDGEMENTS |
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Received 25 July 2002;
revised 12 August 2002;
accepted 11 September 2002.