Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Anne Drumond Villela,Luiz Augusto Basso,Valnês S Rodrigues-Junior,Antônio Frederico Michel Pinto,Paula Eichler,Zilpa Adriana Sánchez-Quitian,Diógenes Santiago Santos,Virgínia Carla De Almeida Falcão,Cristiano Valim Bizarro

doi:10.1590/0074-02760170105

Abstract

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Highlights

Tuberculosis is an infectious disease caused mainly by Mycobacterium tuberculosis
We describe the construction of an M. tuberculosis knockout strain for cdd gene (KO), evaluation of mRNA expression of cdd, deoA and add genes, assessment of protein expression by Multidimentional Protein Identification Technology (MudPIT) LC-MS/ MS, in vitro growth studies, and analysis of cdd deletion in M. tuberculosis invasion and growth in a macrophage model of infection
Anne Drumond Villela et al Fig. 1: genomic environment of cdd gene in Mycobacterium tuberculosis (A), regions cloned into pPR27xylE vector (B), agarose gel electrophoresis of polymerase chain reaction (PCR) products from knockout clones (C), and mRNA expression of cdd, deoA, and add genes (D). (A) Genomic region of cdd gene (402 bp) containing unique internal NotI site and flanking genes; the operon cdd-add is indicated. (B) The cdd gene and flanking regions were amplified by PCR from M. tuberculosis H37Rv genomic DNA, and the cdd gene was disrupted by the insertion of a kanamycin cassette into NotI site

Summary

Introduction

Tuberculosis is an infectious disease caused mainly by Mycobacterium tuberculosis. The World Health Organization estimated that 10.4 million people developed tuberculosis in 2015, resulting in 1.8 million deaths (WHO 2016). We describe the construction of an M. tuberculosis knockout strain for cdd gene (KO), evaluation of mRNA expression of cdd, deoA and add genes, assessment of protein expression by MudPIT LC-MS/ MS, in vitro growth studies, and analysis of cdd deletion in M. tuberculosis invasion and growth in a macrophage model of infection.

Results

Conclusion