Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice

Abstract

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

FIG 1

(A, B, and C) Biochemical characterization of rPLC_Ma. (A) Purified rPLC_Ma was loaded on 12% SDS-PAGE. Molecular weight standards are on the left. Lane 1 contained purified rPLC_Ma (10 μg). The sample was loaded under reducing conditions, and the gel was then stained with Coomassie brilliant blue. (B) Time course hydrolysis of phosphatidylcholine (PC) by 60 μg of rPLC_Ma (■) or 5 ng of recombinant B. cereus PC-PLC (○). The release of phosphocholine was measured indirectly by the fluorescence measurement of resorufin released using the Amplex red phosphatidylcholine kit and continuously monitored at a λ_exc of 510 nm and λ_em of 590 nm, (C) Substrate preference of rPLC_Ma and recombinant B. cereus PC-PLC. Competition assays between PC and phosphatidylethanolamine (PE) (or phosphatidylinositol [PI]) were carried out using different phospholipid ratios (PE [or PI]/PC = 2 or 5) and pure PC. A final PC quantity of 0.1 μmol was used in the pure-PC assay, and 0.2 μmol and 0.5 μmol of PE (or PI) were used for the other PE (PI)/PC ratios. The PC-PLC activity was continuously measured using the Amplex red PC-PLC kit. The relative activity (percent) of PC hydrolysis was calculated from the ratio of PC activity in the presence of PE (or PI) over PC activity in the absence of PE (or PI). (D, E, and F) Cytotoxic effects of rPLC_Ma on mouse macrophages. (D) Macrophage cellular state after 24 h of incubation, with no recombinant PLC (buffer only [negative control]), with 50 μg of purified rPLC_Ma (rPLC_Ma), and with 50 μg of heat-inactivated purified rPLC_Ma (rPLC_Ma, heat inactivated), as shown by light microscopy (magnification, ×200; bar, 25 μm). (E) Purified rPLC_Ma (50 μg; ■) and 15 μg of PC-PLC from B. cereus (○) were incubated with 1 × 10⁶ RAW264.7 mouse macrophages. The values are shown as percent lysis, in which the amount of LDH released in wells with macrophages incubated with enzymes was compared to total LDH released in control wells in which all of the macrophages had been deliberately lysed. LDH released was quantified at 16, 24, and 48 h. The values are the means for triplicate samples. (F) Autoradiography of TLC plate showing the release of radiolabeled DAG, the product of phospholipid hydrolysis (indicated by the black arrow), after incubation of rPLC_Ma (50 μg, lane 2) and PC-PLC from B. cereus (15 μg, lane 3) with radiolabeled macrophages. For the negative control (lane 1), only the buffer without any pure enzyme was added in the incubation medium. Abbreviations: DAG; diacylglycerol; FA, free fatty acid; DPPC, dipalmitoyl-glycerophosphocholine.

FIG 2

Construction of the M. abscessus plc KO mutant by homologous recombination (HR). (A) MAB_0554 and MAB_0555 are separated by 4 bp. The M. abscessus plc KO mutant was thus constructed by amplifying a nearly 2-kbp M. abscessus fragment encompassing this region and cloning the zeocin resistance gene (S. hindustanus ble) into the HindIII (position 731 in MAB_0554)-ClaI (position 590 in MAB_0555) sites. The entire fragment was then electroporated into M. abscessus CIP-S containing the pJV53 plasmid inserted by HR into the M. abscessus chromosome. (B) Southern blotting analysis was performed after genomic DNA restriction by KpnI and gel electrophoresis. A 532-bp probe targeting the 3′ conserved region of MAB_0555 was used for hybridization with DNA fragments: a 2,704-bp band is observed with the WT strain (lane 1), and a 2,635-bp band is observed with the M. abscessus KO mutant (lane 2). (C) Respective PLC activity of the different constructed M. abscessus strains. Phospholipase C activity was measured with p-NPPC for the CIP-S, CIP-S plc KO, CIP-S plc KO plc-complemented, and CIP-S-hsp60-plc (M. abscessus pMVZ361-hsp60pro-MAB_0555, used as a control for PLC activity) strains. (D) In vitro growth curves estimated by spectrophotometry (OD₆₀₀) of CIP-S (circles), CIP-S plc KO (squares), and CIP-S plc KO plc-complemented (triangles) strains. (E) Glycolipid and phospholipid patterns of the different constructed M. abscessus strains. Total lipid contents of the WT M. abscessus strain (lanes 1) and the plc KO M. abscessus mutant (lanes 2) were analyzed by TLC using CHCl₃-CH₃OH (90:10, vol/vol) (left) and CHCl₃-CH₃OH-H₂O (60:35:8, vol/vol/vol) (right) as the solvent systems and anthrone revelation (GPLs, glycopeptidolipids; TDM, trehalose dimycolate; TMM, trehalose monomycolate; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIMs, phosphatidylinositol mannosides).

FIG 3

Survival of plc M. abscessus mutant in eukaryotic cells. (A) Growth of mycobacterial strains within BMDMs recorded by CFU evaluation after 1, 3, and 6 days of coculture. The CIP-S (circles), CIP-S plc KO (squares), and CIP-S plc KO plc-complemented (triangles) strains were used. (B) Growth of mycobacterial strains within amoebae recorded by CFU evaluation after 1.5 h and 1, 2, and 4 days of coculture. CIP-S (white bars), CIP-S plc KO (black bars), and CIP-S plc KO plc-complemented (gray bars) strains were used. Experiments were repeated five times in triplicate at different times for both panels A and B (***, P < 0.001). (C) mRNA plc/sigA ratio (in arbitrary units) for the CIP-S, CIP-S plc KO, and CIP-S plc KO plc-complemented strains cocultivated with macrophages for 5 days. (D) mRNA plc/sigA ratio (in arbitrary units) for the CIP-S, CIP-S plc KO, and CIP-S plc KO plc-complemented strains cultivated in rich medium (7H9) at 30°C or 37°C. The results are representative of two independent experiments (C and D). (E) Western blot analysis of PLC expression during coculture of mycobacterial strains with A. castellanii. Lane 1, total extract (30 μg) of CIP-S cultivated in 7H9 medium; lane 2, total extract (30 μg) of amoebae cultivated for 96 h in PAS buffer in the absence of mycobacteria; lanes 3 to 7, total extract (30 μg) of amoebae cocultivated for 3 h (lane 3), 24 h (lane 4), 48 h (lane 5), 72 h (lane 6), or 96 h (lane 7) in PAS buffer in the presence of CIP-S; lane 8, total extract (30 μg) of amoebae cocultivated for 48 h in PAS buffer in the presence of the CIP-S plc KO plc-complemented strain. This picture is representative of three independent experiments.

FIG 4

Effect of coculture of mycobacterial strains within amoebae on virulence in mice. (A) BALB/c mice were aerosolized with the CIP-S (white bars), CIP-S plc KO (black bars), and CIP-S plc KO plc-complemented (gray bars) strains cultivated in 7H9 medium. Mice were challenged with 10⁸ mycobacteria and sacrificed at days 1, 7, 14, 21, and 28. Lungs, livers, and spleens were collected, homogenized, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted after 5 days of growth. Twenty-five mice per group were challenged. (B) BALB/c mice were aerosolized with CIP-S cultivated in 7H9 medium (○) or obtained after 3 days of coculture in amoebae (■). Mice were sacrificed at days 3 and 7. Lungs were collected, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted for the different dilutions after 5 days of growth. Ten mice per group were challenged. (****, P < 0.0001). (C) BALB/c mice were aerosolized with the CIP-S (empty bars) or CIP-S plc KO (black bars) strain cultivated in 7H9 medium or obtained after 3 days of coculture within amoebae. Mice were sacrificed at days 3 and 7. Lungs were collected, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted after 5 days of growth. Ten mice per group were challenged. (**, P < 0.01; ***, P < 0.001).