Date Published: October 31, 2018
Publisher: Public Library of Science
Author(s): Nicola Horstmann, Chau Nguyen Tran, Chelcy Brumlow, Sruti DebRoy, Hui Yao, Graciela Nogueras Gonzalez, Nishanth Makthal, Muthiah Kumaraswami, Samuel A. Shelburne, Michael R. Wessels.
The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.
The ability of bacteria to modify gene expression levels in adaptation to external influences is key to many aspects of bacterial pathogenesis . Two-component regulatory systems (TCS) are a major mechanism by which bacteria detect and respond to diverse environmental factors [2, 3]. TCS are absent in humans but abundant in a wide range of bacteria. They usually consist of a membrane-embedded histidine kinase that determines the regulatory activity of its cognate response regulator by altering its phosphorylation status [2, 4].
Although phosphorylation of response regulator proteins is critical for bacterial pathogenesis, there remains limited understanding of how variation in response regulator phosphorylation impacts bacterial virulence at diverse infection sites. Herein we employed transcriptome and virulence assays of an array of isoallelic serotype M1 and M3 GAS strains to assess the impact of multiple, distinct phosphorylation levels of the key response regulator CovR on GAS pathophysiology.