Non-coding RNAs of the Q fever agent, Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterial pathogen that undergoes a biphasic developmental cycle, alternating between a small cell variant (SCV) and a large cell variant (LCV). Despite the remarkable niche and life cycle of C. burnetii, little is known about its modes of regulation and the roles that non-coding RNAs play in its growth and development. One such element is the intervening sequence (IVS); a 444-nt RNA element that is inserted within helix 45 of CoxiellaÆs precursor 23S rRNA. C. burnetii may have acquired IVS through horizontal transfer, and it has been subsequently maintained by all strains of C. burnetii through vertical transfer. The IVS of C. burnetii contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). However, our data show that the S23p-encoding ORF is probably undergoing reductive evolution and therefore not expressed in vivo. Additionally, we observed that following RNase III-mediated excision, IVS RNA is degraded and levels of the resulting fragments of 23S rRNA differ significantly from each other and the 16S rRNA. Since the fragment of 23S rRNA that is lowest in quantity may dictate the number of mature ribosomes that are ultimately formed, we hypothesize that the biological role of IVS is to moderate CoxiellaÆs growth by fragmentation of its 23S rRNA thereby fostering CoxiellaÆs tendency towards slow growth and chronic infection. Further, we identified fifteen novel Coxiella burnetii sRNAs (CbSRs) using RNA-seq, which were verified using Northern analyses. Additionally, some of these CbSRs were upregulated in LCVs or during intracellular growth, suggesting adaptive roles in those contexts. Furthermore, we also identified and characterized the 6S RNA of C. burnetii and found that it accumulated during the SCV phase of the bacterium. The location of ssrS gene and the secondary structure of 6S RNA were similar to those of other eubacteria, indicating functionality. We also demonstrated that the 6S RNA of C. burnetii interacts specifically with RNA polymerase (RNAP). Finally, 6S RNA was highly expressed during intracellular growth of C. burnetii indicating that it probably regulates stress response by interacting with RNAP during transcription

Coxiella Burnetii's Infection-Specific Small RNA 12 (CBSR12) Targets Cara and Metk Transcripts

Coxiella burnetii is an obligate intracellular bacterial pathogen and the etiological agent of Q fever. Previous transcriptome analysis of C. burnetii by our group revealed several novel small RNAs (sRNAs) of varying sizes and expression patterns. Sequence alignments of these sRNA’s across all strains of C. burnetii show strong conservation, indicating a functional role for these RNA’s in C. burnetii’s intracellular lifestyle. Furthermore, during C. burnetii’s biphasic life cycle of metabolically active (LCV) and inactive (SCV) states, several of the sRNA’s have shown differential expression in SCV and LCV cells via RNA-Seq and Northern blot analyses. One such sRNA, termed CbsR12, showed a marked upregulation in infected Vero host cells when compared to bacteria grown in axenic media. Additionally, RNA-Seq data and qRT-PCR analyses show a marked upregulation of CbsR12 in LCV cells compared to SCV cells. Here, we show that C. burnetii RNase III cleaves CbsR12 into two fragments, an observation supported by both in silico and 5’ RACE analyses. in silico sRNA target prediction programs were used to determine possible mRNA targets of Cbsr12. We subsequently determined through in vitro electrophoretic mobility shift assays (EMSAs) and in vivo luciferase reporter assays, that CbsR12 binds carA transcripts, which codes for carbamoyl-phosphate synthase subunit A, and metK transcripts, which codes for S-adenosyl methionine synthetase. These genes code for essential enzymes involved in pyrimidine biosynthesis and the methionine cycle, respectively

Nivel de adherencia al tratamiento y calidad de vida relacionada a la salud en pacientes con VIH/sida, de un hospital MINSA nivel II-1 de Ferreñafe – Lambayeque, durante agosto – diciembre, 2017

Se ha observado que pacientes infectados por el virus de inmunodeficiencia humana y síndrome de inmunodeficiencia adquirida (VIH/SIDA) que no toman en cuenta las indicaciones del médico para su tratamiento, pueden generar una propagación constante y continua, dando paso a un problema de salud más grave a nivel mundial. Por ello se realizó una investigación no experimental - descriptiva, con el propósito de determinar los niveles de adherencia al tratamiento y calidad de vida relacionada a la salud según sexo y grado de instrucción, además de describir las dimensiones sociodemográficas en pacientes con VIH/SIDA, de un Hospital MINSA nivel II-1 de Ferreñafe - Lambayeque, durante agosto a diciembre del 2017. Se utilizó los cuestionarios CEAT-VIH para hallar los niveles de adherencia al tratamiento y MOS-SF 30 para determinar los niveles de calidad de vida relacionada a la salud, estimando su validez y confiabilidad en una población similar, siendo válidos y fiables. Además, en cuanto la variable sexo, se obtuvo que los varones tienen una adecuada adherencia al tratamiento, sin embargo para calidad de vida relacionada a la salud ambos sexos se encontraron en un nivel bajo. Por ende la población estudiada presenta baja adherencia al tratamiento y calidad de vida relacionada a la salud. Con respecto a los aspectos sociodemográficos, el 53% es de sexo masculino, el 62% es soltera, siendo la vía sexual (96%) la principal forma de transmisión del virus del VIH. Además el 75% lleva un tiempo mayor a 12 meses de tratamiento en el servicio de TARGA.Tesi

Identification of Novel Small RNAs and Characterization of the 6S RNA of \u3ci\u3eCoxiella burnetii\u3c/i\u3e

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated ($2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 59 UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription

The Intervening Sequence of \u3ci\u3eCoxiella burnetii\u3c/i\u3e: Characterization and Evolution

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii\u27s logarithmic growth phase (1–5 d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii\u27s divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness

The intervening sequence of Coxiella burnetii: characterization and evolution

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii’s logarithmic growth phase (1-5d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii’s divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness

RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms

The Transcriptional landscape of Streptococcus pneumoniae TIGR4 reveals a complex operon architecture and abundant riboregulation critical for growth and virulence.

Efficient and highly organized regulation of transcription is fundamental to an organism's ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5'-untranslated regions (5'-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5'-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance