Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Sub-lethal Rifampicin effects on the Mycobacterium smegmatis cell wall and on survival in macrophage infection models (#435)

Jonathan Blackburn 1 , Alexander Giddey 1 , Nelson Soares 1
  1. University of Cape Town, Cape Town, WESTERN CAPE, South Africa

Background: Tuberculosis disease, caused by Mycobacterium tuberculosis, is one of the leading causes of mortality globally. Mycobacterial drug resistance is a growing concern with frontline drugs such as rifampicin in danger of becoming ineffective. We report the effects of sub-lethal rifampicin exposure on the cell wall proteome and lipidome of both susceptible (WT) and drug-resistant (SL) isogenic engineered strains of Mycobacterium smegmatis and validate a data derived hypothesis regards bacterial survival.

Methods: Both rifampicin susceptible and resistant Mycobacterium smegmatis strains were treated with sub-lethal doses of rifampicin. The lysate was enriched for the cell wall, cytosolic, and cellular debris fractions; peptides prepared via FASP and analysed on our Q-Exactive by LC-MS/MS. Proteins were identified and quantified via MaxQuant and statistically analysed in R for dysregulation and pathway enrichment via STRINGdb.

Results: We identified a total of 2632 proteins with 646 and 258 found to be dysregulated in susceptible and resistant strains respectively. GO term enrichment showed enrichment for 75 and 46 KEGG pathways respectively with ABC Transporters enriched in the top 10 terms, with majority of proteins decreased abundance, in both strains. Porphyrin and chlorophyll metabolism was among the top 25 terms in both with dysregulation of specific enzymes conserved across drug-sensitivity. Both strains showed evidence for downregulation of mammalian cell entry proteins known to be important in infection of some cell types and survival of mycobacteria in macrophages. Pre-treatment with sub-lethal concentrations of rifampicin showed reduced uptake and survival (24 hours) of rifampicin susceptible mycobacteria in a macrophage infection model.

Conclusions: Mycobacteria showed conserved responses to sub-lethal rifampicin treatment across drug-sensitivity suggesting a specific, non-stress-induced {adaptive} response to this important front-line drug. Mycobacterium smegmatis appears to respond to rifampicin treatment by reducing cell wall permeability and trans-cell wall transport with the consequence of impaired infectivity and survival in macrophages.