The growth phase-dependent regulation of the pilus locus genes by two-component system TCS08 in Streptococcus pneumoniae q Xin-Ming Song a, * , Wayne Connor a , Karsten Hokamp b , Lorne A. Babiuk c , Andrew A. Potter a a Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, S7N 5E3 Canada b Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland c University of Alberta, Edmonton, Alberta, T6G 2J9 Canada article info Article history: Received 31 July 2008 Received in revised form 2 October 2008 Accepted 7 October 2008 Available online 17 October 2008 Keywords: Streptococcus pneumoniae Two-component system Pilus Adhesion Invasion Epithelial cells abstract The two-component system TCS08 of Streptococcus pneumoniae contributes to the virulence in vivo and regulates phosphotransferase system (PTS) genes in an avirulent strain. However, its role in pathogenic strains and virulence mechanism are largely unknown. In this study, we constructed TCS08 knockout mutants in a serotype 4 encapsulated pathogenic strain TIGR4, and investigated target genes regulated by TCS08 through transcriptional profile analysis. Compared to TIGR4, expression of the rlrA islet genes (SP0461–SP0468) encoding pneumococcal pili was found to be up-regulated in the rr08 mutant (Drr08). Further quantitative real-time PCR (qRT-PCR) analysis revealed that such induction was more significant when the strains were grown to late-logarithmic phase. In phenotype analyses, disruption of both hk08 and rr08 genes (DTCS08) resulted in increased adherence to human lung epithelial cells (A549) at 3 h at late-logarithmic and stationary phases. However, the invasion level of DTCS08 was reduced at different growth phases. Similar phenotype changes, though less significant, were also observed when the assays were performed on human nasopharyngeal epithelial cells (Detroit 562). These data suggest that TCS08 is involved in adhesion and invasion of host epithelial cells, which is likely mediated via regulation of the pilus locus genes in a growth phase-dependent manner. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Despite the existence of antibiotics and vaccines, Streptococcus pneumoniae is still one of major infectious disease killers (together with HIV, malaria and tuberculosis) in the world, causing respira- tory tract diseases and systemic infections such as pneumonia, bronchitis, otitis media, bacteremia, sepsis and meningitis [1]. During the course of an infection, the S. pneumoniae bacterium encounters a number of different environmental niches such as the nasopharynx, lungs, blood, middle ear and potentially the brain. To persist and establish an infection at these various host niches, the pneumococcus needs to adapt and orchestrate its gene expression. Two-component systems (TCS) in bacteria play a central role in sensing environmental changes and regulating gene expressions accordingly. A typical TCS is composed of two different types of proteins: an hk encoded histidine kinase sensor protein (HK) located on the membrane, and an rr encoded response regulator protein (RR) located in the cytoplasm. Stimulation of HK results in activation of RR through phosphorylation, which subsequently controls expression of target genes via binding to the promoter regions of the regulated target genes. During infection, TCS modulates a variety of cellular responses in bacteria, such as osmoregulation, chemotaxis, sporulation, photosynthesis and pathogenicity [2]. In S. pneumoniae, a total of 13 TCSs and one orphan RR have been identified [3,4], and many of them are involved in virulence via regulating expression of different target genes [5]. For example, TCS02 and TCS06 regulate the expression of choline-binding proteins PspA [6] and CbpA [7,8], respectively. Both virulence factors are also regulated by TCS06 through different mechanisms [9]. TCS04 controls the expression of pneumococcal surface adhesin A (PsaA) [10], while TCS05 (CiaRH) is linked to HtrA, a virulence factor that contributes to nasopharyngeal colonization [11]. In animal experiments, up to 10 TCS systems have been claimed to be associated with virulence [4,5], indicating the importance of TCS in pathogenicity. Among these systems, TCS08 plays an essential role for pneumococcal growth and survival in vivo [4]. In an unencap- sulated avirulent strain, TCS08 was found to be involved in regu- lation of phosphotransferase system (PTS) genes [12]. However, the roles of TCS08 in encapsulated wild-type strains and its virulence mechanism are still largely unknown. In this study, we compared transcriptional profiles of an rr08 knockout mutant and the parent wild-type strain, and characterized q Published with permission of the Director of VIDO as journal series No. 508. * Corresponding author. Tel.: þ1 306 966 7483; fax: þ1 306 966 7478. E-mail address: xinming.song@usask.ca (X.-M. Song). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.10.006 Microbial Pathogenesis 46 (2009) 28–35