Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

Abstract

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

Chlamydia trachomatisregulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets

“…Inclusions were imaged by confocal microscopy. Similar to our previous findings (Recuero‐Checa et al, ), Figure S1a shows that LDs are visible in WT and SKO cells. Although we did not observe LDs in SKO cells treated with T863, we did notice that there were some LDs in DKO cells.…”

“…Although others have claimed that LDs are localised within the inclusion and treatment of Ct infected cells with triacsin C, which pharmacologically inhibits LD formation, impairs Ct growth (Cocchiaro et al, ), we found that the effect was due to inhibition of the ACSL family of proteins by triacsin C with no evidence of LDs localising within the inclusion (Recuero‐Checa et al, ). Interestingly, we found that treatment with OA resulted in an increase in Ct inclusion formation even in triacsin C treated cells where LD formation remained inhibited.…”

“…WT, SKO, and DKO MEF cells were a gift from Dr. Robert V. Farese (Harvard Medical School, Boston, MA). HeLa 229 cells were grown at 37 °C, 5% CO 2 , in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% heat‐inactivated fetal bovine serum (Hyclone, GE Healthcare, Logan, UT) and supplemented with gentamicin (Gibco, Carlsbad, CA) and vancomycin (Sigma Aldrich, St. Louis, MO) as previously described (Recuero‐Checa et al, ; Somboonna et al, ).…”

“…LD formation was induced with 100 μM OA (Sigma‐Aldrich) as described (Recuero‐Checa et al, ). Twenty micromolar of T863 (Sigma‐Aldrich) was used to abrogate LD formation in SKO cells as described (Cao et al, ).…”

Exogenous fatty acid metabolism in bacteria

“…Additional reports propose that endogenously synthesize fatty acids are exported out of the C. trachomatis cell and into the chlamydial inclusion, converted to acyl-CoA by the host acyl-CoA synthetase, and used to acylate the deacylated host phospholipids in the inclusion [57,58]. These models for phospholipid synthesis are unprecedented in literature and are not consistent with other experimental results.…”