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Trigger for group A streptococcal M1T1 invasive disease

Jason N. Cole^*, Jason D. McArthur^*, Fiona C. McKay^*, Martina L. Sanderson-Smith^*, Amanda J. Cork^*, Marie Ranson^*, Manfred Rohde, Andreas Itzek, Hongmin Sun, David Ginsburg^,,||,#, Malak Kotb^**, Victor Nizet, G. S. Chhatwal and Mark J. Walker^*,1

^* School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia;

Department of Microbial Pathogenesis and Vaccine Development, German National Centre for Biotechnology, Braunschweig, Germany;

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA;

Howard Hughes Medical Institute,

^|| Department of Internal Medicine and

^# Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA;

^** Department of Molecular Science, University of Tennessee, Memphis, Tennessee, USA; and

Department of Pediatrics, UCSD, La Jolla, California, USA

¹Correspondence: School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. E-mail: mwalker{at}uow.edu.au

SPECIFIC AIMS

The globally disseminated M1T1 clone of Streptococcus pyogenes (group A Streptococcus; GAS) causes a number of highly invasive human diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. Although the transition from local to systemic infection occurs by an unknown mechanism, invasive M1T1 clinical isolates express significantly less cysteine protease SpeB than M1T1 isolates from localized infections. Subversion of the host plasminogen activation system by GAS may also play a key role in systemic disease initiation. In this study, we utilize the representative wild-type (WT) clonal M1T1 isolate 5448 and the isogenic mutant speB to investigate the interaction between SpeB and human plasminogen in GAS invasive disease. The specific aims were: 1) to characterize capsule expression, SpeB protease activity, plasminogen-binding capacity, and accumulation of surface-bound plasmin activity in WT and speB strains; 2) to examine the virulence of WT and speB strains in a humanized plasminogen transgenic mouse model of subcutaneous (s.c.) infection; and 3) to analyze the SpeB activity of WT colonies isolated from the skin lesions and blood of infected plasminogen-humanized mice.

PRINCIPAL FINDINGS

1. Characterization of M1T1 5448 and speB
The WT and speB mutant expressed identical amounts of hyaluronic acid capsule (Fig. 1 a), excluding a pleiotropic mutation that confounded earlier studies of SpeB in GAS pathogenesis. SpeB protease activity was undetectable in the speB mutant (P0.05; Fig. 1b ), and SpeB zymogen was identified only at the ExPortal microdomain of the WT strain (Fig. 1c ). Nonetheless, equivalent amounts of human plasminogen were bound by the WT and speB mutant (Fig. 1d ), suggesting that SpeB does not affect the capacity of M1T1 GAS to bind human plasminogen. Compared with the WT strain, speB accumulated 75-fold higher levels of plasmin activity on the bacterial surface following incubation in human plasma (P0.05; Fig. 1e ). These data suggest that SpeB may interfere with the accumulation of plasmin on the GAS cell surface.

View larger version (19K): [in this window] [in a new window]   Figure 1. In vitro characterization of M1T1 S. pyogenes strain 5448 (WT) and the isogenic speB mutant. a) 5448 and the speB mutant express equivalent amounts of capsular hyaluronic acid (mean±SD). b) Secreted SpeB protease activity (mean±SD) is abolished in the speB mutant compared with 5448. c) Electron microscopic analyses detected SpeB zymogen at the ExPortal microdomain of 5448, but not speB (data not shown). d) Equivalent amounts of purified human 125I-labeled plasminogen (mean±SD) are bound by 5448 and speB. e) The speB mutant cell surface accrues a 75-fold higher concentration of human plasma activity (mean±SD) compared with strain 5448.

2. Infection of humanized plasminogen transgenic mice
Compared with nontransgenic control mice (Tg^–), the virulence of the WT strain was significantly enhanced in humanized plasminogen transgenic mice (Tg⁺) (P0.05; 10% vs. 80% mortality) (Fig. 2 a), indicating that human plasminogen is essential for M1T1 virulence. In comparison to the WT GAS parent strain, the speB mutant was attenuated for virulence in the Tg⁺ mice (P0.05; 80% vs. 20% mortality) (Fig. 2a, b ) and displayed significantly fewer bacterial counts in the blood (P0.05; Fig. 2c ). These data support a role for SpeB in GAS survival at the site of local skin infection.

View larger version (10K): [in this window] [in a new window]   Figure 2. Survival curves, bacterial counts and SpeB expression phenotype following s.c. infection of humanized plasminogen transgenic mice (Tg+) and nontransgenic control mice (Tg–) with S. pyogenes strain 5448 (WT) or the isogenic speB mutant. a) The virulence of 5448 is increased in transgenic mice compared with the nontransgenic control. b) The virulence of the speB mutant was attenuated in both transgenic and control mice. c) Bacterial counts in the bloodstream of Tg+ mice 72 h postinfection are significantly higher for 5448 compared with speB. d) Compared with the SpeB activity of 5448 colonies isolated from the inoculum, the 5448 Tg+ lesion isolates exhibit a varied SpeB-expression phenotype, while the 5448 Tg+ blood isolates have significantly reduced SpeB activity.

3. SpeB activity of 5448 in vivo isolates
The WT strain used in the initial challenge uniformly expressed high levels of SpeB protease activity (100% of inoculum colonies was SpeB-positive; n=100). WT bacteria recovered from the lesions of four Tg⁺ mice expressed a mixed SpeB phenotype (74%, 66%, 63% and 31% of lesion colonies examined were SpeB-negative). However, WT bacteria recovered from the blood unvaryingly demonstrated very low SpeB activity (100% of blood colonies examined were SpeB-negative) (Fig. 2d ). This finding is consistent with the epidemiologic observation that M1T1 clinical isolates from human invasive disease express reduced levels of SpeB.

CONCLUSIONS AND SIGNIFICANCE

The mechanism utilized by GAS to switch from localized to systemic infection is presently unknown. In this study, we have shown that the absence of cysteine protease SpeB activity is required for the accumulation of plasmin activity on the cell surface of M1T1 GAS. In a humanized plasminogen transgenic mouse model of s.c. infection, the loss of SpeB activity at the site of infection triggers the systemic dissemination of M1T1 GAS in vivo. These data support the proposal that human plasminogen plays a critical role in the initiation of GAS invasive disease.

Our accumulated data suggest that SpeB, while contributing to localized infection, simultaneously mitigates the potential interaction of M1T1 GAS with the human plasminogen activation system. The ablation of SpeB expression in WT M1T1 GAS in the blood of plasminogen-humanized Tg⁺ mice indicates that the loss of SpeB activity in a subpopulation of bacteria occurs at the site of infection, allowing accumulation of surface plasmin activity. This subpopulation thus gain enhanced invasive propensity, favoring transition of GAS from the site of infection to the blood. In our model, vascular leakage induced by M protein complexing with fibrinogen to activate heparin binding protein release from neutrophils, may provide a source of plasminogen at the site of infection (Fig. 3 ).

View larger version (71K): [in this window] [in a new window]   Figure 3. Proposed model for group A streptococcal systemic disease initiation. a) S. pyogenes M1T1 (blue) gain entry through the skin and a host innate immune response is initiated. b) During the initial stages of infection, S. pyogenes M1T1 express SpeB to combat the host response. c) Loss of SpeB activity in a subpopulation of group A streptococci (green) leads to the accumulation of surface plasmin activity. d) Transition of S. pyogenes M1T1 is facilitated by surface plasmin activity, resulting in systemic infection.

The expression of the streptococcal cysteine protease SpeB and human invasive disease severity are inversely related in M1T1 clonal isolates. The model we propose accounts for this previously perplexing clinical observation and describes a mechanism by which systemic disease initiation occurs. The elucidation of the mechanism by which GAS causes human invasive disease will inform future treatment and prevention strategies.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5804fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5804fjev1 20/10/1745    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cole, J. N. Articles by Walker, M. J. Search for Related Content PubMed PubMed Citation Articles by Cole, J. N. Articles by Walker, M. J.