FUN_04

Functional evaluation of M. tuberculosis Rv2179c, the founding member of a new ribonuclease family.

 

THE PROJECT PROPOSAL IS PRESENTED HERE AND THE FINAL REPORT IS PRESENTED BELOW.

SSGCID Functional Project Proposal:  PI, Christoph Grundner Ph.D., Seattle BioMed. Co-investigators: Vic Arcus, Ph.D., University of Waikato, New Zealand, Jan Abendroth, Ph.D., Emerald Bio

 

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. Many RNAs require processing for function, and degradation of RNA together with new transcription determines the availability of transcript. In M. tuberculosis (Mtb), only few RNases have been identified to date.

In a recent chemical biology screen, we identified ATP binding proteins in the Mtb proteome. The activity-based ATP analogue used as a probe also recognized RNA binding proteins by virtue of their ability to bind ATP in the context of RNA. The hypothetical protein Rv2179c was among the hits with no similarity to known ATP binding proteins, suggesting that it may be a divergent RNA binding protein. The apo and co-crystal structures of Rv2179c solved by the SSGCID indeed identified Rv2179c as a DEDD family RNase and a close structural ortholog of RNase T. Although the sequence identity to known RNases is minimal (16% identical to E. coli), all catalytic residues are conserved and structurally superpose with those in other family members. The co-crystal structure of Rv2179c with AMP shows that AMP binds in the position of RNA in other RNAse-RNA structures. Thus, we identified a novel, highly divergent RNAse with many orthologs in other bacterial species, defining an entire new family of RNases in bacteria. Rv2179c is predicted to be essential, suggesting a central function in Mtb RNA homeostasis. To determine the catalytic function of Rv2179c and to understand its role in Mtb physiology, we propose the following functional studies:

Specific Aim 1: Defining the substrate specificity of Rv2179c.

Work to be performed in the lab of C.  Grundner and V. Arcus at Seattle Biomed and the University of Waikato, New Zealand and will be carried out during Q1-Q2. Work at Emerald Bio will take place in Q3-Q4.

In collaboration with Vic Arcus, we will define the sequence specificity of Rv2179s using an in vitro, mass spectrometric RNase assay. This assay uses recombinant RNase and a library of pentaprobes, RNAs encoding every combination of five bases. The specific cleavage products are then analyzed by MALDI-TOF mass spectrometry, giving a detailed specificity profile for endoribonucleases. To test for exoribonuclease activity, we will use primers with 3’ or 5’ overhangs to determine cleavage selectivity. These studies will reveal likely in vivo substrates and provide RNA sequences amenable to cocrystallization.

Specific Aim 2: Determining the cellular role of Rv2179c.

Work to be performed in the lab of C. Grundner at Seattle BioMed and will be carried out during Q1-Q4.

We will define the cellular function of Rv2179c by regulated protein degradation. This approach, only recently developed for Mtb, allows for depletion of essential Mtb gene products. First, we will use Rv2179c depletion to confirm its essentiality. Cell death will be measured by a colony forming units assay, testing Mtb survival in response to Rv2179c reduction. Also, reduced levels of Rv2179c are expected to lead to substrate accumulation, providing a tool to identify the physiologic substrates. In addition to reducing Rv2179c levels, we will overexpress Rv2179c using a tet-regulated overexpression system. We will identify the substrate(s) accumulating in the depletion mutant by RNASeq, identifying RNAs underrepresented in the overexpression mutant, and overrepresented in the knockdown strain. To
further understand Rv2179c’s cellular role, we will phenotype the loss of function and overexpression mutants. We will determine viability in rich broth culture, starvation, hypoxia, and in infected macrophages, conditions that lead to global gene expression changes in Mtb and that require extensive RNA turnover.

Specific Aim 3: Define the phylogenetic extend of the Rv2179c family of RNases.

Work to be performed in the lab of C. Grundner at Seattle BioMed and will be carried out during Q3-Q4.

Sequence analysis revels that Rv2179c has hundreds of orthologs in bacteria, suggesting that Rv2179c is the founding member of a large, novel RNase family. Orthologs are found in Actinobacteria such as Corynebacterium, Nocardia, and Rhodococcus, but more distant members are also found in phylogenetically distant bacteria, including gram-negative pathogens such as Pseudomonas and Burkholderia. To test if RNase function is conserved throughout the family, we will clone and express members from the most distant parts of the phylogenetic tree and test for RNase activity. Together, these studies will characterize a new family of bacterial RNases, dissect the cellular function of the essential Rv2179c in Mtb RNA homeostasis, and help annotate hundreds of hypothetical proteins from Actinobacteria to Protobacteria.

FINAL REPORT

Functional Study 4 (FUN_04): Functional evaluation of M. tuberculosis Rv2179c, the founding member of a new ribonuclease family.

Project lead: Christoph Grundner, Seattle BioMed

Project collaborators: Christopher Sassetti, U Mass, Massachusetts; Paul Agris, RNA Institute, University of New York, Albany, New York.

Status: Completed

Timeline:

ORIGINAL
  TIMELINE

ORIGINAL   MILESTONE

REVISED   MILESTONE

ACHIEVED

Quarter 1

09.2012-11.2012

  • Design synthetic   RNA substrates (~6)
  • Test substrates for   binding and cleavage by Rv2179c
  • Further characterize   Rv2179c by mutagenesis
  • Test substrates for   binding and cleavage by Rv2179c
  • Further   characterize Rv2179c by mutagenesis
  • Test for   essentiality
  • Publication

X

Quarter 2

12.2012-02.2013

  • Generate regulated   protein degradation mutant of Rv2179c in M. smegmatis   and Mtb.
  • Co-crystallization   of Rv2179c with substrate
  • Generate regulated   knockout (KO) mutant of Rv2179c in M. tuberculosis.

X

Quarter 3

03.2013-05.2013

  • Phenotype Rv2179c knock-down   mutants
  • Phenotype Rv2179c   knockdown mutants
  • Co-crystallization   of Rv2179c with substrate

(X)

Quarter 4

06.2013-08.2013

  • Generate Rv2179c   orthologue knock-down in P. putida   (and/or a related species)
  • Write manuscript
  • Generate Rv2179c   orthologue knock-down in P. putida   (and/or a related species)

 

Summary:  M. tuberculosis Rv2179c encodes a putative novel RNA-binding protein ATP-binding protein identified in a chemical biology screen performed by Dr. Grundner. The apo- and co-crystal structures of Rv2179c were solved by the SSGCID and confirmed Rv2179c as a DEDD family RNase with structural homology to RNase T.  Rv2179c was predicted to be essential, suggesting a central function in Mtb RNA homeostasis.  This FUN project planned to determine the catalytic function of Rv2179c and to understand its role in Mtb physiology.  During Year 7, we completed two of the three Specific Aims, as detailed below.  We were able to generate the Rv2179c knockout strain in Mtb, correcting the prediction that Rv2179c is essential for Mtb growth.  The project saw a major re-orientation in Quarter 4 in response to a publication (Structure, 2014, 22:719-30) that described the functional characterization of the M. marinum Rv2179c orthologue.  Although this study pre-empted our substrate identification efforts, it raised the exciting possibility that the Rv2179c protein also has an in vivo attenuation phenotype in Mtb.  To test this idea, we infected human macrophages with WT and Rv2179c knockout strains and assayed for bacterial growth in naïve and IFGγ-activated macrophages, which  is a good predictor for in vivo phenotypes of Mtb in the mouse model of infection.  However, we observed no growth attenuation the Mtb-human macrophage system, suggesting that the M. marinum phenotype is specific to that system, and that in Mtb Rv2179c, although likely processing mRNA and thus controlling mRNA turnover and stability, does not affect growth in vitro or in vivo.  Because of this lack of a clear phenotype, potential redundancy, and the finding of the Rv2179c substrate by another group, Dr. Grundner does not plan further work on this project.  A paper describing the structure of Rv2179c as a founding member of a large new RNase sub-family has been published in Journal of Biological Chemistry (2013, 289(4):2139-47)

Specific Aim 1: Defining the substrate specificity of Rv2179c.

Completed.  During Quarter 1, RNA substrate testing confirmed that Rv2179c has 3'-exoribonuclease activity.  Testing for activity on DNA substrates was also performed and none of the DNAs tested (3', 5' overhang, blunt end DNA) were found to be substrates.  Since publication of a study describing the M. marinum orthologue (MMAR_3223) of Rv2179c confirmed our annotation of Rv2179c as a 3’-5’ exoribonuclease and presented evidence that Rv2179c processes the polyA tail on mRNAs, we decided to not further pursue our own substrate identification efforts, since they would likely duplicate this existing work.

Specific Aim 2: Determining the cellular role of Rv2179c.

Completed.  In Quarter 1, Dr. Grundner and his collaborator Christopher Sassetti analyzed essentiality of Rv2179c using deep sequencing data from an Mtb transposon screen and showed it to be not essential.  This was a new milestone, not originally planned for the project.  The milestone for design of synthetic RNA substrates was moved to the 2nd quarter.  Dr. Grundner also generated a Rv2179c knockout strain  and confirmed the finding Rv2179c is not essential in Mtb.  Dr. Grunder’s group began phenotyping of the Rv2179c mutants during Quarter 3.  The Rv2179c knockout strain showed in vitro growth similar to the Mtb WT strain under normal, aerated culture conditions.  To test if Rv2179c was conditionally required, he assessed the survival of Rv2179 knockout strain in hypoxia; a growth-limiting stress Mtb encounters during infection.  Rv2179c’s survival was comparable to WT Mtb as determined by colony forming units assay; thus Rv2179c is dispensable for survival of hypoxic stress.  Since the M. marinum transposon mutant of MMR_3223 showed dramatically decreased virulence in the zebrafish model of infection, we decided to test whether the Mtb Rv2179 KO reduced survival in a human macrophage infection model.  We observed similar macrophage viability between WT and Rv2179 KO Mtb in naïve and IFN-treated human macrophages.  Since the human macrophage is a good predictor of in vivo phenotypes, these data make it unlikely that the Mtb Rv2179c has an effect on in vivo survival of Mtb.   

The Mtb Rv2179c knockout has no effect on Mtb survival in human macrophages.  Infection of human macrophages with Rv2179c knockout strain in naïve (top) and IFNγ-activated macrophages shows only small differences in bacterial loads.

Specific Aim 3:  Define the phylogenetic extent of the Rv2179c family of RNases.

Not completed.  Sequence analysis reveals that Rv2179c has hundreds of orthologs in bacteria, suggesting that Rv2179c is the founding member of a large, novel RNase family.  Unfortunately, time and resources did not allow Dr. Grundner to generate knock-down mutants of the Rv2179c orthologue in Pseudomas putida and/or a related species.

 

 

 

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