c Streptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus (GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1 gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the ⌬scl mutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.

Streptococcus pyogenes is a bacterial pathogen that can cause a range of infections from pharyngitis to necrotizing fasciitis. During an infection, the phagocytes of the host engulf the bacteria and fuse the phagosome with a lysosome to degrade the bacteria and prevent further infection. Previous experiments using a pH‐sensitive GFP construct demonstrated that while bacteria are localized to the lysosomal compartment of THP‐1 monocytes, the construct was not degraded. Because we determined the GFP construct was indeed pH‐sensitive, one possible explanation is that S. pyogenes is able to survive the low pH conditions of a lysosome. We tested the growth of S. pyogenes in different pH medias, ranging from pH = 4 ‐ 7. Our results indicated that within the pH range of a lysosome, bacteria persisted, but did not proliferate. Upon more detailed examination of the bacteria in a narrower pH range (pH = 4 ‐ 5), we found that bacteria were able to survive at pH = 4.5 ‐ 4.7. These results were corroborated with fluorescence microscopy of pH‐sensitive GFP‐expressing bacteria incubated in low pH media. Thus, S. pyogenes can survive the low pH‐environment of the lysosome. Our data do not however exclude the possibility that S. pyogenes can also prevent the acidification of the lysosome and future experiments will be aimed at measuring the pH of the lysosome in an infected cell as well as elucidating bacterial factors involved in pH resistance.

Group A Streptococcus (GAS) has evolved multiple immune evasion strategies in order to successfully establish infections in otherwise healthy individuals. In this study, we sought to create a molecular tool to study intracellular trafficking and degradation of GAS by host phagocytic cells. We created a construct using a tandem red fluorescent protein linked to a pH‐sensitive green fluorescent protein (mWASABI), which degrades upon contact with the low pH environment of the lysosome. We confirmed successful cloning and expression of the construct into GAS and degradation of the green fluorescent protein under low pH conditions. However, to our surprise, infection of THP‐1 macrophage cells with the RFP‐mWASABI expressing bacteria in combination with immunofluorescent assays indicates the failure of the GFP to degrade in host cells despite bacterial colocalization with lysosomes. Our data suggests GAS either disables lysosomal acidification, thus preventing the activation of lysosomal degradative enzymes, or withstands the low pH environment and proteolytic enzymes present in the lysosomes. Current work focuses on elucidating specific bacterial factors involved in lysosomal resistance mechanisms. These studies contribute to our understanding of how GAS has evolved to circumvent cellular defense mechanisms and will aid in improving treatment options for GAS infections.

Group A Streptococcus (GAS) is a Gram‐positive bacterium that is among the top 10 causes of infection‐related mortality in humans, with infections ranging from minor illnesses such as pharyngitis, to life threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. As the first response to infection, the innate immune system recruits leukocytes such as macrophages to the site of GAS infection, which are able to phagocytose GAS. Once GAS is inside the phagosome, the lysosome fuses with the phagosome to form the phagolysosome. To degrade pathogens such as GAS, the phagolysosome reduces its internal pH to activate proteolytic enzymes. To monitor this process, we infected THP‐1 macrophages with M1 serotype GAS transformed with a plasmid (pWASABI) that expresses a pH‐sensitive green fluorescent protein that is degraded at low pH. Our results showed that GAS expressing pWASABI has a persistent fluorescent signal when bacteria were present in phagolysosomes. In comparison, the non‐pathogenic Gram‐positive species Lactococcus lactis expressing pWASABI had an appropriate decreased fluorescence signal. These results suggest that GAS inhibits the acidification of the phagolysosome. To further confirm the pH of the phagolysosome, we stained infected macrophages with Lysotracker, an acidotropic fluorescent dye. The preliminary results of this experiment confirmed that phagolysosomes containing GAS are not acidified. Previous research has suggested that pore‐forming toxins such as streptolysin O (SLO) and streptolysin S (SLS) induce phagolysosomal leakage of protons. To examine this, we transformed a mutant strain lacking SLO (ΔSLO) with pWASABI. Our preliminary results showed no difference in fluorescence signal when the ΔSLO strain was present in phagolysosomes, suggesting that SLO may not be inducing lysosomal leakage. We are currently exploring whether pore formation by SLS plays a role in lysosomal leakage. The results of these experiments will help provide a clearer image of the molecular mechanism of innate immune resistance of GAS, and may aid in the development of therapeutics aimed to enhance the innate immune system's ability to degrade GAS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.