Phospholipid composition of purified Chlamydia trachomatis mimics that of the eucaryotic host cell

Abstract

Chlamydia trachomatis is an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells. Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite. In this report, we examine the phospholipid composition of C. trachomatis purified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered. A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect on C. trachomatis growth. However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine. Brefeldin A, fumonisin B1, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin. None of the agents had a significant effect on C. trachomatis replication. Treatment with fumonisin B1 and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin. This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content. Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae. Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or to C. trachomatis. Thus, the final phospholipid composition of purified C. trachomatis mimics that of the host cell in which it is grown.

FIG. 1

Incorporation of [U-¹⁴C]isoleucine into glycerophospholipids of C. trachomatis-infected wild-type CHO K1 (A) and thermolabile CDP-choline synthetase CHO strain 58 (B) cells at 33°C (open bars) and 40°C (hatched bars). The host cells were held at their respective temperatures for 60 h prior to infection. The temperature was maintained after infection. Infected cultures were radiolabelled at 20 h p.i. and harvested 5 h later. The results for each phospholipid are expressed as percentages of the total radioactivity incorporated into phospholipids. CL, cardiolipin. Results are expressed as means ± standard deviations of results from three separate experiments.

FIG. 2

Phospholipid composition of mock-infected CHO strain 58 cells (A), C. trachomatis-infected CHO strain 58 cells at 40 h p.i. (B), and highly purified EBs prepared from infected CHO strain 58 cells at 40 h p.i. (C) grown at 33°C (open bars) or 40°C (hatched bars). Phospholipids were quantitated by measuring the phosphorous associated with a given phospholipid and are expressed as percentages of the total phospholipid phosphorous. Results are averages of results from two separate experiments; duplicate results varied by less than 10%. CL, cardiolipin.

FIG. 3

Incorporation of [¹⁴C]isoleucine into glycerophospholipids of C. trachomatis-infected mouse L cells in the absence (open bars) and presence (hatched bars) of 20 μM fumonisin B₁. Fumonisin B₁ was added at the start of the infection (2 h p.i.), and radiolabelled isoleucine was added at 20 h p.i. The cells were harvested 5 h later. Results are expressed as means ± standard deviations of results from three separate experiments. CL, cardiolipin.

FIG. 4

Incorporation of [¹⁴C]serine into phospholipids of mock-infected mouse L cells (A) and C. trachomatis-infected mouse L cells (B) in the absence (open bars) and presence (hatched bars) of 20 μM fumonisin B₁. Fumonisin B₁ was added at the start of the infection (2 h p.i.), and radiolabelled serine was added at 20 h p.i. The cells were harvested 5 h later. Results are expressed as means ± standard deviations of results from three separate experiments.

FIG. 5

SM composition of mock-infected mouse L cells (MI), C. trachomatis-infected mouse L cells at 40 h p.i. (L2), and highly purified EBs prepared from infected mouse L cells at 40 h p.i. (EB) grown in the absence (open bars) or presence (hatched bars) of 20 μM fumonisin B₁ (A), 5 μg of brefeldin A ml⁻¹ (B), or 0.05 U of sphingomyelinase ml⁻¹ (C). Phospholipids were quantitated by measuring the phosphorous associated with a given phospholipid. Results for SM are expressed as percentages of the total phospholipid phosphorous. Results are averages of results from two separate experiments; duplicate results varied by less than 10%.

FIG. 6

Incorporation of [¹⁴C]serine into phospholipids of mock-infected mouse L cells (A) and C. trachomatis-infected mouse L cells (B) in the absence (open bars) and presence (hatched bars) of 5 μg of brefeldin A ml⁻¹. Brefeldin A was added at the start of the infection (2 h p.i.), and radiolabelled serine was added at 20 h p.i. The cells were harvested 5 h later. Results are expressed as means ± standard deviations of results from three separate experiments.

FIG. 7

Incorporation of [¹⁴C]serine into phospholipids of mock-infected mouse L cells (A) and C. trachomatis-infected mouse L cells (B) in the absence (open bars) and presence (hatched bars) of 0.05 U of sphingomyelinase ml⁻¹. Sphingomyelinase was added at the start of the infection (2 h p.i.), and radiolabelled serine was added at 20 h p.i. The cells were harvested 5 h later. Results are expressed as means ± standard deviations of results from three separate experiments.

FIG. 8

(A) Incorporation of [³H]choline into SM of mock-infected mouse L cells (MI) and C. trachomatis L2-infected mouse L cells (L2) grown in the absence (open bars) and presence (hatched bars) of 5 μg of brefeldin A ml⁻¹. [³H]SM synthesis was 18,347 dpm mg⁻¹ in mock-infected control cells and 16,926 dpm mg⁻¹ in L2-infected cells. (B) Transport of natural [³H]choline-labeled SM to the surfaces of mock-infected mouse L cells and C. trachomatis L2-infected mouse L cells grown in the absence (open bars) and presence (hatched bars) of 5 μg of brefeldin A ml⁻¹. Hydrolyzable cell surface SM was quantitated with an exogenous sphingomyelinase treatment as described in Materials and Methods. Results are averages of results from two separate experiments; duplicate results varied by less than 10%.

FIG. 9

Summary of the effects of brefeldin A, fumonisin B₁, and sphingomyelinase (SMase) treatment on SM content of mock-infected mouse L cells (open bars), C. trachomatis-infected mouse L cells at 40 h p.i. (upward hatched bars), and highly purified EBs prepared from infected mouse L cells at 40 h p.i. (downward hatched bars).