Temporal changes in lipid composition during the chlamydial infection cycle

Analysis of lipids at a single time point in the developmental cycle of C. trachomatis (24 h p.i.) can not sufficiently reveal temporal variations of the lipid composition and does not cover changes occuring at later stages. Therefore, time course experiments were performed to cover the complete chlamydial developmental cycle. Lipid extracts from four different time points of infection and two uninfected samples were analyzed (Figure 3-32).

Figure 3-32. Workflow scheme of time course lipid analysis. Lipids were extracted from uninfected and infected HeLa cells (C. trachomatis L2, MOI 2.5) according to Folch et al., 1957 and subsequently analyzed by MALDI-TOF mass spectrometry.

Cells were infected for 12 h, 24 h, 36 h, and 44 h to cover the complete chlamydial developmental cycle. Lipids from uninfected cells (start) and uninfected cells (end) reflect the lipid composition at the beginning and at the end of the experiment, respectively.

Cells were infected at different time points; lipids were extracted at the same time point (Figure 3-32). To differentiate between cell type specific and cell type independent effects on the lipid composition, two different cell lines, HeLa cells and HEp-2 cells, were used for the time course

Results and discussion: part II

experiments (Figure 3-33 A). Extracted lipids amounts were normalized based on cell numbers, which were microscopically determined prior to MALDI-TOF mass spectrometry measurement (Figure 3-33 B).

Figure 3-33. Cell numbers during infection time course. (A) Microscopy pictures of cells infected with C. trachomatis. HeLa cells and HEp-2 cells were infected for 12 h, 24 h, 36 h, and 44 h. White arrows mark inclusions. (B) Quantification of cell numbers from Figure 3-33 A. Cell numbers were normalized to the 24 h time point. Results are from two independent biological experiments.

Negative ion mode MALDI-TOF measurement of all time points revealed major changes in PI and CL lipid composition during the chlamydial developmental cycle in HeLa cells. Focussing on lipids with lower masses (m/z < 900), PI species with comparably long fatty acid residues (PI (18:0/20:4), PI (18:0/20:3)) decreased during the progression of the cycle, while PI species with shorter fatty acid residues (PI (16:0/16:1), PI (16:0/18:2), PI (16:0/18:1), PI (18:0/18:2), PI (18:0/186:1)) increased over time (Figure 3-34 A). These changes are also reflected in the quantification of Figure 3-34 A (Figure 3-34 B).

Results and discussion: part II

Figure 3-34. Lipid profile of infection time course in HeLa cells (intermediate masses). (A) Negative ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HeLa cells. HeLa cells were infected for 12 h, 24 h, 36 h, and 44 h. PI species with long fatty acid residues decrease over time, while PI species with short fatty acid residues increase.

Figure shows representative mass spectra of two independent biological experiments. (B) Bar diagram quantifying changes in PI composition during infection of HeLa cells with C. trachomatis (Figure 3-34 A). Values are normalized to PI (18:0/20:3).

Results are from two independent biological experiments. Error bars indicate SE.

Negative ion mode MALDI-TOF measurement of HEp-2 cells infected with C. trachomatis resulted in the detection of the same lipid species with lower masses (m/z < 900) as in infected HeLa cells.

Furthermore, the same tendency of changes in the lipid profile was observed (Figure 3-35).

Results and discussion: part II

Figure 3-35. Lipid profile of infection time course in HEp-2 cells (intermediate masses). (A) Negative ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HEp-2 cells. HEp-2 cells were infected for 12 h, 24 h, 36 h, and 44 h. PI species with long fatty acid residues decrease over time, while PI species with short fatty acid residues increase. Figure shows representative mass spectra of two independent biological experiments. (B) Bar diagram quantifying changes in PI composition during infection of HEp-2 cells with C. trachomatis (Figure 3-35 A). Values are normalized to PI (18:0/20:3). Results are from two independent biological experiments. Error bars indicate SE.

Among the lipids with higher masses (m/z > 1300), CL levels were found to be affected during progression of the infection in HeLa cells. While a core set of CL species (CL (2x16:0/2x18:2), CL (16:0/18:1/2x18:2), CL (16:0/2x18:1/18:2), CL (2x18:1/2x18:2), CL (3x18:1/18:2)) remained

Results and discussion: part II

unchanged during infection, CL species with shorter fatty acid residues (CL (4x16:0), CL (14:0/16:0/2x18:2)) increased over time (Figure 3-36). Furthermore, additional unassigned peaks (m/z = 1331.9, m/z = 1345.9, m/z = 1359.9) increased during the infectious cycle. Interestingly, these peaks differ from the mass of CL (4x16:0) by a multitude of 14 Da (Figure 3-36 A).

Figure 3-36. Lipid profile of infection time course in HeLa cells (high masses). (A) Negative ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HeLa cells. HeLa cells were infected for 12 h, 24 h, 36 h, and 44 h. CL species with short fatty acid residues increase over time. Red marks indicate unassigned mass peaks increasing during the developmental cycle. Figure shows representative mass spectra of two independent biological experiments. (B) Bar diagram quantifying changes in CL composition during infection of HeLa cells with C. trachomatis (Figure 3-36 A). Values are normalized to CL (3x18:1/18:2). Results are from two independent biological experiments. Error bars indicate SE.

Results and discussion: part II

Similar changes were detected during C. trachomatis infection of HEp-2 cells. CL species with shorter fatty acid residues (CL (4x16:0), CL (14:0/16:0/2x18:2)) were found to increase during the infection cycle (Figure 3-37). Again, peaks at m/z = 1331.9, m/z = 1345.9, and m/z = 1359.9 were found to increase during the infectious cycle (Figure 3-37 A).

Figure 3-37. Lipid profile of infection time course in HEp-2 cells (high masses). (A) Negative ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HEp-2 cells. HEp-2 cells were infected for 12 h, 24 h, 36 h, and 44 h.

CL species with short fatty acid residues increase over time. Red marks indicate unassigned mass peaks increasing during the developmental cycle. Figure shows representative mass spectra of two independent biological experiments. (B) Bar diagram quantifying changes in CL composition during infection of HEp-2 cells with C. trachomatis (Figure 3-37 A). Values are normalized to CL (3x18:1/18:2). Results are from two independent biological experiments. Error bars indicate SE.

Results and discussion: part II

Among lipid species with m/z < 800, PE species (PE (14:0/16:0), PE (2x16:0), PE (16:0/18:1), PE (18:0/18:2), PE (18:0/18:1), PE (18:0/20:4)), and PG species (PG (14:0/16:0), PG (2x16:0), PG (16:0/18:1), PE (18:0/18:2)) were detected, while short fatty acid lipids (PE (14:0/16:0), PE (2x16:0), PG (14:0/16:0), PG (2x16:0)) were found mainly at later infection time points (Figure 3-38). Interestingly, peaks at m/z = 676.5, m/z = 702.5, and m/z = 707.5 were observed, with a distinct 14 Da difference compared to PE (2x16:0), PE (16:0/18:1), and PG (2x16:0), respectively.

Figure 3-38. Lipid profile of infection time course in HeLa and HEp-2 cells (low masses) (part I). Negative ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HeLa and HEp-2 cells. Cells were infected for 12 h, 24 h, 36 h, and 44 h. Red marks indicate unassigned mass peaks increasing during the developmental cycle. Figure shows representative mass spectra of two independent biological experiments.

Results and discussion: part II

Positive ion mode MALDI-TOF spectra of all time points of the developmental cycle revealed SM (16:0) and several PC species with nearly no changes during progression of the infection (Figure 3-39). Remarkably, additional peaks at m/z = 740.5, m/z = 742.5, and m/z = 768.6 were detected, with a distinct 14 Da difference compared to PC (16:0/16:1) + Na⁺, PC (16:0/16:0) + Na⁺, and PC (16:0/18:1) + Na⁺, respectively.

Figure 3-39. Lipid profile of infection time course in HeLa and HEp-2 cells (low masses) (part II). Positive ion mode mass spectra of lipid extracts from C. trachomatis infection time course in HeLa and HEp-2 cells. Cells were infected for 12 h, 24 h, 36 h, and 44 h. Red marks indicate unassigned mass peaks increasing during the developmental cycle. Figure shows representative mass spectra of two independent biological experiments.

Results and discussion: part II

Taken together, these data show that C. trachomatis changes the overall lipid composition of infected cells. During the infectious cycle, PI species with long fatty acid residues decreased, while short chain PI and CL species increased. Interestingly, LPC species are only detected in infected cells.

Furthermore, peaks with a characteristic 14 Da shift compared to their related lipids were observed exclusively in infected cells.