GASAUTOPHAGY · Influence of Streptococcal virulence factors on host autophagy regulation

Influence of Streptococcal virulence factors on host autophagy regulation.

Streptococcus pyogenes, also called group A streptococci (GAS), are bacteria that cause many different types of human infections, ranging from non-life-threatening, superficial infections like “strep throat” to more severe, life-threatening, invasive infections like “flesh eating disease.” GAS cause over 700 million superficial infections and 650,000 invasive infections worldwide annually, with 3,500 cases reported in the UK each year. These high-risk invasive infections afflict otherwise healthy people, and ultimately kill 20-30% of patients. To cause invasive infection GAS must first penetrate the skin, which acts as a protective barrier between delicate internal tissues and the external world. Skin cells, also called epithelial cells, have their own immune defenses to kill GAS. In the last decade, a new immune defense system in the epithelial cells was identified, which is autophagy. Autophagy was initially defined as “self-eating,” bulk degradation system for cytoplasmic components. It is essential for clearance and recycling of intracellular contents and was originally described as a starvation-induced response that provides nutrients by degrading long-living proteins and by recycling intracellular organelles. Additionally, it was reported that epithelial cells can clear intracellular invading GAS by autophagy. Besides numbers of human immune system targeting GAS, GAS still causes invasive infections. GAS uses bacterial proteins called virulence factors to evade host defenses, dodging the immune system to invade the body and cause serious, life-threatening infection. The main objective of this project was to identify new virulence factors that enable evasion of the newly discovered immune system autophagy. This project also aimed to discover the roles of autophagy in immune defenses against other bacteria such as Group C or Group G streptococcus. The main result of this project has been the discovery of the novel role of GAS protein SpyCEP, a GAS serine protease that was previously described to cleave and inactivates human cytokine IL-8. Dr. Uchiyama found that the GAS M1 protease SpyCEP activates the host protease calpain, which is a known endogenous autophagy inhibitor. As a result, GAS M1 inhibits autophagy and avoids capture in autophagosomes. Several lines of evidence support this conclusion. (1) Knocking out spyCEP in GAS M1 wt (M1 ΔcepA) decreased activation of host calpain, increased activation of autophagy and increased association of M1 ΔcepA with the LC3 autophagosome marker. (2) Inhibition of calpain with calpeptin resulted in increased activation of autophagy, and increased association of intracellular M1 wt with LC3. (3) Purified SpyCEP activated calpain and inhibited autophagy. (4) Over-expression of SpyCEP in the autophagy susceptible GAS JRS4 wt (JRS4 pcepA) strain resulted in host calpain activation and resistance of JRS4 pcepA to autophagy. In conclusion we report a new role for a well-known GAS virulence factor, SpyCEP. By activating host calpain and inhibiting autophagy, the GAS M1 strain evades autophagy mediated killing in epithelial cells. This may be a critical mechanism for establishing invasive infection and SpyCEP can be a possible target for treatment of invasive GAS infections. Another important objective reached in this project has been the investigation of the role of autophagy against Group C streptococcus (GCS) and Group G streptococcus (GGS). These pathogens which were originally grouped as “zoonotic streptococci,” are of increasing clinical interest for causing serious infections in humans. All three GAS, GCS and GGS share virulence factors that may have been transferred between streptococcal species via extensive horizontal gene-transfer. GCS and GGS had never been studied in relation with host autophagy. In this project, Dr.Uchiyama have discovered that autophagy in host epithelial cells clear both GCS and GGS efficiently. We could show this in a visualization assays using immunofluorescence microscope, and functional assays analyzing autophagy mediated killing of intracellular bacteria. The results obtained in this project have high relevance in the field of host-pathogen interaction of intracellular bacteria, showing a novel mechanism of autophagy evasion by intracellular bacteria. GAS SpyCEP can be a potential target for treatment in the future, letting increased autophagy mediated clearance of intracellular GAS. In addition to the scientific results, this project has also enabled great personal and professional development of the grantee. Dr. Uchiyama have experienced a top research environment, learned to lead a science project and had possibility to supervise student and a postdoc. These experiences and career trainings enabled Dr. Uchiyama to be appointed for a junior faculty position as an assistant project scientist in the University of California San Diego. This research work has also built extensive scientific communication across Europe, Japan and US which lead to several invited seminars in different countries.

Data: CORDIS, © European Union