Presentation Title

Group A Streptococcus ruptures the phagolysosomal membrane in THP-1 macrophages using pore-forming toxins.

Faculty Mentor

Cheryl Y. Okumura

Start Date

17-11-2018 10:15 AM

End Date

17-11-2018 10:30 AM

Location

C162

Session

Oral 2

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Group A Streptococcus (GAS) is a gram-positive bacterial pathogen responsible for approximately 750 million human-specific infections worldwide every year, ranging in severity from mild pharyngitis to necrotizing fasciitis, which illustrates that GAS has evolved several mechanisms to counter the innate immune response. To defend against invasive pathogens, such as GAS, the innate immune system typically deploys macrophages to phagocytose and eradicate the pathogen via engulfment into the phagosome, fusion with lysosomes, phagolysosomal acidification, and activation of proteolytic enzymes. We have previously demonstrated that GAS persists within macrophages despite trafficking normally through the phagocytic pathway. However, because GAS is not capable of surviving highly acidic conditions, this suggests that GAS instead prevents lysosomal acidification. Here, we investigated the possibility that GAS prevents acidification by physically rupturing the phagolysosomal membrane, inhibiting generation of the acidifying proton gradient necessary for activation of proteolytic enzymes. Immunofluorescence data suggest that phagosomal leakage of fluorescent dyes as large as 40 kD occurs in THP-1 macrophages infected with wild type (WT) GAS. Furthermore, GAS-infected macrophages display significant staining of phagolysosomes when probed with galectin-3, a molecular marker for severe phagosomal membrane damage. An additional investigation to determine the specific virulence factors employed for membrane disruption suggests that the pore-forming toxin streptolysin O (SLO) provides a crucial role in the mechanism for phagolysosomal membrane disruption. Overall, we have concluded that GAS induces phagolysosomal membrane damage via a SLO-dependent mechanism. The information gained through our investigations provides important insights into the specific mechanisms of how GAS has evolved to evade the innate immune response, which may lead to the development of more effective therapies targeting this pathogenic mechanism.

Summary of research results to be presented

In order to investigate the possibility that GAS prevents acidification by physically rupturing the phagolysosomal membrane, macrophage phagolysosomes were loaded with multiple sized fluorescent dyes (i.e. 500 D, 10 kD, 40 kD) prior to infection with GAS. The immunofluorescence data suggest that phagosomal leakage of even the 40 kD fluorescent dye occurs when infected with wild type (WT) GAS. To indicate severity of phagolysosomal leakage (i.e. lysosomal membrane damage), GAS-infected macrophages were then probed for galectin-3 staining, which is a molecular marker indicating severe phagosomal membrane damage. Here, GAS-infected macrophages display significant galectin-3 staining, suggesting a high severity of phagolysosomal membrane damage and subsequent leakage. Finally, to identify specific virulence factors, these experiments were repeated by infecting macrophages with the following different strains of GAS: Heat-killed GAS (HK), Streptolysin O (SLO) knockout, and Streptolysin S (SLS) knockout. While SLS displayed significant 40 kD leakage and galectin-3 staining, similar to WT GAS, HK and SLO knockout appeared to inhibit phagolysosomal leakage and damage. This indicated that the pore-forming toxin streptolysin O (SLO) plays a crucial role in the mechanism for phagolysosomal membrane disruption. As a result, we have concluded that GAS induces severe phagolysosomal membrane damage via a SLO-dependent mechanism.

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Nov 17th, 10:15 AM Nov 17th, 10:30 AM

Group A Streptococcus ruptures the phagolysosomal membrane in THP-1 macrophages using pore-forming toxins.

C162

Group A Streptococcus (GAS) is a gram-positive bacterial pathogen responsible for approximately 750 million human-specific infections worldwide every year, ranging in severity from mild pharyngitis to necrotizing fasciitis, which illustrates that GAS has evolved several mechanisms to counter the innate immune response. To defend against invasive pathogens, such as GAS, the innate immune system typically deploys macrophages to phagocytose and eradicate the pathogen via engulfment into the phagosome, fusion with lysosomes, phagolysosomal acidification, and activation of proteolytic enzymes. We have previously demonstrated that GAS persists within macrophages despite trafficking normally through the phagocytic pathway. However, because GAS is not capable of surviving highly acidic conditions, this suggests that GAS instead prevents lysosomal acidification. Here, we investigated the possibility that GAS prevents acidification by physically rupturing the phagolysosomal membrane, inhibiting generation of the acidifying proton gradient necessary for activation of proteolytic enzymes. Immunofluorescence data suggest that phagosomal leakage of fluorescent dyes as large as 40 kD occurs in THP-1 macrophages infected with wild type (WT) GAS. Furthermore, GAS-infected macrophages display significant staining of phagolysosomes when probed with galectin-3, a molecular marker for severe phagosomal membrane damage. An additional investigation to determine the specific virulence factors employed for membrane disruption suggests that the pore-forming toxin streptolysin O (SLO) provides a crucial role in the mechanism for phagolysosomal membrane disruption. Overall, we have concluded that GAS induces phagolysosomal membrane damage via a SLO-dependent mechanism. The information gained through our investigations provides important insights into the specific mechanisms of how GAS has evolved to evade the innate immune response, which may lead to the development of more effective therapies targeting this pathogenic mechanism.