Study On Pathogenesis Of Group A Streptococcus CovRS Two-Component System

Group A Streptococcus (GAS) is one of the most important human bacterial pathogens. According to the data in2011, it shows that700million of mild, local infection cases occurred worldwide each year, in which650,000cases turn into serious invasive infections (severe systemic infection). One quarter of these invasive infection cases eventually lead to death. GAS can cause relatively mild infections including pharyngitis, acute tonsillitis, scarlet fever, skin infections and impetigo. It can also cause life-threatening invasive infections, such as toxic shock syndrome (TSS) and necrotizing fasciitis. Besides, GAS is also responsible for apyogenous sequelae, such as rheumatic heart disease and acute glomerulonephritis.There is currently no commercial vaccine against GAS because of its diverse serotype complexity and vaccine safety concerning. In recent years, there is an increase in cases of scarlet fever and severe systemic infections caused by GAS, which aroused greater concern for streptococcal infections. Despite there have been a lot of researches on virulence factors and pathogenesis of GAS, the pathogenic mechanism of GAS invasive infections and the molecular mechanism how local infections transit to invasive infections are still unclear. Therefore, further study on the mechanism of innate immunity evasion during invasive infections and the phase-switching mechanism could help to discover new anti-infection targets and establish early-stage diagnostic methods of invasive diseases. These progresses will be helpful to prevent and control severe invasive diseases.In this study, we selected representative strains of MIT1serotype to elucidate the pathogenesis of invasive infections. M1T1is the serotype which is mostly related with life-threatening conditions, such as streptococcal toxic shock syndrome and necrotizing fasciitis. First, our study found that GAS could inhibit the recruitment of neutrophils to infection sites via regulating the two-component system CovRS, in which way GAS increases the invasive capacity. This explains natural null mutation of CovRS in hypervirulent GAS strain MGAS5005is responsible for the enhancement of its invasiveness and virulence. Second, the importance of three reported factors, which are regulated by CovRS and involved in the inhibition of neutrophil recruitment, in the course of invasive infections are evaluated in invasive infection model. Third, neutrophils play an important role during the course, in which local infections caused by M1T1strains transit to invasive infections. Fourth, the molecular model of local invasive infections caused by M1T1serotype strains switching to invasive infections is further established. The main contents are as follows:1. Infection model of invasive infection caused by M1T1GAS strains is established.In this study, mice were subcutaneously infected with clinical isolates of invasive representative strain MGAS5005and pharyngitis representative strain MGAS2221to establish the infection model of invasive infection. Compared with pharyngitis representative strain MGAS2221, invasive representative strain MGAS5005displays more virulence and invasive capacity in soft tissue. In addition, the infection sites of MGAS5005was severely lack of recruited neutrophils, which is consistent with clinical manifestation that in cases of severe invasive infections-necrotizing fasciitis, infection sites also lacks of neutrophil. This indicates that the animal model successfully simulates the process of invasive infections, thus it laid the foundation for the study of pathogenesis of invasive infections.2. M1T1GAS strains inhibit the neutrophil recruitment to infection sites to increase the invasive ability and virulence via regulation of two-component system CovRS.Homologous replacement of natural null covS△1bp in MGAS5005with wild-type covS resulted in MGAS5005wtcovS. In animal infection experiments, MGAS5005wtcovS showed reduced virulence as well as invasive ability. Besides, it enhanced neutrophil ingress to infection sites, where the level of neutrophils was similar to that at MGAS2221sites. On the contrary, MGAS2221△covS mutant showed greatly enhanced virulence and invasion capacity. The level of neutrophils at MGAS2221△covS sites was similar to that at MGAS5005infection sites. Therefore, these results indicate that GAS employs the two-component system CovRS to inhibit neutrophil recruitment in infection sites, in which way increases invasive capacity and virulence. It also explains that covS null mutation is responsible for the enhancement of the inhibition of neutrophil recruitment and virulence in invasive strain MGAS5005. Detecting the mutation state of covRS in patients’infection sites is promising to develop into an auxiliary prediction method of invasive infections.3. Streptococcal secreted esterase (SsE) is the critical neutrophil recruitment inhibitor and virulence factor during invasive infections.Previous results demonstrated two-component system CovRS is closely related to inhibition of neutrophil recruitment in invasive infections. CovRS functions via regulating down-stream effectors. Interleukin-8/CXC chemokine peptidase (SpyCEP), C5a peptidase (ScpA), and streptococcal secreted esterase (SsE) are reported to be negatively regulated by CovRS and involved in inhibition of neutrophil recruitment during GAS infections. To evaluate and compare the importance of these factors during the invasive infection process, we constructed single mutants, double mutant AspyCEPAscpA, AscpAAsse, AspyCEPAsse and triple mutant△spyCEPAscpAAsse of invasive representative strain MGAS5005. In invasive infection model, comparing the single, double and triple mutants, a total of seven mutants we found that:all of the mutants with sse deletion (including single, double and triple mutants) decreased inhibition capacity of neutrophil recruitment and virulence; on the contrary, AspyCEP, AscpA and AspyCEPAscpA lead to similar overall pathology with parent strain MGAS5005; these three factors have no synergistic effect on inhibition of neutrophil infiltration and virulence enhancement. These results demonstrate that SsE is not only the key factor required for GAS inhibition of neutrophil infiltration during invasive infections, but also an important virulence factor of GAS. Development of drug and therapeutic monoclonal antibodies against SsE is promising for the treatment of invasive infections. Besides, association the expression level of SsE with the invasive infection severity has the potential to develop as an evaluation indicator of disease severity.4. Neutrophil plays a critical role during the phase of GAS in vivo transition from pharyngitis strain to invasive strain.Some studies reported that covRS spontaneously mutates in some mouse-passaged derivatives, which could not secret streptococcal pyrogenic exotoxin (SpeB). In this study, we refer these strains as SpeB negative (SpeB-) strains. Mice were subcutaneously infected with pharyngitis infection representative strain MGAS2221(SpeB positive). It was discovered that SpeB negative (SpeB-) mutants recovered from infection sites increased with the passage of time, which indicates that SpeB-mutants have survival advantage in infection sites compared to MGAS2221. Based on the result of in vitro growth curve and in vivo competitive growth assay, the survival advantage of SpeB-is not due to a growth advantage but the increasing resistance against inflammatory cells-mediated clearance. By RT-PCR analysis, we found the expression of important factors, such as spyCEP, hasA and sse, increased in SpeB-strains. Compared with MGAS2221, SpeB-strain showed enhanced virulence, capacity of neutrophil recruitment inhibition and invasiveness in mice soft tissue infection model. These results indicate that passage in mice could induce the pharyngitis representative strain MGAS2221to transit into SpeB strain with enhanced capacity of neutrophil recruitment inhibition, invasiveness and virulenc.To find the selective pressure favoring the phase-switching, mice model lacking neutrophil is used in this study. First, anti-Gr-1and anti-Ly6G monoclonal antibodies are used to deplete neutrophils in mice infection sites. The results verified that neutrophil involves in the phase-switching process. Second, the results with gp91phox-/-deficient mice, which are defective in neutrophil-mediated killing, further support that neutrophil plays a critical role during the phase of GAS in vivo transition. Besides, to exclude the effect exerted by inflammatory monocytes, CCR2-/-deficient mice, in which inflammatory monocytes could not emigrate to bloodstream from bone marrow, was used. The results showed that inflammatory monocytes didn’t contribute critically to this process.5. This study improved the theoretical model of local infections caused by M1T1switching to invasive infections(1) At the beginning of infection establishment, GAS needs wild type CovRS two-component system to sense environmental change and regulate downstream gene expression, as well as SpeB to destroy the host first barrier (such as the throat or skin epithelial cells).(2) After bacteria break through the first barrier, host will recruit a large number of neutrophils to the infection sites to clear the bacteria. In responding, GAS employs multiple mechanisms to evade from immunity clearance.(3) Partial bacteria mutated in covRS, which leads to increasing expression of.immunity evasion factors. These strains with enhanced capacity of innate immunity evasion take survival advantage in vivo.(4) GAS which succeeds in immunity evasion diffuses into the blood and cause invasive disease.