Evolution, purification, and characterization of RC0497: A peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

Jignesh G. Patel, Hema P. Narra, Krishna Mohan Sepuru, Abha Sahni, Sandhya Golla, Aishwarya Sahni, Amber Singh, Casey L.C. Schroeder, Imran H. Chowdhury, Vsevolod Popov, Sanjeev Sahni

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Abstract

Rickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized R. conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. Homology based search of rickettsial genomes reveals that RC0497 homologs, containing amidase-2 family and peptidoglycan binding domains, are highly conserved among spotted fever group rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

Original languageEnglish (US)
JournalBiological Chemistry
DOIs
StatePublished - Jan 1 2019

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Keywords

  • endothelial cells
  • N-acetylmuramoyl-L-alanine amidase
  • peptidoglycan
  • RC0497
  • Rickettsia
  • spotted fever group

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Clinical Biochemistry

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