Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages - PubMed (original) (raw)

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Alexander Speer et al. Mol Microbiol. 2015 Sep.

Abstract

Sphingomyelinases secreted by pathogenic bacteria play important roles in host-pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface-exposed C-terminal sphingomyelinase domain and a putative N-terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sphingomyelinase of Mycobacterium tuberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5- and 100-fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.

© 2015 John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1

Fig. 1. Rv0888 mediates glucose uptake in mycobacteria

(A) Growth of M. bovis BCG strains ML383 (wt, black), ML386 (cpnT::Tn, red) and wt expressing rv0888 (pML2102) (blue) in HdB medium supplemented with 1% glucose. OD600 of ML383 and wt expressing rv0888 is significantly higher (p-value < 0.001) compared to the cpnT::Tn mutant in every time point starting day 5. The data was analysed by one-way ANOVA followed by a Tukey test. (B) Western blot analysis of M. bovis BCG wt and the cpnT::Tn mutant. Rv0888 was detected with an Rv0888-specific antiserum. M. bovis BCG RNA polymerase (RNApol) was detected on the same blot with monoclonal antibodies. Lanes: 1, ML383 (wt); 2, ML386 (cpnT::Tn). (C) Western blot analysis of M. smegmatis SMR5 (wt) and porin deletion strain ML16 (Δ_mspA,_ Δ_mspC,_ Δ_mspD_) expressing mspA, rv0888 or the enzymatic mutant rv0888*. Rv0888 was detected with an Rv0888-specific antiserum. M. smegmatis RNA polymerase (RNApol) and MspA were detected on the same blot. Lanes: 1, SMR5/pMS2kan (wt, empty vector); 2, ML16/pMS2kan (empty vector); 3, ML16/pML632 (+mspA); 4, ML16/pML2118 (+rv0888); 5, ML16/pML3108 (+rv0888*). (D) Accumulation of 14C-glucose by M. smegmatis strains SMR5/pMS2kan (wt, empty vector, black), ML16/pMS2kan (empty vector, red), ML16/pML632 (+mspA, green), ML16/pML2118 (+rv0888, yellow) and ML16/pML3108 (+rv0888*, blue) was measured. The uptake rate is expressed as pmol of glucose per milligram of dried cells. Mean values of biological triplicates are shown with standard deviations. The uptake of glucose in all tested strains is significantly higher (p-value < 0.001) compared to ML16 (pMS2kan) after 1 min. The p-values were calculated by one-way ANOVA followed by a Tukey test.

Fig. 2

Fig. 2. Rv0888 is an outer membrane protein of M. tuberculosis

(A) Subcellular localization of Rv0888. Mtb mc26206 (wt) cells were lysed (WC) and water soluble (SN) and insoluble proteins (P) were separated by centrifugation. The culture supernatant was concentrated 500-fold (CF). Proteins from each fraction were analyzed by Western blot. Rv0888 was detected with an Rv0888-specific antiserum. GlpX, Rv1698 and CFP10 served as marker proteins for soluble proteins (SN), membrane associated proteins (P) and secreted proteins (CF), respectively. (B) Surface accessibility of Rv0888. Cells of Mtb ML1528 (H37Rv L5::pCV125), ML1566 (rv0888::loxP), ML923 (rv0888::loxP, L5::rv0888) and ML925 (rv0888::loxP, L5::rv0888*) were incubated with a monoclonal anti-Rv0888 antibody (10E2.7) followed by detection with anti-mouse FITC-labeled antibody. The fluorescence of surface-stained Mtb cells was measured by flow cytometry and is displayed as histograms (MFI, mean fluorescence intensity). The MFI of strains ML923 and ML925 is significant higher (p-value < 0.05, Chi-Squared T(X) > 500) when compared to ML1528 or ML1566**. (C)** Whole cells of Mtb mc26206 (wt) were biotinylated. Cells were lysed and biotinylated proteins were purified using a Strep-Avidin column. An untreated sample served as a control for unspecific binding. After elution samples were analyzed by Western blot. Proteins of known subcellular localization served as control for cytosolic proteins (RNApol, IdeR), inner membrane associated proteins (MmpS5, EccB5) and surface assessable proteins (LpqH).

Fig. 3

Fig. 3. Rv0888 has a C-terminal sphingomyelinase domain

(A) Predicted domain organization of Rv0888. Rv0888 encodes for a Sec-signal sequence which is cleaved after residue 31. The secondary structure of Rv0888 between residues 31–229 is predicted to have a high content of hydrophobic β-sheets. The C-terminus (residue 230–490) contains conserved residues found in neutral sphingomyelinases. (B) Sphingomyelinase activity of M. smegmatis strains using TLC analysis. Whole cell lysates of M. smegmatis porin mutant ML375 (Δ_mspA,_ Δ_mspC,_ Δ_mspD ΔgroEL-5′_) and ML375 expressing rv0888 (pML2100) or the enzymatic mutant rv0888* (pML3109) were incubated with 100 ng sphingomyelin (SM). After 2 h of incubation amounts corresponding to 5 ng SM were transferred onto a TLC plate (origin) and resolved using chloroform/methanol/water (65:20:1, vol/vol). Phospholipids were visualized by the Dittmer-Lester reagent. Lanes: 1, SM in reaction buffer; 2, ML375; 3, ML375/pML2100 (+rv0888); 4, ML375/pML3109 (+rv0888*). PC; phosphocholine. (C) Western blot analysis of Mtb rv0888 deletion strain. Mtb strains H37Rv (wt), Δ_rv0888_ and Δ_rv0888_ expressing rv0888 were analyzed by Western blot after growing in 7H9/OADC or in HdB supplemented with 0.75 mM sphingomyelin (HdB/SM) or 0.75 mM sphingomyelin and 0.2% glucose (HdB/SM/glucose). Rv0888 was detected with monoclonal anti-Rv0888 antibody (10E2.7), Mtb RNA polymerase (RNApol) served as a loading control. The signals were quantified by pixel-densitometry. Lanes: 1, ML1528 (H37Rv L5::pCV125; wt); 2, ML1566 (rv0888::loxP); 3, ML923 (rv0888::loxP, L5::rv0888); 4, ML1528; 5, ML1566; 6, ML923; 7, ML1528. (D) Mtb whole cell lysates (WC) and concentrated culture filtrates of the corresponding strain were incubated with 7 μM of 14C- SM for 48 h at 37°C before analysis by thin layer chromatography (TLC). Sphingomyelinase of S. aureus served as a positive control. PC: phosphocholine. (E) Rv0888 protein purified from E. coli (14.6 pM) was incubated with 0.18, 0.35, 1.8, 3.5, 18 and 35 μM 14C-sphingomyelin (SM). The reaction products were separated by TLC and the amount of cleaved phosphocholine (PC) was determined by densitometry. The SM hydrolysis rates were blotted against the substrate concentration. The Michaelis-Menten parameters vmax and Km were calculated by non-linear regression to be 15.72 μM/min and 671 μM, respectively. A detailed data analysis is shown in Fig. S6. (F) Enzymatic sphingomyelinase assay using purified proteins. 1.5 μg of purified Rv0888 and Rv0888* proteins were analyzed using the fluorometric Amplex Red sphingomyelinase assay. SM hydrolysis is indicated by fluorescence in arbitrary units (a.u.). The activity of Rv0888 protein was calculated using the slopes of the fastest SM hydrolysis by Rv0888 and the S. aureus sphingomyelinase (0.04 units/mL).

Fig. 4

Fig. 4. The sphingomyelinase Rv0888 mediates contact-dependent hemolysis

Bacterial cells of M. smegmatis and Mtb were incubated with human blood (MOI 500) at 37°C for 18 h and 24 h, respectively. The intact erythrocytes and bacterial cells were removed by centrifugation and the supernatants containing the released hemoglobin were transferred into cuvettes and quantified by determining the absorbance at 540 nm. A bacteria free sample served as a control for autolysis of erythrocytes. (A) Supernatant of erythrocytes incubated with the M. smegmatis porin mutant ML16 (Δ_mspA,_ Δ_mspC,_ Δ_mspD_), ML16 expressing rv0888 (pML2118) or the enzymatic mutant rv0888* (pML3108) was quantified spectroscopically (B). (C) Hemolysis caused by Mtb ML1528 (H37Rv L5::pCV125; wt), ML1566 (rv0888::loxP) and ML923 (rv0888::loxP, L5::rv0888). Wt Mtb and M. smegmatis cells lysed 50% of all erythrocytes as determined by complete lysis with 0.5% SDS. Mean values of biological triplicates are shown with standard deviations. Lysis is expressed in relative values normalized to wt Mtb and wt M. smegmatis cells as 100%. The P values were calculated by a one-way ANOVA in combination with a Tukey test and are denoted as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001). (D) Western blot analysis of Mtb wt cells in contact with erythrocytes for 24 h. Rv0888 was detected with monoclonal anti-Rv0888 antibody (10E2.7), Mtb RNA polymerase (RNApol) served as a loading control.

Fig. 5

Fig. 5. Sphingomyelinase activity of Rv0888 is required for growth of Mtb on sphingomyelin

(A/B) Growth of Mtb strains ML1528 (H37Rv L5::pCV125; wt; red), ML1566 (rv0888::loxP, green), ML923 (rv0888::loxP, L5::rv0888, yellow) and ML925 (rv0888::loxP, L5::rv0888*, blue) in HdB medium with different carbon sources. (A) Mtb strains were grown in HdB with 0.75 mM sphingomyelin as sole carbon source. As control served Mtb ML1528 grown in HdB medium without sphingomyelin to ensure the absence of other carbon source (black). Growth was monitored by changes in optical density (OD600). Average values of technical triplicates are shown with standard deviations. An OD600 of ML1528 and ML923 that is significantly higher compared to ML1528 (no SM), ML1566 and ML925 is indicated with an asterik (p-value < 0.001). (B) Mtb strains were grown in HdB medium with 0.38 mM sphingomyelin, 0.38 mM phosphocholine or 15 mM glucose and 15 mM glycerol as carbon sources in a 96-well plate for two weeks. Growth of bacteria was determined by fluorescence after addition of the dye resazurin and is indicated by arbitrary units (a.u.). Average values of technical triplicates are shown with standard deviations. Values marked with asterisks are significantly different compared to the carbon source free culture. The data was logarithmically transformed to achieve normal distribution and a one-way ANOVA in combination with a Tukey test was performed. The p values are * (p < 0.05); ** (p < 0.01); *** (p < 0.001).

Fig. 6

Fig. 6. Rv0888 enables utilization of sphingomyelin cleavage products as nutrient sources

Phosphocholine utilization of M. smegmatis SMR5 (wt), the M. smegmatis porin mutant ML16 (Δ_mspA,_ Δ_mspC,_ Δ_mspD_) and ML16 expressing mspA, rv0888 or rv0888*. Ten-fold serial diluted cultures were dropped on (A) low-phosphate HdB/kan plates with regular amounts of phosphate (12 mM), no phosphate (trace phosphorus) and HdB plates with trace amounts of phosphorus supplemented with 0.5 mM phosphocholine. (B) Diluted cells were transferred on low-nitrogen HdB/kan plates with regular amounts of ammonium sulfate (15 mM), no ammonium sulfate (trace nitrogen) and HdB plates with trace amounts of nitrogen supplemented with 20 mM phosphocholine. The concentration of phosphocholine in these experiments was determined empirically. Lanes: 1, SMR5/pMS2kan (wt, empty vector); 2, ML16/pMS2kan (empty vector); 3, ML16/pML632 (+mspA); 4, ML16/pML2118 (+rv0888); 5, ML16/pML3108 (+rv0888*).

Fig. 7

Fig. 7. Expression of rv0888 enhances intracellular replication of M. tuberculosis in macrophages

Differentiated THP-1 macrophages were infected with Mtb ML1528 (H37Rv L5::pCV125; wt), ML1566 (rv0888::loxP), ML923 (rv0888::loxP, L5::rv0888) and ML925 (rv0888::loxP, L5::rv0888*) at an MOI of 1. Bacterial strains were grown in HdB medium supplemented with 0.75 mM sphingomyelin and 0.2% glucose to logarithmic phase before infection. Macrophages were lysed after 4 h and 5 days post-infection and the number of viable bacteria was counted as colony forming units (CFU) on agar plates. Mean values of biological triplicates are shown with standard deviations. The data was logarithmically transformed to achieve normal distribution and a one-way ANOVA in combination with a Tukey test was performed. The p values are * (p < 0.05); ** (p < 0.01); *** (p < 0.001).

Fig. 8

Fig. 8. Role of SpmT (Rv0888) in utilization of sphingomyelin as a versatile nutrient source by M. tuberculosis

SpmT (Rv0888) is an outer membrane protein of Mtb and consists of two domains. The C-terminal domain is a cell-surface sphingomyelinases which catalyzes the hydrolysis of SM into phosphocholine and ceramide. The N-terminal domain inserts into the outer membrane (OM) and may facilitate uptake of phosphocholine. Ceramide is utilized by Mtb as a carbon source after uptake by an unknown mechanism. The phosphate group of phosphocholine might be cleaved off by the alkaline phosphatase in the periplasm and taken up by an inner membrane (IM) phosphate transporter. It is unclear how choline is taken up and utilized as a nitrogen source. This model visualizes how the sphingomyelinase SpmT enables Mtb to utilize sphingomyelin as a nutrient source for carbon, nitrogen and phosphorus.

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