Regulation of type 3 fimbria expression by RstA affects biofilm formation and virulence in <i>Klebsiella pneumoniae</i> ATCC43816

<i>Klebsiella pneumoniae</i> causes both community-acquired and healthcare-associated infections, presenting a major therapeutic challenge to global public health. RstBA is a common two-component regulatory system that controls downstream gene expression in certain <i>Enterobacteriaceae</i> species. However, the role of RstBA in <i>K. pneumoniae</i> infection remains unknown. To determine its function, a wild-type <i>K. pneumoniae</i> strain (ATCC43816) and <i>rstA</i> mutant and complementation strains were constructed. Phenotypic experiments and <i>in vivo</i> animal infection assays demonstrated that deletion of <i>rstA</i> decreased virulence and biofilm formation. RNA sequencing analysis of ATCC43816 and <i>rstA</i> mutant strains was performed to study the regulatory mechanisms, revealing differential expression of genes involved in arginine and proline metabolism, phenylalanine metabolism, and quorum sensing. In addition, the <i>mrkI</i> and the <i>mrkABCDF</i> gene cluster, which regulates and encodes type 3 fimbriae, exhibited lower expression in the absence of <i>rstA</i>, possibly related to decreased virulence and biofilm formation. Quantitative real-time reverse transcription PCR, promoter activity assays, and electrophoretic mobility shift assays were conducted to identify the transcriptional regulation of <i>mrkI</i> and <i>mrkABCDF</i> by <i>rstA</i>. Our findings show that <i>rstA</i> regulates type 3 fimbriae expression by regulating <i>mrkI</i> indirectly and regulating <i>mrkA</i> directly by binding to its promoter. This study provides new insights into the functional importance of RstA in regulating biofilm formation and virulence in <i>K. pneumoniae</i>.IMPORTANCE<i>Klebsiella pneumoniae</i> is an opportunistic pathogen that has become a significant cause of community-acquired and nosocomial infections. The rise of hypervirulent and multi-drug-resistant <i>K. pneumoniae</i> poses a significant threat to public health. The two-component regulatory system is a typical signal-sensing and stress-response system widely distributed in bacteria, playing a critical regulatory role in bacterial infection. Through <i>in vivo</i> and <i>in vitro</i> experiments, we demonstrate that <i>rstA</i> regulates the expression of type 3 fimbriae by regulating <i>mrkI</i> indirectly and <i>mrkA</i> directly, thereby playing an essential role in the virulence and biofilm formation of <i>K. pneumoniae</i>. Understanding the regulatory mechanism of RstA in <i>K. pneumoniae</i> provides a proof-of-concept for identifying new genetic targets for controlling <i>K. pneumoniae</i> infection, which may aid in the development of therapeutic drugs.