Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

Figure 1. Construction of M. tuberculosis H37RvplcABC KO (H37RvΔPLC).

(A) Schematic representation of genomic organization of plc genes in M. tuberculosis H37Rv wild type and H37RvΔPLC strains ; (B) AvrII restriction fragment profiles of M. tuberculosis WT and KO strains separated by agarose gel electrophoresis; (C) Pattern obtained from genomic DNAs digested with AvrII and hybridized with a probe specific for the plcC downstream region; Lanes: 1 (second lane from left), negative control (pYUB412 vector); 2, positive control pYUB412::plcABC; 3 and 4, M. tuberculosis H37Rv WT, 5 and 6, M. tuberculosis H37RvΔplcABC; 7, M, Smart Ladder (Eurobio).

Figure 2. Phospholipase C enzymatic assay.

This assay is based on the detection of the hydrolysis of p-nitrophenylphosphorylcholine (p-NPPC) to p-nitrophenol. While the substrate, p-NPPC, is colourless, the product p-nitrophenol due to its ability to absorb light at 410 nm, is yellow. About 500 μg of total protein were used in the assay and measurements were performed in triplicates. (A) Crude extracts of 4 day-old cultures from different M. tuberculosis strains were incubated with 5 mmol.l⁻¹ of p-nitrophenol phosphorylcholine; (B) Measurement of phospholipase activity of different M. tuberculosis strains over 3 timepoints. Measurements were performed in duplicates.

Figure 3. Phagosomal rupture by M. tuberculosis mutants.

Capacity of different M. tuberculosis strains and mutants to induce phagosomal rupture inTHP-1 cells, monitored by CCF-4 staining and flow cytometric analysis. Non infected (gray), infected (MOI = 1:10) with different strains of M. tuberculosis (purple) using a recently developed approach. Results shown are representative of 2 independent experiments. The shift towards blue emission (447 nm) of CCF-4 is due to the inhibition of FRET and is proportional to the mycobacteria-induced phagosomal rupture/cytosolic access.

Figure 4. Growth kinetics of M. tuberculosis strains in THP-1 derived macrophages.

Number of colony forming units (CFU) obtained at different time points after infection. MOI was 1:20 (bacteria/cells). The figures show the means and the standard deviations obtained in 3 independent experiments.

Figure 5. Virulence evaluation of M. tuberculosis strains in different mouse infection models.

Number of colony forming units (CFU) 3 weeks days after intravenous infection with M. tuberculosis WT and mutant strains in (A) lungs; and (B) spleens of SCID mice. (C) Panel C shows the in vitro growth characteristics of the same panel of strains as above, in C57BL/6 mice 6 weeks after infection. Results shown are representative of 2 independent experiments.

Figure 6. plcA-egfp fusion gene expression of M. tuberculosis H37RvΔPLC::Pr_plcA-egfp during growth in phosphate limiting conditions.

(A) The curve shows the phosphate concentration in samples over time of in vitro growth. Histogram represents increase of the culture fluorescence intensity due to GFP expression over time. Results shown are representative of 2 independent experiments. Note that towards the end of the experiment the phosphate concentration slightly increased, which is plausibly due to lysis of some of the older bacterial cells. (B) Measurement of fluorescence divided into GFP and red fluorescence in a culture of H37RvΔPLC::Pr_plcA-egfp complemented with a DsRed expressing plasmid. DS-red is expressed via a constitutive promoter while GFP expression is dependent on plcA promoter activity. (C) Promoter activity of M. tuberculosis H37RvΔPLC::Pr_plcA-egfp in presence of decreasing phosphate concentration due to in vitro growth of culture during 7 days at 37 °C under shaking conditions. Measures show the ratio between fluorescence and absorbance, the first reflecting GFP expression levels and the latter reflecting cell density. (D) Survival of M. tuberculosis H37Rv WT and mutant strains in broth that provides phosphatidylcholine as the sole phosphate source. Results shown represent two different experiments. For each experiment the different strains tested were plated and counted in triplicate.