Redefining the Small Regulatory RNA Transcriptome in Streptococcus pneumoniae Serotype 2 Strain D39

ABSTRACT Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by posttranscriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in the S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs has been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery’s virulent S. pneumoniae strain D39 using two independent RNA sequencing (RNA-seq)-based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program toward identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4, including ones previously implicated in virulence, are not present in the strain D39 genome, suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 66 new sRNA candidates in strain D39, 30 of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112. IMPORTANCE Recent work has shown that sRNAs play crucial roles in S. pneumoniae pathogenesis, as inactivation of nearly one-third of the putative sRNA genes identified in one study led to reduced fitness or virulence in a murine model. Yet our understanding of sRNA-mediated gene regulation in S. pneumoniae has been hindered by limited knowledge about these regulatory RNAs, including which sRNAs are synthesized by different S. pneumoniae strains. We sought to address this problem by developing a sensitive sRNA detection technique to identify sRNAs in S. pneumoniae D39. A comparison of our data set reported here to those of other RNA-seq studies for S. pneumoniae strain D39 and TIGR4 has provided new insights into the S. pneumoniae sRNA transcriptome.

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Molecular Evolution Perspectives on Intraspecific Lateral DNA Transfer of Topoisomerase and Gyrase Loci in Streptococcus pneumoniae, with Implications for Fluoroquinolone Resistance Development and Spread

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.10.4315-4326.2005 ◽

2005 ◽

Vol 49 (10) ◽

pp. 4315-4326 ◽

Cited By ~ 16

Author(s):

Michael J. Stanhope ◽

Stacey L. Walsh ◽

Julie A. Becker ◽

Michael J. Italia ◽

Karen A. Ingraham ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Transfer ◽

Lateral Transfer ◽

Respiratory Pathogen ◽

Fluoroquinolone Resistance ◽

Data Set ◽

History Of ◽

Important Means ◽

Viridans Group Streptococci

ABSTRACT Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.

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Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that thedltLocus Promotes Inflammation and Transmission

mBio ◽

10.1128/mbio.01032-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 7

Author(s):

M. Ammar Zafar ◽

Alexandria J. Hammond ◽

Shigeto Hamaguchi ◽

Weisheng Wu ◽

Masamitsu Kono ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Respiratory Tract ◽

Capsular Polysaccharide ◽

Inflammatory Responses ◽

Transposon Mutagenesis ◽

Respiratory Pathogen ◽

Invasive Infection ◽

Upper Respiratory Tract ◽

Content Type ◽

Upper Respiratory

ABSTRACTHost-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogenStreptococcus pneumoniaegenerated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism’s transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was thedltlocus, which addsd-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence ofd-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of thedltlocus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in alysM−/−host, there was no longer an effect of thedltlocus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCEStreptococcus pneumoniae(the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism’s capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiasedin vivotransposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by thedltlocus, previously shown to addd-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

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TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.02707-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 863-875 ◽

Cited By ~ 41

Author(s):

Alexandria A. Reinhart ◽

Daniel A. Powell ◽

Angela T. Nguyen ◽

Maura O'Neill ◽

Louise Djapgne ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Iron Homeostasis ◽

Opportunistic Pathogen ◽

Wild Type ◽

Content Type ◽

Genes Encoding ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Heme Regulation ◽

Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

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Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

10.1101/2021.08.13.456236 ◽

2021 ◽

Author(s):

Kumari Kavita ◽

Aixia Zhang ◽

Chin-Hsien Tai ◽

Nadim Majdalani ◽

Gisela Storz ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Binding ◽

Class Ii ◽

Rna Chaperone ◽

Terminal Domain ◽

C Terminus ◽

Bacterial Rna ◽

Small Regulatory Rnas ◽

Regulatory Rnas

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

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SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis

10.1101/021915 ◽

2015 ◽

Author(s):

Benjamin K Johnson ◽

Matthew B Scholz ◽

Tracy K Teal ◽

Robert B Abramovitch

Keyword(s):

Gene Expression ◽

Differential Gene Expression ◽

Quality Analysis ◽

Rna Seq ◽

Sequencing Data ◽

Data Set ◽

Bacterial Rna ◽

Analysis Workflow ◽

Differential Gene ◽

Reference Counting

Summary: SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. The workflow is implemented in Python for file management and sequential execution of each analysis step and is available for Mac OS X, Microsoft Windows, and Linux. To promote the use of SPARTA as a teaching platform, a web-based tutorial is available explaining how RNA-seq data are processed and analyzed by the software. Availability and Implementation: Tutorial and workflow can be found at sparta.readthedocs.org. Teaching materials are located at sparta-teaching.readthedocs.org. Source code can be downloaded at www.github.com/abramovitchMSU/, implemented in Python and supported on Mac OS X, Linux, and MS Windows. Contact: Robert B. Abramovitch ([email protected]) Supplemental Information: Supplementary data are available online

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Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space

mSystems ◽

10.1128/msystems.00216-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 6

Author(s):

Neil D. Ritchie ◽

Tom J. Evans

Keyword(s):

Streptococcus Pneumoniae ◽

Small Rnas ◽

Pneumococcal Infection ◽

Pleural Space ◽

Type I Interferons ◽

Type I ◽

Bacterial Cells ◽

Rna Seq ◽

Bacterial Killing ◽

Content Type

ABSTRACT Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease. IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.

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Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00026-16 ◽

2016 ◽

Vol 80 (4) ◽

pp. 1029-1057 ◽

Cited By ~ 19

Author(s):

Ruben A. T. Mars ◽

Pierre Nicolas ◽

Emma L. Denham ◽

Jan Maarten van Dijl

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Regulation Of Gene Expression ◽

Regulatory Rna ◽

Gram Positive ◽

Content Type ◽

Rna Molecules ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

The Stability

SUMMARYBacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules includetrans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5′ untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such asEscherichia coliandSalmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacteriumBacillus subtilis. A recent study identified 1,583 putative regulatory RNAs inB. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation inB. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation inB. subtilismostly involves elements at the 5′ ends of mRNA molecules. These can include 5′ secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs inB. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions inB. subtilis.

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The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES

Microbiology Spectrum ◽

10.1128/spectrum.01095-21 ◽

2021 ◽

Author(s):

Joanna Houghton ◽

Angela Rodgers ◽

Graham Rose ◽

Alexandre D’Halluin ◽

Terry Kipkorir ◽

...

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Differential Gene Expression ◽

Control Of Gene Expression ◽

Content Type ◽

Infection Models ◽

Small Regulatory Rnas ◽

Differential Gene ◽

Regulatory Rnas

Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis . Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA.

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ThePseudomonas aeruginosaPrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of theantRmRNA

Journal of Bacteriology ◽

10.1128/jb.00704-17 ◽

2018 ◽

Vol 200 (10) ◽

Cited By ~ 16

Author(s):

Louise Djapgne ◽

Subrata Panja ◽

Luke K. Brewer ◽

Jonathan H. Gans ◽

Maureen A. Kane ◽

...

Keyword(s):

Quorum Sensing ◽

Iron Homeostasis ◽

Molecular Mechanisms ◽

Biological Activities ◽

Content Type ◽

Downstream Effects ◽

Infection Models ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Translational Start

ABSTRACTPseudomonas aeruginosais an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions,P. aeruginosatranscribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation ofantR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation ofantR, demonstrating that the PrrF sRNAs interact with theantR5′ untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. TheP. aeruginosaHfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd[dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to theantRmRNA (0.3 μM), and PrrF was able to bind toantRmRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to theantRUTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, inP. aeruginosa.IMPORTANCEThe iron-responsive PrrF sRNAs play a central role in regulatingP. aeruginosairon homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and theantRmRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target inP. aeruginosa.

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