Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria, which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival and passage by transovarial/transstadial transmission in tick vectors in contrast to the ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sRs) which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were found to be differentially upregulated (>2-fold) in AAE2 cells and HMECs, respectively. Furthermore, enrichment for primary transcripts by treatment with Terminator 5-Phosphate-dependent Exonuclease resulted in identification of 3903 and 2555 transcription start sites (TSS) including 214 and 188 primary TSS in R. conorii during the infection to tick and human host cells, respectively. Seventy five coding genes exhibited different TSSs depending on the host environment. Most strikingly, we observed differential expression of 6S RNA during host-pathogen and vector-pathogen interaction, in vitro, and implicate a role for this noncoding RNA in regulation of rickettsial transcriptome depending on host niche. In sum, our data authenticates the presence of novel Rc_sRs in R. conorii, reveals the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in regulation of coding transcriptome during tripartite host-pathogen-vector interaction.