Heterogeneous fitness landscape cues, pknG high expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth - 02/02/22

Doi : 10.1016/j.tube.2021.102156

Édgar Rodríguez–Beltrán ^a,^{^{1
Master of Science – Microbiology program, Instituto de Biotecnología, The National University of Colombia, AV CR 30 45–03, Bogotá D.C. 111321, Colombia.},}^{^{2
Present address: Studies in Translational Microbiology and Emerging Diseases Research Group (MICROS), School of Medicine and Health Sciences, Universidad del Rosario, CR 24 63C-69, Bogotá D.C. 111221, Colombia.}}, Gerson-Dirceu López ^b, Juan Manuel Anzola ^c,^d, Juan Germán Rodríguez–Castillo ^a, Chiara Carazzone ^b, Martha I. Murcia ^a,^⁎
^a MicobacUN Group, Microbiology Department, The National University of Colombia (NUC) School of Medicine, AV CR 30 45-03, Bogotá, D.C, 111321, Colombia
^b Laboratory of Advanced Analytical Techniques in Natural Products (LATNAP), Chemistry Department, Universidad de los Andes, CR 1 18A-12, Bogotá, D.C, 111711, Colombia
^c Corpogen, CR 4 20-41, Bogotá, D.C, 110311, Colombia
^d Universidad Central, CR 5 21-38, Bogotá, D.C, 110311, Colombia

^∗Corresponding author. Facultad de Medicina, Departamento de Microbiología, Universidad de Nacional de Colombia, AV CR 30 45-03, Edificio 471, Oficina 312, Bogotá, D.C, 111321, Colombia.Facultad de MedicinaDepartamento de MicrobiologíaUniversidad de Nacional de ColombiaAV CR 30 45-03Edificio 471Oficina 312BogotáD.C111321Colombia

Abstract

Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 μg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

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Keywords : Tuberculosis, Rifampicin, rpoB, Fitness, Virulence, PDIM

Plan

Introduction

Materials and methods

Strains and cultures

Genotyping by GenoType MTBDRplus 2.0 and sequencing

Fitness assays

qRT-PCR for virulence-resistance genes

Lipid extraction and HPLC-MS/MS

Results

GenoType MTBDRplus 2.0 and sequencing

Fitness assays

qRT-PCR for virulence-resistance genes

Lipid extraction and HPLC-MS/MS

Discussion

In Middlebrook 7H10/OADC, selected ATCC25618 rpoB-S450L mutants have intermediate fitness and low prevalence, whereas mutations in H445 have clues of fitness heterogeneity and the greatest fitness

ATCC 25618 rpoB-S450L has diminished noncompetitive and competitive fitness against rpoB-wt in rich medium, but this hides some clues of heterogeneous fitness landscape with mixed instances of high fitness and low fitness when compared with rpoB-wt

rpoB S450L shows significant pknG 3.8 × overexpression vs rpoB-wt, and rpoB wt has increased polyketide synthase ppsA expression vs rpoB-S450L

rpoB S450L shows diminished PDIM production compared with rpoB-wt in concordance with diminished ppsA

Conclusions

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Vol 132

Article 102156- janvier 2022 Retour au numéro

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Heterogeneous fitness landscape cues, pknG high expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth - 02/02/22

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