Keywords

Mycobacterium abscessus, hypoxia, nitrate assimilation, transcriptional regulation

Abstract

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting immunocompromised patients and individuals with underlying comordibities such as Cystic Fibrosis (CF). Treatment strategies are limited due to inherent antibiotic resistance and restricted accessibility of Mab to antibiotics within macrophage phagosomes, granuloma lesions, and the mucus laden CF airways. Transcriptional adaptation to stresses encountered in these niches such as hypoxia, reactive nitrogen intermediates (RNI) and elevated nitrate and nitrite are not well-understood. In Mycobacterium tuberculosis (Mtb) hypoxia adaptation and nitrate metabolism are linked via induction of the two-component system (TCS) DosRS and nitrate metabolism genes. DosRSMtb induces a 50-gene regulon necessary for hypoxia adaptation including the NarK2 transporter facilitating nitrate use as a terminal electron acceptor via a respiratory nitrate reductase. The role of the Mab DosRS ortholog in hypoxia adaptation and nitrate metabolism is not-well defined. To address this knowledge gap, we developed an in vitro hypoxia model to examine differential gene expression and phenotype of a Mab dosRS mutant. RNAseq revealed hypoxic induction of >1000 genes including 127 DosR-dependent genes many of which are species-specific. Absence of DosRSMab led to attenuated growth in hypoxia highlighting the necessity of DosRSMab for hypoxic adaptation. Nitrate metabolism genes including a putative nitrate/nitrite regulator (nnaR) were downregulated in hypoxia suggesting a novel interplay between hypoxia and nitrate metabolism in Mab. To assess the role of NnaR in the regulation of nitrogen metabolism, we constructed a Mab nnaR knockout mutant. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a nitrate transporter, a role distinct from other mycobacteria. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides a first look at species-specific transcriptional adaptations to hypoxia and nitrate metabolism driven by DosRS and NnaR in Mab.

Completion Date

2024

Semester

Spring

Committee Chair

Rohde, Kyle

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Format

application/pdf

Language

English

Rights

In copyright

Release Date

November 2024

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

Campus Location

Health Sciences Campus

Accessibility Status

Meets minimum standards for ETDs/HUTs

Additional Files
Table S1.xlsx (1145 kB)
Table S2.xlsx (20 kB)
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