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Abstract
Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.
Highlights
Mycobacterium tuberculosis (Mtb) is a major human pathogen and is the causative agent of tuberculosis (TB)
To determine if the uspA-uspB-uspC genes are co-transcribed, total RNA was extracted from either M. smegmatis or M. bovis BCG as a replacement for Mtb since comparison with the sequence of uspABC region in the Mtb genome identified that the intergenic regions in M. bovis BCG are identical to Mtb and show only two non-synonymous single nucleotide polymorphism (SNPs) changes in the uspA and uspC coding sequences
In order to confirm the in silico prediction and determine the transmembrane topology in intact mycobacterial cells, Mtb uspA, Mtb uspB and Mtb uspC N- and Cterminal fusions were generated to the truncated periplasmic β -lacta mase reporter BlaTEM-1 as a selectable extracellular reporter (Fig. 2) (McCann et al, 2007; Perkowski et al, 2017)
Summary
Mycobacterium tuberculosis (Mtb) is a major human pathogen and is the causative agent of tuberculosis (TB). Mtb is an extremely successful pathogen and TB is one of the leading causes of death world-wide from a bacterial pathogen (World Health Organisation, 2020). In 2019 alone, approximately 1.5 million people died from TB and over 10 million new TB cases were reported (World Health Organisation, 2020). TB is treatable and curable, effective treatment regimens have been threatened and rendered ineffective by the emergence of drug-resistant strains of Mtb. there is an urgent need to identify novel targets and pathways within Mtb to develop new therapeutic interventions to address this global health problem. Mtb is an intracellular pathogen with a remarkable ability to survive for decades in a nutrient restricted environment within the human host
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