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.2015 Dec 18:16:1075.
doi: 10.1186/s12864-015-2293-7.

Bacterial small RNAs in the Genus Rickettsia

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Bacterial small RNAs in the Genus Rickettsia

Casey L C Schroeder et al. BMC Genomics..

Abstract

Background: Rickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either "junk DNA" or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche.

Results: We employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii.

Conclusions: Our results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms.

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Figures

Fig. 1
Fig. 1
sRNA promoter frequencies. Conservation diagrams illustrating the probability of a nucleotide in a specific promoter motif position. The left side demonstrates the −10 promoter motif, while the right side is the −35 promoter motif. The upper portion displays the typhus group, while the lower displays the spotted fever group. Both groups have −10 motifs similar to theE. coli consensus sequence (TATAAT). On the other hand, the −35 motifs vary when compared to theE. coli consensus sequence (TTGACA)
Fig. 2
Fig. 2
Alignment ofR. rickettsii strain Sheila Smith sRNA candidate #71. The sRNA candidate #71, predicted only in R.rickettsii strain Sheila Smith but not in strain Iowa and the upstream 150 bp region of predicted sRNA were aligned with the corresponding genomic region from strain Iowa. The predicted −10 box (orange), −35 box (blue), and sRNA sequence (green) are highlighted. A 20bp deletion observed in the genomic sequence of strain Iowa is shown by the dotted line
Fig. 3
Fig. 3
Alignment ofR. rickettsii strain Iowa sRNA candidate #118. The sRNA candidate #118, predicted only in R.rickettsii strain Iowa but not in strain Sheila Smith and the 150 bp up- and downstream regions of predicted sRNA were aligned with the corresponding genomic region from strain Sheila Smith. The predicted −10 box (orange), −35 box (blue), sRNA sequence (green) and the Rho independent terminator (yellow) are highlighted. A nucleotide sequence absent in the genomic sequence of strain Sheila Smith and mapping to the predicted sRNA and the Rho independent terminator in strain Iowa is shown by the dotted line
Fig. 4
Fig. 4
6S RNA (ssrS) expression during host cell infection.R. prowazekii strain Brienl 6S RNA (ssrS) expression was measured during the infection of HMECs over a course of 72 h (n = 5). The expression was normalized to 16S rRNA (endogenous control) and baselined to 1.5h post infection. Significant increase was observed starting at 6h post infection. Data is represented as Mean ± SEM. **p < 0.01
Fig. 5
Fig. 5
Genomic location ofR. prowazekii strain Brienl sRNAs. Schematic representation of sRNAs identified to be expressed inR. prowazekii strain Brienl during the infection of HMECs. Green arrows represent the orientation of flanking ORFs in relation to the sRNA depicted by blue arrows. The nucleotide distance between the sRNA and the flanking ORF is shown above the brace
Fig. 6
Fig. 6
Expression ofR. prowazekii strain Brienl candidate sRNAs during host cell infection. TheR. prowazekii strain Brienl sRNA candidates #1,#5, #9, #10, #24, and #25 were tested for their expression during infection of HMEC by RT-PCR (n = 3). The band sizes shown on the left side correspond to the 100 bp DNA ladder (New England Biolabs). The lane 2 (-RT) is a “no reverse transcriptase” control, while the lane 3 (Crtl) is an uninfected HMEC control. Lanes 4 through 10 are the samples fromR. prowazekii strain Brienl infected HMECs from 1.5h to 72h post infection. All the tested sRNA candidates showed expression during host cell infection
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