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Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis.

ABSTRACT: Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.

SUBMITTER: Marra A

PROVIDER: S-EPMC127766 | biostudies-literature | 2002 Mar

REPOSITORIES: biostudies-literature

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Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis.

Marra Andrea A Asundi Jyoti J Bartilson Magdalena M Lawson Stacey S Fang Flora F Christine Jillian J Wiesner Cedric C Brigham Daniel D Schneider William P WP Hromockyj Alexander E AE

Infection and immunity 20020301 3

Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen on ...[more]

PMID: 11854229

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Project description:RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis, as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that the abundances of nearly 200 mRNA transcripts were significantly increased, whereas those of several pneumococcal small regulatory RNAs (sRNAs), including the Ccn (CiaR-controlled noncoding RNA) sRNAs, were altered in the Δrny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Δpnp mutants showed no growth defect in vitro but differentially expressed a total of 40 transcripts, including the tryptophan biosynthesis operon genes and numerous 5' cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Together, our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase mediates virulence phenotypes, likely through sRNA regulation. IMPORTANCE Streptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. RNases are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes, including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled noncoding (Ccn) sRNAs.

Project description:Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of Lactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a spxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803-813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.

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Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis.

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Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis.

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