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Principal Investigator: Michelle Margaret Meyer
Organization: BOSTON COLLEGE
Fiscal Year: 2024
Award: $193,750
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
Streptococcus pneumoniae is a leading cause of pneumonia, acute otitis media (ear infection), and
meningitis in the United States. S. pneumoniae remains an important pathogen despite a vaccine and
effective antibiotic treatments due to increasing levels of antibiotic resistance and vaccine evasion
through serotype switching. Virulence in S. pneumoniae is mediated by a complex gene regulatory
program that is only partially understood. In many bacteria, trans-acting small non-coding RNAs
(sRNAs) play key roles in regulating stress and virulence responses. While many sRNAs have been
identified in S. pneumoniae via high-throughput sequencing studies, biological function has been
assigned to only a few. The goal of this exploratory proposal is to identify the mRNA targets for sRNA
candidates in S. pneumoniae, focusing specifically on those implicated in mediating virulence. We have
two aims to reach this goal: First, we will determine the mRNA targets for a set of prioritized sRNA
candidates implicated in S. pneumoniae virulence during mouse infections. Second, we will
characterize the entire S. pneumoniae sRNA:mRNA interaction network. To achieve the first aim, we
will use MS2-afinity purification coupled with RNA sequencing (MAPS) to identify mRNA targets for a
set of prioritized sRNAs associated with fitness defects in mouse infections. MAPS tags each sRNA
individually to allow specific capture of sRNA targets, but allows unbiased identification of potential
mRNA partners via high-throughput sequencing of the captured RNAs. This approach will be combined
with computational tools that predict the targets of sRNAs using thermodynamic properties. A set of 5-
10 of the most promising mRNA targets identified from the combined approaches will be assessed
using reporter gene assays to validate the sRNA impact on gene expression. To achieve the second
aim, we will use RIL-seq, a technique which captures mRNA:sRNA pairing through crosslinking
followed by ligation. Subsequent sequencing identifies chimeric reads corresponding two interacting
RNAs. To enrich for sRNA:mRNA pairs, a FLAG-tagged RNA-binding protein partner to
immunoprecipitated the sRNAs. In S. pneumoniae, which lacks an RNA chaperone, we will use Cbf1,
an exonuclease shown to process sRNAs, to enrich our population. Taken together, our two aims will
identify mRNA targets for sRNA candidates associated with virulence, and move the field from
cataloging sRNA presence toward identification of sRNA biological function, and thus therapeutic
intervention.
Terms: <0-11 years old><Acute><Affinity Chromatography><Antibiotic Resistance><Antibiotic Therapy><Antibiotic Treatment><Assay><Bacteremia><Bacteria><Bacterial Physiology><Bioassay><Biological Assay><Biological Function><Biological Process><Biology><Carbon><Cataloging><Chaperone><Child><Child Youth><Children (0-21)><Closure by Ligation><Competence><Complex><Computational toolkit><Coupled><D pneumoniae><D. pneumoniae><Data><Defect><Diplococcus pneumoniae><E coli><E. coli><Escherichia coli><Exhibits><Exonuclease><Functional RNA><Gene Expression><Genes><Goals><Gram-Negative Bacteria><Gram-Positive Bacteria><High-Throughput Nucleotide Sequencing><High-Throughput Sequencing><Immune Precipitation><Immunoprecipitation><Individual><Infection><Ligation><Mediating><Mediator><Meningitis><Messenger RNA><Mice><Mice Mammals><Modeling><Molecular Chaperones><Murine><Mus><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Nucleotides><Organism><Otitis Media><Outcome><Pathogenicity><Play><Pneumococcus><Pneumonia><Population><Prevalence><Process><Property><Proteins><RNA><RNA Gene Products><RNA Seq><RNA sequencing><RNA-Binding Proteins><RNAseq><Regulator Genes><Regulatory Pathway><Reporter><Reporter Genes><Research><Resistance to antibiotics><Resistant to antibiotics><Ribonucleic Acid><Role><S enterica><S pneumoniae><S. enterica><S. pneumoniae><Salmonella enterica><Serotyping><Small RNA><Streptococcus pneumoniae><Stress><Survey Instrument><Surveys><Techniques><Therapeutic Intervention><Thermodynamic><Thermodynamics><Tn-seq><Tnseq><Transcriptional Regulatory Elements><United States><Untranslated RNA><Vaccines><Validation><Virulence><Work><affinity purification><antibiotic drug resistance><antibiotic resistant><bacteraemia><bacterial disease treatment><bacterial infectious disease treatment><bacterial sepsis><biological adaptation to stress><computational toolbox><computational tools><computational toolset><computerized tools><crosslink><ear infection><experiment><experimental research><experimental study><experiments><fitness><intervention therapy><kids><living system><mRNA><middle ear infection><noncoding><pathogen><prediction algorithm><predictive tools><priority pathogen><programs><reaction; crisis><regulatory gene><response><social role><stress response><stress; reaction><trans acting element><transcriptome sequencing><transcriptomic sequencing><transposon insertion sequencing><transposon sequencing><validations><youngster>