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

BACKGROUND: 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 in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their 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. RESULTS: 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_sR's), 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 differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5'-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs 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. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. CONCLUSIONS: In sum, the findings of this study authenticate the presence of novel Rc_sR's in R. conorii, reveal 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 the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii.

Rickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro.

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.

Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium.

Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during R. conorii infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of R. rickettsii invasion into ECs. Consistent with these findings, R. rickettsii infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of ß-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.

Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions.

Natural pathogen transmission of Rickettsia prowazekii, the etiologic agent of epidemic typhus, to humans is associated with arthropods, including human body lice, ticks, and ectoparasites of eastern flying squirrel. Recently, we have documented the presence of small RNAs in Rickettsia species and expression of R. prowazekii sRNAs during infection of cultured human microvascular endothelial cells (HMECs), which represent the primary target cells during human infections. Bacterial noncoding transcripts are now well established as critical post-transcriptional regulators of virulence and adaptation mechanisms in varying host environments. Despite their importance, little is known about the expression profile and regulatory activities of R. prowazekii sRNAs (Rp_sRs) in different host cells encountered as part of the natural life-cycle. To investigate the sRNA expression profile of R. prowazekii during infection of arthropod host cells, we employed an approach combining in vitro infection, bioinformatics, RNA sequencing, and PCR-based quantitation. Global analysis of R. prowazekii transcriptome by strand-specific RNA sequencing enabled us to identify 67 cis-acting (antisense) and 26 trans-acting (intergenic) Rp_sRs expressed during the infection of Amblyomma americanum (AAE2) cells. Comparative evaluation of expression during R. prowazekii infection of HMECs and AAE2 cells by quantitative RT-PCR demonstrated significantly higher expression of four selected Rp_sRs in tick AAE2 cells. Examination of the coding transcriptome revealed differential up-regulation of >150 rickettsial genes in either HMECs or AAE2 cells and yielded evidence for host cell-dependent utilization of alternative transcription start sites by 18 rickettsial genes. Our results thus suggest noticeable differences in the expression of both Rp_sRs as well as the coding transcriptome and the exploitation of multiple transcription initiation sites for select genes during the infection of human endothelium and tick vector cells as the host and yield new insights into rickettsial virulence and transmission mechanisms.

Small Regulatory RNAs of Rickettsia conorii.

Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed 'Rc_sR's). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA.

Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii.

Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs (RNaseP_bact_a, α-tmRNA, 4.5S RNA, 6S RNA) was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5' RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. 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.

Bacterial small RNAs in the Genus Rickettsia.

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.