An investigation of how manipulation of Mycobacterium tuberculosis lipid body content impacts early infection of macrophages

Background: Lipid bodies (LBs), intracellular accumulations of triacylglycerol (TAG) are prominent features of Mycobacterium tuberculosis (Mtb) in the sputum of tuberculosis patients. TAG LBs form with growth arrest and are assimilated on regrowth. TAG is also found in the cell envelope and changes may impact cell surface hydrophobicity (CSH). How TAG LB content, CSH, and growth state affect early interactions between Mtb and macrophages is unknown.

Methods: Mtb CDC1551 wild-type (WT) and transposon mutants with disrupted genes involved in TAG accumulation (tgs1::Tn) and assimilation (lipY::Tn) were investigated. Strains were assessed during culture and following treatment with nitric oxide/oleic acid (NO/OA) to promote LB accumulation. LB content (%LB), assessed by fluorescence microscopy, and CSH by hexadecane partitioning, were used to characterise the strains. These were then used to infect THP-1 macrophages with an assessment of uptake and early bacterial growth.

Results: Mtb CDC1551 WT responded to NO/OA by increasing tgs1 expression, %LB and CSH, with levels returning to baseline 24h post-induction. Tgs1::Tn showed reduced growth with a similar %LB content as WT, however, it failed to accumulate LB on NO/OA induction and had reduced CSH. Tgs1::Tn prepared from growing Sauton’s culture, showed greater intracellular growth than WT, however, following NO/OA induction, the growth of both strains was similar. LipY::Tn had an increased growth rate and %LB compared with WT in Sauton’s culture. LipY::Tn increased %LB content and CSH with NO/OA induction, but unlike WT, did not show a return to baseline levels 24h post-induction. LipY::Tn from growing culture, or 24h post-NO/OA exposure, had greater intracellular growth compared with the WT. Although transcriptional complementation of tgs1 and lipY was demonstrated, restoration to the WT phenotype was not observed.

Conclusions: Tgs1 and lipY mutation had a significant impact on growth, %LB and response to NO/OA. CSH and %LB did not relate to macrophage uptake and intracellular growth.