HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria

Jie Yin Grace Ngan; Swathi Pasunooti; Wilford Tse; Wei Meng; So Fong Cam Ngan; Huan Jia; Jian Qing Lin; Sze Wai Ng; Muhammad Taufiq Jaafa; Su Lei Sharol Cho; Jieling Lim; Hui Qi Vanessa Koh; Noradibah Abdul Ghani; Kevin Pethe; Siu Kwan Sze; Julien Lescar; Sylvie Alonso

doi:10.1073/pnas.2006717118

HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria

Proc Natl Acad Sci U S A. 2021 Mar 23;118(12):e2006717118. doi: 10.1073/pnas.2006717118.

Authors

Jie Yin Grace Ngan^{1

2}, Swathi Pasunooti³, Wilford Tse³, Wei Meng³, So Fong Cam Ngan³, Huan Jia³, Jian Qing Lin³, Sze Wai Ng^{1

2}, Muhammad Taufiq Jaafa^{1

2}, Su Lei Sharol Cho^{1

2}, Jieling Lim^{1

2}, Hui Qi Vanessa Koh^{1

2}, Noradibah Abdul Ghani^{1

2}, Kevin Pethe^{3

4}, Siu Kwan Sze³, Julien Lescar³, Sylvie Alonso^{5

2}

Affiliations

¹ Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 117456 Singapore.
² Life Sciences Institute, Immunology Programme, National University of Singapore, 117456 Singapore.
³ School of Biological Sciences, Nanyang Technological University, 637551 Singapore.
⁴ Lee Kong Chian School of Medicine, Nanyang Technological University, 636921 Singapore.
⁵ Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 117456 Singapore; [email protected].

Abstract

GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.

Keywords: HflX; hypoxia; mycobacteria; ribosome-splitting factor; tuberculosis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
DNA Replication*
Escherichia coli Proteins / genetics
Escherichia coli Proteins / metabolism
GTP Phosphohydrolases / genetics
GTP Phosphohydrolases / metabolism*
GTP-Binding Proteins / genetics
GTP-Binding Proteins / metabolism
Hypoxia / genetics*
Hypoxia / metabolism*
Mice
Mutation
Mycobacterium / genetics*
Mycobacterium / metabolism*
Mycobacterium bovis / genetics
Mycobacterium bovis / metabolism
Ribosomes / metabolism

Substances

Escherichia coli Proteins
GTP Phosphohydrolases
GTP-Binding Proteins
HflX protein, E coli