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Recent Streptolysin O Research

This page is intended to serve as a brief summary of several recent papers investigating the role of a virulence factor - streptolysin O - in infections caused by the bacteria Streptococcus pyogenes. Streptococcus pyogenes is a gram-positive bacterium that causes group A streptococcal (GAS) infections which affect up to 700 million people per year. While the mortality rate for these infections is relatively low, in recent decades there has been a significant increase in the number of invasive GAS infections which incur a mortality rate of up to 25%, thus demonstrating the importance of studying this organism in order to curb future disease-related fatalities. S. pyogenes have a number of toxins and other virulence factors which contribute to their pathogenic phenotype; one of particular interest is the exotoxin streptolysin O (SLO). SLO binds to the cholesterol in eukaryotic cell membranes leading to the formation of pores which, at sufficient SLO concentrations, promote the lysis of the target cell.

By lysing immune cells such as macrophages and neutrophils, S. pyogenes is able to better evade the innate defenses of its host organisms, promoting bacterial survival and contributing to a more invasive disease phenotype. Only recently have studies indicated that in macrophages this SLO-induced lysis is not a simple osmotic lysis, but is in fact a form of caspase-mediated apoptosis. Indeed, SLO has been shown to be necessary and sufficient to cause caspase-mediated macrophage apoptosis in vitro, and mutant S. pyogenes lacking SLO were significantly less virulent in vivo. These results demonstrate that research into the functionality of SLO is ongoing, making it an ideal virulence factor for further study. Ultimately the continued study of SLO will both yield strategies for combating invasive GAS infections and provide generalized insight into cytolysin functionality and corresponding host defense mechanisms.

Paper 1

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

In this paper the authors investigated the specificity of the S. pyogenes-derived cytolysin streptolysin O (SLO) in contributing to the cytolysin-mediated translocation (CMT) of effector proteins (SPN) into target cells. They first demonstrated that perfringolysin O (PFO), a largely homologous protein to SLO, is not CMT-competent despite its ability to cause poration. They next identified a domain found in SLO but not in PFO and created a mutant lacking this domain which was found not to be CMT-competent. When a PFO chimera containing this SLO-derived domain was constructed it was still not CMT-competent, the authors concluded that both this domain and a yet-unidentified domain are essential for SLO-mediated CMT.

The strength of this paper lies in the authors' ability to address alternative explanations of experimental results by providing evidence to support their hypothesis. For example, they suggest that the frequency of PFO poration might allow SPN to circulate back into the culture media, and they then show data demonstrating that SPN levels in the culture media are not above background in PFO-expressing strains. The main weakness of this paper is its reliance on a single protocol for the majority of the data presented therein. The overall conclusions may have been strengthened by employing alternative means to assess SPN translocation and host cell poration. As such alternatives may not be readily available, this does not significantly detract from the quality of this paper and the results are not in question. Further attempts to discover the additional domains of SLO required for CMT-competency could yield important insight into SLO functionality. Additionally, further study of the mechanisms underlying the CMT process may provide valuable information regarding this virulence property present in certain Gram positive bacteria.

Paper 2

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

In this paper, written by the same lab as the above paper, the authors investigated whether or not the pore-forming capacity of SLO is required for CMT to occur. To do so they created strains expressing mutant forms of SLO incapable of forming functional pores but still able to bind target cell membrane. Using these cells they determined that CMT is independent of pore formation, TLR4 function, and host cell clathrin-mediated endocytosis. They additionally concluded that both pore formation and CMT were required for cytotoxicity.

One main strength of this paper lies in its thorough experimental design. The authors carefully controlled experiments by using SLO mutants which simulated different stages of pore formation, and they were careful to rule out alternative pathways of SPN internalization. The greatest weakness of this paper is its brevity. Had the authors constructed more mutant slo genes and/or mutated host cell proteins, they would have been able to begin to delve into the biological processes responsible for their observed results, providing a more robust scientific article. The authors stress the fact that unlike PFO and other cholesterol dependent cytolysins, SLO is able to bind to cholesterol-poor membranes. They suggest that SLO may thus have an alternative membrane receptor which should be screened for in future experiments, thereby expanding current understanding of CMT functionality.

Paper 3

  • 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.

The authors of this important paper sought to understand the role of bacterial toxins in streptococcal toxic shock syndrome (STSS), an invasive form of S. pyogenes infection with a 30-70% mortality rate. They found that SLO is necessary to promote the formation of platelet/ neutrophil complexes which aggregate and occlude capillaries, leading to the tissue necrosis characteristic of STSS. They demonstrated that neutralization of SLO was sufficient to prevent aggregation and subsequent necrosis, and additionally presented data indicating that P-selectin was necessary for platelet/neutrophil aggregation.

This paper is strengthened significantly by its use of multiple techniques to confirm the importance of SLO in aggregate formation. The authors neutralized SLO activity with LDL, tested an S. pyogenes strain that does not produce SLO, and worked with recombinant SLO. The results from these experiments all ultimately supported the importance of SLO in STSS-linked platelet/neutrophil aggregation. The greatest weakness of this paper is its frequent use of the phrase “data not shown”. Several interesting results are mentioned without corresponding figures, making them somewhat less satisfying. This is no doubt due to page/figure restrictions on the part of the journal, and as such it only slightly detracts from the paper as a whole. While this paper demonstrates a clear role for SLO in STSS and thus introduces novel potential avenues of STSS diagnosis/treatment, it fails to delve into the mechanisms underlying these findings. Consequently, it opens the door to a number of SLO-linked future investigations which may ultimately lead to an improved STSS prognosis by allowing for an understanding of how SLO mediates this aggregation in vivo.

Paper 4

  • Goldmann O, et al. Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway. Cellular Microbiology. 2009; 11(1): 138-155.

This paper studied the S. pyogenes-induced killing of host macrophages in an attempt to better understand the basis for this pathogenic mechanism. The authors first determined that this macrophage death was most similar to an oncotic phenotype, and that this oncosis was dependent upon SLO/SLS expression. They found that the ensuing lysis was not an osmotic lysis, and that it coincided with a loss of mitochondrial membrane potential, an increase in intracellular ROS production, and could be reduced by inhibiting calcium-dependent endopeptidases.

The greatest strength of this paper is the comprehensive approach taken by the authors to confirm their results. By employing techniques including FACS, TEM/SEM, fluorescence microscopy, and in vivo analysis, the authors are able to achieve an improved understanding of the underlying process. While this wealth of data undeniably strengthens the results of this study, it also makes it harder to follow the progression of the paper due to the sheer number and variety of experiments. As a whole the results of the paper appear sound, however readability suffers and might be benefitted by a streamlining of the article as a whole. Future studies relating to this paper could focus on a number of topics, such as the mechanism by which SLO/SLS activity and ROS production/calcium signaling act to promote macrophage oncosis. Additional research would likely yield essential clues that would improve clinical treatments for those suffering from severe S. pyogenes infections.

Paper 5

  • Timmer AM, et al. Streptolysin O Promotes Group A Streptococcus Immune Evasion by Accelerated Macrophage Apoptosis. J Biol Chem. 2009; 284(2): 862-871.

This paper, published within 1 month of the Goldmann et al. paper, investigates the same S. pyogenes-induced macrophage death, but ultimately comes to different conclusions about certain aspects of the process. The authors demonstrated that intracellular S. pyogenes leads to caspase-dependent macrophage apoptosis, and that SLO is necessary and sufficient to induce this apoptosis. They additionally observed reduced mitochondrial membrane potential in moribund macrophages, and found that SLO expression reduced immune activation and promoted S. pyogenes survival both in vitro and in vivo.

This paper is particularly strong due to its clear, comprehensive experimental progression. The authors began by establishing the effects of S. pyogenes on macrophages in vitro before proceeding to identify SLO as the causative virulence factor via complementation experiments. They then went on to expand their study, concluding with an in vivo assessment that clearly demonstrated the significance of their research. As a whole this paper has no clear weaknesses, with the possible exception of its overabundance of similar-looking figures. The vast majority of the figures are nearly identical bar graphs which present data very effectively, but lend parts of the paper a somewhat cluttered and confusing appearance. Even so, as a whole this paper is comprehensive and coherent, leaving little room for complaint. As some of the results of this paper conflict with those of Goldmann et al., future experiments ought to try to determine whether this is due to the differences in macrophage/ bacterial strains used or due to a misinterpretation of data. This paper additionally demonstrates the clear clinical relevance of SLO in the context of S. pyogenes virulence in vivo, making the underlying mechanisms a clear target for future pharmaceutical study.

Paper 6

  • 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.

The authors of this paper sought to investigate the mechanisms by which S. pyogenes interacts with the innate immune system. They first demonstrated that S. pyogenes stimulated IL-1β secretion from macrophages in a caspase-1 and SLO dependent manner. They then found that S. pyogenes activates the intracellular Nlrp3 inflammasome, but that TLR signaling, rather than Nlrp3 activation, is necessary for caspase-1/pro IL-1β induction. They concluded with an experiment indicating that TLR signaling is only required in the context of recombinant SLO, and that a S. pyogenes infection is able to activate Nlrp3/caspase-1 activity through an unknown mechanism.

This paper is strengthened by its clear and methodical approach to experimentation. The authors fully explain each method and make it apparent as to how each experiment precipitated the next. This paper suffers somewhat due to its failure to fully clarify its conclusions. The authors did not emphasize the relevance of their TLR signaling results, which varied between experiments with recombinant SLO and live S. pyogenes. This experimental result is not surprising given the complexities of host-pathogen interactions in vivo, but it still served to somewhat detract from the clarity of the paper as a whole by obscuring the conclusions. The authors of this paper stress that much future work is needed to understand the full importance of inflammasome activation as a part of the innate immune response. To that end further research should examine the role of Nlrp3 in S. pyogenes infection in vivo. Similarly, establishing the mechanism by which SLO induces Nlrp3 activation may provide valuable insight into the pathogenic mechanisms of S. pyogenes and the corresponding host defenses.

Paper 7

  • 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.

The authors of this paper chose to investigate the mechanisms by which host cells cope with SLO-induced perforation of the cellular membrane. Through their experimentation they demonstrated that some cells are able to resist SLO-induced loss of cytoplasmic contents via a mechanism which may involve the membrane localization of Annexin A1, which may work to seal membrane pores. They additionally concluded that there is a critical intracellular Ca2+ concentration above which cells are unable to recover from SLO-perforation, ultimately leading to cell death and promoting bacterial invasion.

This paper is improved by its use of relatively novel means of fluorimetric analysis to analyze intracellular Ca2+ activity by using time-course imaging of Annexins labeled with calcium-sensitive fluorophores in SLO inoculated human embryonic kidney cells. This approach allowed for effective characterization of Annexin and Ca2+-linked responses to SLO, enabling the authors to clearly test their hypotheses. This paper does, however, suffer due to the fact that the aforementioned assay is the only experimental approach employed therein. The inclusion of other supporting results would better illustrate their conclusions and could improve the overall significance of the paper. In their concluding paragraphs, the authors suggest that Annexin A1 may operate as one mechanism of SLO removal from membranes of perforated cells. While this is speculative, it serves as a potential area for future research which could serve to both improve understanding of Annexin functionality and of the ways in which host cells are able to resist the cytolytic actions of SLO in the context of a complex S. pyogenes infection.

Paper 8

  • 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.

The authors of this paper studied the behavior of intracellular S. pyogenes prior to its elimination via autophagic degradation. They first observed that S. pyogenes was able to escape from early endosomes prior to being enveloped by autophagosomes. 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.

The strength of this paper is its ability to clarify its proposed model of S. pyogenes invasion and subsequent degradation in HeLa cells. The authors include a diagram of this model in order to emphasize the roles of Rab5, Rab7, and SLO in this process, thereby enhancing the clarity and quality of the paper as a whole. This paper does not have any significant weaknesses that detract from overall quality. Small changes could have been made, such as repositioning of the summary figure earlier in the paper to serve as a framework for reader understanding, but as a whole the paper is effective and contains appropriate knockout/complementation experiments which serve to confirm the findings presented therein.

This paper demonstrates that autophagic vesicles serve to eliminate S. pyogenes from cells following endosomal escape, but does not establish the mechanisms underlying the induction of this process. Consequently, future studies should seek to elucidate the means by which these bacteria induce autophagy, and should establish whether this process is nonspecific or relies upon certain bacterial epitopes and/or host pattern recognition receptors. Such research would provide valuable insight into the mechanisms underlying intracellular host/pathogen interactions and could implicate therapeutic targets for modulation of the autophagic response.

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