The Role of SLO in Streptococcus pyogenes Infections

Abstract

Streptococcus pyogenes is a pathogen which annually infects ~10% of the world's population, extolling an economic burden of up to $500 million/year in the United States alone. Approximately 0.1% of these group A streptococcal, GAS, infections take the form of invasive diseases which have a devastating mortality rate of 25%. As invasive GAS infections have been undergoing a resurgence during the past 30 years, there is a clear need for extensive research in order to identify new methods to combat this widespread pathogen.

A significant body of recent research focused on the pleiotropic virulence factor streptolysin O, SLO, a cytolytic exotoxin produced by almost all known strains of S. pyogenes. This toxin has been identified as having essential roles in the killing of macrophages in host organisms, thereby allowing for immune evasion and increasing the virulence of these bacteria. SLO has also been implicated in a process whereby toxins and other effector proteins are injected into target cells in order to promote pathogenicity.

The aforementioned mechanisms underscore the value of studying SLO in the context of GAS infections. Future research should seek to clarify the means by which SLO mediates macrophage death and effector protein translocation into host cells, as these properties significantly increase the pathogenicity of S. pyogenes. Any technique capable of modulating these mechanisms could help to identify valuable drug treatments, which would reduce the mortality and morbidity of this widely disseminated microorganism and improve prognosis for infected individuals worldwide.

Introduction

Streptococcus pyogenes is a Gram-positive bacterium that causes group A streptococcal (GAS) infections, which occur in approximately 700 million people each year [1]. These bacteria are best known for causing streptococcal pharyngitis, a disease which has an estimated annual economic burden of $225-$539 million in the United States and an associated mortality of less than 0.1% [1,2]. In the past 30 years there has been an increase in the incidence of invasive GAS infections, with an estimated 633,000 cases/year and a 25% mortality rate [3]. The significant mortality and morbidity associated with GAS infections underscores the importance of researching the virulence properties of this pathogen further in order to curb future rates of infection and death.

S. pyogenes has several virulence factors which work synergistically to promote pathogenesis; one protein which has been of particular interest in recent years is the exotoxin streptolysin O (SLO). This monomeric protein binds to the cholesterol of eukaryotic cell membranes and oligomerizes to form 20-100 member transmembrane rings, ultimately leading to the lysis of target cells [4,5]. While the mechanisms underlying SLO functionality have been known for ~20 years, the true disease-relevance of this toxin has only been thoroughly investigated within the past decade. Recent research has revealed pleiotropic roles for SLO in GAS pathogenesis. The cytolytic properties of SLO allow it to kill macrophages thereby promoting immune evasion, whereas sublytic SLO concentrations have been associated with other GAS virulence properties [4]. As SLO is a critical virulence factor of pathogenic GAS strains, ongoing studies are working to unravel its functionality in the context of disease. The ultimate goal of using the newfound knowledge of GAS virulence is to reduce disease-associated mortality and morbidity across the world.

State of Field

Since it is often described as a “pore-forming cytolysin”, it is not surprising that SLO kills the macrophages of its host by forming transmembrane channels in the plasma membrane. Initially it was suggested that these pores led to a simple osmotic lysis of target cells, however recent research into SLO-mediated macrophage death has implicated a more complex lytic mechanism.

A 2009 study by Goldmann et al. demonstrated that during the course of a GAS infection, dying macrophages display an oncotic phenotype, which is dependent upon the expression of SLO by infecting S. pyogenes [6]. This oncotic phenotype was said to be more similar to a necrotic than to an apoptotic phenotype, as demonstrated by the fact that no caspases were active in lytic macrophages following GAS infection. They further showed that osmoprotectants did not reduce cytotoxicity and that cell death coincided with a loss of mitochondrial membrane potential, a hallmark of programmed cell death [6]. They speculated that SLO-induced pores may lead to a depletion of cellular ATP in host cells thereby causing the opening of “death channels” in the plasma membrane, ultimately leading to cell lysis [6].

A separate study published in 2009 by Timmer et al. came to different conclusion regarding macrophage cell death during GAS infections. Whereas Goldmann et al. had suggested that the SLO-mediated cell death was oncotic, Timmer indicated that it is a classical apoptotic cell death [6,7]. Timmer et al. found that SLO was necessary and sufficient to induce macrophage apoptosis, and that this apoptosis was partially caspase-dependent and was associated with a loss of mitochondrial membrane potential [7]. They also expanded on their research by demonstrating that SLO+ GAS strains exhibit macrophage apoptosis and reduced immune activation in vivo as compared to ΔSLO mutant strains [7]. This finding emphasizes the disease-relevance of SLO as a virulence factor and suggests that SLO-induced macrophage apoptosis may be an important means of immune evasion utilized by pathogenic S. pyogenes.

While the findings of Goldmann et al. and Timmer et al. do conflict on some points, they agree on the core conclusion that SLO expression is necessary for GAS to induce the programmed cell death of host macrophages, and that this death is marked by a loss of mitochondrial membrane potential. The observed differences in macrophage morphology and caspase activation may not be due to failures in experimental methodology on the part of either research group, but may rather be due to certain variables not taken into account during experimentation. One possible explanation is touched on in a 2009 study by Harder et al.. which examined the role of Nlrp3 inflammasome activation in caspase-1 activity during the course of GAS infections [8].

Harder et al. found that S. pyogenes induces IL-1β in a caspase-1 and SLO-dependent manner, which is consistent with the work done by Timmer et al [7,8]. They additionally found that activation of the Nlrp3 inflammasome and NF-κB signaling were required for caspase-1 activation, and that TLR signaling played an important role in this process [8]. Interestingly, they noted that while Nlrp3 was required for caspase-1 activation, a lack of Nlrp3 did not affect host susceptibility to infection with S. pyogenes. This result suggests that S. pyogenes may be able to infect hosts via multiple mechanisms, potentially explaining why in one case SLO may induce caspase-1-independent macrophage oncosis while in another it may induce caspase-1-dependent macrophage apoptosis. Differences in other experimental factors may have led to differential Nlrp3 activation/ NF-κB signaling in the experiments of Goldmann et al. leading to this different experimental result. Further experiments are necessary to fully clarify the basis for this variation in disease phenotype.

In 2009 Babiychuk et al. chose to examine the cellular aspects of SLO-induced lysis by studying the role of Ca2+ in this process. They found that extracellular Ca2+ was protective against SLO-induced cell death, and that the intracellular concentration of Ca2+ following perforation of the cell membrane was responsible for establishing whether or not a particular cell proceeded to cell death [9]. They established that there is a critical intracellular concentration of Ca2+ above which SLO-perforated cells die and below which they have the potential to proceed to repair and survival [9]. While it did not directly assess the functionality of SLO in the context of target cell death, this study revealed important new information with regard to the downstream effects of the perforation of the plasma membrane. An integration of these results with the aforementioned studies on macrophage death in the context of GAS infections may help to yield a more complete picture of the complete process responsible for this destructive virulence property of S. pyogenes.

SLO has also been implicated as being essential to cytolysin-mediated translocation (CMT), which is a process whereby S. pyogenes is able to promote the uptake of effector proteins by target cells in lieu of the type three secretion system present in many Gram-negative bacteria. S. pyogenes utilizes CMT in order to induce the uptake of S. pyogenes nicotinamide adenine dinucleotide-glycohydrolase (SPN) resulting in an increase in cytotoxicity [10]. Mutant strains lacking SPN exhibit a marked reduction in virulence, suggesting that CMT is a critical virulence process, which is likely active during pathogenic GAS infections [11].

Because SLO is essential for CMT, early reports speculated that the presence of SLO-induced transmembrane pores allowed for the simple diffusion of SPN into target cells. In order to investigate this hypothesis, Meehl and Caparon performed experiments in which they introduced a plasmid carrying the gene for the functionally similar cytolysin perfringolysin O (PFO) into a ΔSLO S. pyogenes strain [10]. They discovered that PFO does not complement the ΔSLO mutation, and that at no point during infection did SPN translocate into PFO perforated cells [10]. As a result of this finding, they created a mutant strain expressing a form of SLO which contained a deletion of an N-terminal domain not found in the otherwise highly homologous PFO protein. By deleting this region they were able to eliminate CMT while still allowing SLO to perforate target cell membranes, suggesting that the pore-forming functionality of SLO may not be essential to this process [10]. In a final experiment, these researchers introduced the N-terminal domain of SLO into PFO and determined that it is necessary but not sufficient to induce CMT, leaving the mechanisms underlying CMT largely undetermined [10].

In an effort to further investigate these mechanisms, researchers from the same laboratory carried out a study in 2010 in which they sought to determine whether pore formation was at all necessary for SLO-mediated CMT. In order to test this they created several SLO mutants that were “locked” at different stages in the oligomerization process, which occurs during proper pore formation [11]. They discovered that these SLO mutants were still able to associate with target cell membranes and promote CMT, leading to the conclusion that pore formation is not required for CMT to occur [11]. Nonetheless, they noted that CMT alone did not promote cytotoxicity, and that only pore forming CMT-competent S. pyogenes were highly lethal. This conclusion underscores the importance of the pleiotropic effects of SLO in the promotion of S. pyogenes virulence, however it fails to elucidate the mechanisms responsible for CMT and as a result future experiments will be necessary to fully understand this process.

In addition to these two important roles played by SLO in GAS infections, this protein has also been shown to be essential to other processes which promote invasive forms of disease. For example, in 2005 Bryant et al. published a paper investigating the importance of SLO in the formation of platelet/neutrophil complexes during streptococcal toxic shock syndrome (STSS), an invasive GAS infection with a 30-70% mortality rate [12]. They found that SLO is necessary to promote the formation of these complexes, which aggregate and occlude capillaries, leading to the tissue necrosis characteristic of STSS [12]. They also demonstrated that neutralization of SLO was sufficient to prevent aggregation and subsequent necrosis, and presented data indicating that P-selectin was necessary for platelet/neutrophil aggregation [12]. This work serves to highlight the devastating role, which SLO can have in the progression of disease, making it a clearly valuable target for future research.

In 2010 Sakurai et al. investigated another aspect of S. pyogenes invasiveness, which had been relatively poorly characterized; they examined the invasion and subsequent degradation of these bacteria via the autophagic machinery in HeLa cells. They first observed that S. pyogenes was able to escape from early endosomes prior to being enveloped by autophagosomes [13]. They additionally noted that the cellular Rab5 and Rab7 proteins were necessary for proper endosomal maturation in S. pyogenes infected cells, and that an accumulation of SLO-induced pores in the early endosome was necessary for bacterial escape prior to subsequent autophagic degradation [13]. They ultimately concluded that in this aspect of the infective process ΔSLO mutants were able to survive longer than WT S. pyogenes, however these mutants were unable to escape the endosomes and were eventually degraded. This research revealed an interesting role played by SLO which may not enhance virulence in healthy individuals, but which provides unique insight into one of this proteins numerous activities in vivo.

As these abovementioned studies clearly demonstrate, SLO is a virulence factor, which plays myriad roles in the progression of both invasive and noninvasive GAS infections. Despite its importance in the context of pathogenicity, much remains to be studied regarding the mechanisms underlying SLO functionality.

Future Directions

Despite having been a well-known cytolytic exotoxin of S. pyogenes for several decades, the mechanisms underlying the disease-relevance of SLO have only recently been thoroughly investigated. As a result, much remains to be clarified regarding the multiple roles which this protein plays in the progression of GAS infections in vivo. By fully investigating these mechanisms it may be possible to alter them in such a way as to improve disease prognosis, thereby curbing the elevated mortality of invasive GAS infections.

As the studies of Goldmann, Timmer, and Harder demonstrated, the mechanism by which SLO mediates the death of host macrophages during infection has yet to be fully examined and multiple mechanisms may be important to the overall process. A more unified understanding of this topic may be achieved by synthesizing the methods of these three papers into a single study. One may hypothesize that all forms of macrophage death which occur during GAS infections work through a unified series of mechanisms which will be clarified if the experimental approaches of these three papers were to be combined into a single study.

In order to test this hypothesis, the experiments of Goldmann et al. should be replicated in vitro with macrophages derived from mice which are deficient in Nlrp3 activity in order to assess whether or not Nlrp3 plays a role in SLO-induced macrophage oncosis. The activation of NF-κB during these experiments should also be measured, as should the production of IL-1β. If SLO-induced macrophage oncosis but not apoptosis still occurs despite a lack of Nlrp3 activation then this may represent an alternative pathway utilized by S. pyogenes in order to promote immune evasion in vivo. If NF-κB signaling does not play a significant role in this oncotic process then additional experiments should seek to clarify the relevant signaling pathways, as this may help in the identification of the downstream targets, which are responsible for this intermediate cell death phenotype.

Cytolysin mediated translocation of SPN into target cells has been shown to be an important virulence mechanism in pathogenic S. pyogenes strains, yet the mechanism underlying this process is still poorly characterized. Based on previous research, it is likely that there are multiple domains of the SLO protein including the N-terminal domain, which are essential to CMT.

In order to test this hypothesis and to better determine the molecular basis for CMT, an extensive series of SLO mutants should be created which contain point mutations/deletions. These mutants should be introduced into ΔSLO S. pyogenes strains, which should then be screened for their ability to promote pore formation and CMT in A549 adenocarcinoma cells. This process has the potential to identify domains, which are essential to CMT that have not previously been identified. Such identification would yield a wealth of new knowledge regarding CMT and would open several avenues of future experimentation with the ultimate goal of inhibiting/targeting the CMT process in individuals suffering from GAS infections.

The abovementioned experiments will serve as a crucial first step in the fight to eliminate the threat caused by this ubiqutous pathogen. These and other future studies will serve to promote a more complete understanding of the roles played by SLO in the progression of GAS infections. This new information will in turn allow for the development of novel drug treatments which may ultimately help to save thousands of lives each year and to improve the quality of life for countless other individuals across the world.

References

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2. Pfoh E, et al. Burden and Economic Cost of Group A Streptococcal Pharyngitis. Pediatrics. 2008; 121: 229-234.

3. Carapetis JR, et al. The global burden of group A streptococcal diseases. The Lancet - Infectious Diseases. 2005; 5(11): 685-694.

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8. Harder J, et al. Activation of the Nlrp3 Inflammasome by Streptococcus pyogenes Requires Streptolysin O and NF-kB Activation but Proceeds Independently of TLR Signaling and P2X7 Receptor. J Immunol. 2009; 183(9): 5823-5829.

9. Babiychuk EB, et al. Intracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells. Cell Death and Differentiaton. 2009; 16: 1126-1134.

10. Meehl MA, Caparon MG. Specificity of streptolysin O in cytolysin-mediated translocation. Molecular Microbiology. 2004; 52(6): 1665-1676.

11. Magassa N, et al. Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O. EMBO Reports. 2010; 11(5): 400-405.

12. Bryant AE, et al. Vascular Dysfunction and Ischemic Destruction of Tissue in Streptococcus pyogenes Infection: The Role of Streptolysin O-Induced Platelet/Neutrophil Complexes. J Infect Disease. 2005; 192: 1014-1022.

13. Sakurai A, et al. Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7. J Biol Chem. 2010; 285(29): 22666-22675.


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