Construction of yycF-deficient mutant Streptococcus pneumoniae D39 with constitutive expression of pcsB and study on its pathogenicity

Abstract:

[Background] The YycFG two-component regulatory system plays a critical role in the Streptococcus pneumoniae response to the external environment. The response regulator protein YycF(VicR) is essential for the growth of Streptococcus pneumoniae, but the function of YycF in regulating bacterial virulence is unclear. [Objective] To analyze the effect of the response regulator protein YycF on biological characteristics and pathogenicity, the pcsB constitutive-expressing, yycF-deficient mutant strain of Streptococcus pneumoniae was constructed and characterized. [Methods] First, the pcsB constitutive expression strain (Pc-PcsB+) was constructed by janus cassette (JC) counter selection, and the yycF gene in Pc-PcsB+ was replaced with an erythromycin resistance gene (erm). The growth characteristics, the contents of capsular polysaccharide, cell adhesion and invasion abilities, and pathogenicity of D39rpsl41, Pc-PcsB+ and Pc-PcsB+DyycF were assessed. [Results] The yycF-deficient mutant strain (Pc-PcsB+DyycF) was derived from Pc-PcsB+. Compared with Pc-PcsB+, we observed slower growth, abnormal division, increasing amount of capsular polysaccharide in intracellular and smaller molecule capsular polysaccharide in the Pc-PcsB+DyycF. In vitro studies showed that the adherence ability of Pc-PcsB+DyycF was significantly reduced than that of Pc-PcsB+ (P=0.006). The virulence test suggested that all mice infected with D39rpsl41 died, while the mortality rates of mice challenged with Pc-PcsB+, Pc-PcsB+DyycF were decreased to 91.7% and 75% respectively, though no statistical significance between them was observed (P=0.183). The colonization study revealed that the bacterial burden of Pc-PcsB+DyycF in the lung tissue was significantly lower than that of Pc-PcsB+ (P=0.033). [Conclusion] In this study, the yycF-deficient mutant Streptococcus pneumoniae D39 was constructed successfully, and the biological characteristics and pathogenicity of Pc-PcsB+DyycF were identified, which provide a theoretical basis for further study on the regulatory mechanism of YycFG on the pathogenicity of Streptococcus pneumoniae.