Rickettsia prowazekii and its ‘Junk DNA’: The Identification and Characterization of Small RNAs

As the etiologic agents of Rocky Mountain spotted fever and epidemic typhus, Rickettsia are obligate intracellular Gram-negative pathogenic bacteria. Genomic sequencing determined that ~24% of the Rickettsia prowazekii genome was noncoding DNA due to genomic reduction. Regions of bacterial noncoding DNA were found to encode important post-transcriptional regulators of bacterial virulence and growth called bacterial small RNAs (sRNAs). These sRNAs are classified as either trans-acting, whose biogenesis is predominantly linked to intergenic regions, or cis-acting, encoded on the antisense strand of an open reading frame (ORF). Despite being present in a majority of other bacteria, the existence and function of sRNAs are unknown in Rickettsia. In order to explore the possibility of rickettsial sRNAs, a combination of bioinformatics and in vitro techniques was employed. The first objective identified over 1,700 sRNAs using the SIPHT analysis tool to analyze 16 different strains representing 13 rickettsial species. Strong σ70 promoters and Rho-independent terminators were detected in their respective positions for all candidate sRNAs predicted in R. prowazekii, R. typhi, R. conorii, and R. rickettsii. Next generation sequencing (NGS) performed at 3h and 24h on R. prowazekii-infected human microvascular endothelial cells (HMECs) validated the expression of 26 sRNA candidates. Six selected candidates were further confirmed using RT-PCR. The second objective analyzed the NGS data and found the expression of an additional 35 trans- and 23 cis-acting novel sRNAs in addition to well-conserved kingdom-wide sRNAs (6.5S, 4.5S, RNaseP_bact_a, α-tmRNA). Three novel sRNAs and the conserved sRNAs were confirmed using either Northern blot analysis or RT-PCR. Transcriptional start sites were determined using RLM-RACE and secondary structure predicted for five novel sRNAs and 6S RNA. The third objective examined comparison of sRNA expression between HMECs and AAE2 (arthropod host cells), and found that using qRT-PCR, four sRNAs that showed significantly higher levels of expression at 24h. Also, a closer examination of Rp_sR60 found an interaction with H375_0420, in which both sRNA and target were found to be significantly decreased at 3h and 24h in HMECs. During AAE2 infection, both of these targets were found to be consistently expressed at 0.5h, 3h, and 24h. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis.