Clostridium perfringens α-Toxin Impairs Lipid Raft Integrity in Neutrophils

Abstract

Clostridium perfringens type A, a Gram-positive, anaerobic bacterium, causes gas gangrene. Recently, we reported that C. perfringens α-toxin blocked neutrophil differentiation in an enzyme activity-dependent manner to impair host innate immunity, which should be crucial for the pathogenesis of C. perfringens. However, the detailed mechanism remains unclear. Lipid rafts have been reported to be platforms for signaling molecules involved in the regulation of cell differentiation in many different cell types. In this study, we found that cell surface expression of a lipid raft marker, GM1 ganglioside, decreased in association with neutrophil differentiation by flow cytometry analysis and morphological observation. In vitro treatment of isolated mouse bone marrow cells with α-toxin or an α-toxin variant lacking phospholipase C and sphingomyelinase activities revealed that α-toxin increased the cell surface expression of GM1 ganglioside in an enzyme activity-dependent manner. C. perfringens infection also increased GM1 ganglioside levels in bone marrow myeloid cells. Moreover, treatment of bone marrow cells with methyl-β-cyclodextrin, a lipid raft-disrupting agent, impaired neutrophil differentiation. Together, our results suggest that the integrity of lipid rafts should be properly maintained during granulopoiesis, and α-toxin might perturb lipid raft integrity leading to the impairment of neutrophil differentiation.

Clostridium perfringens type A is a Gram-positive, anaerobic bacterium that causes life-threatening gas gangrene, characterized by the destruction of muscle.^1–3) Because gas gangrene caused by C. perfringens type A progresses so rapidly that shock, multiple organ failure, and death of patients occur within a short time.^4,5)

Of many toxins produced by C. perfringens type A, α-toxin, which has two enzyme activities classified as phospholipase C (PLC) and sphingomyelinase (SMase) activities, is a major virulence factor during C. perfringens infection.^4,6–9) A variant α-toxin lacking enzymatic activities (H148G) loses its ability to cause death in mice, demonstrating that these activities are important for the pathogenesis of C. perfringens.¹⁰⁾ Recently, we reported that α-toxin blocks neutrophil differentiation in an enzyme activity-dependent manner, leading to impairment of host innate immunity, which is crucial for the pathogenesis of C. perfringens to promote disease.¹¹⁾ However, the detailed mechanism by which α-toxin blocks neutrophil differentiation remains unclear.

Bacterial SMase disrupts cholesterol-rich plasma membrane microdomains, also known as lipid rafts, in human lymphocytes.¹²⁾ Lipid rafts are known to act as platforms for signaling molecules involved in the regulation of cell differentiation in many different cell types.^13,14) For instance, glycosyl-phosphatidylinositol-linked glial-cell-derived neurotrophic factor (GDNF) receptor-α recruits transmembrane tyrosine kinase to lipid rafts upon stimulation by GDNF family ligands, resulting in intracellular GDNF signaling, which contributes to the development of the nervous system.¹⁵⁾ Moreover, Konstantinidis et al. reported recently that the clustering of lipid rafts at the furrow between incipient reticulocytes and pyrenocytes was necessary during erythroblast enucleation,¹⁶⁾ meaning that lipid rafts are involved in the regulation of erythropoiesis. Thus, lipid rafts are thought to play a role in the differentiation of many types of cell, including hematopoietic cells. However, the role of lipid rafts in granulopoiesis has not been elucidated.

In this study, to clarify whether lipid rafts are involved in α-toxin-induced impairment of neutrophil differentiation, we evaluated cell surface expression and localization of a lipid raft marker, GM1 ganglioside (GM1),¹⁷⁾ in bone marrow-derived neutrophils. Here, we show that α-toxin impairs the integrity of lipid rafts, which provides a mechanism to explain how α-toxin blocks neutrophil differentiation.

MATERIALS AND METHODS

Animals

C57BL/6J mice were kept in a specific pathogen-free animal facility at Tokushima Bunri University. Mice aged more than 8 weeks old were used for all experiments. Mouse experiments were approved by the Animal Care and Use Committee of Tokushima Bunri University. Procedures were performed in accordance with institutional guidelines, and the details about isolation of bone marrow cells and bacterial infection were described below.

Reagents and Strains

Fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or PE-Cy7-conjugated specific antibody against mouse CD3e (clone 145-2C11), CD4 (clone GK1.5), CD8a (clone 53-6.7), CD45R (B220, clone RA3-6B2), TER119 (clone TER-119), CD11b (clone M1/70), Ly6G/6C (Gr-1, clone RB6-8C5), or CD117 (c-kit, clone 2B8), and purified rat anti-mouse CD16/CD32 (Fc Block) were purchased from BD Biosciences. Alexa Fluor 488-conjugated cholera toxin subunit B was from Life Technologies. Methyl-β-cyclodextrin (MβCD) was from Sigma. Giemsa’s azur eosin methylene blue solution was from Merck. All other chemicals were of the highest grade available from commercial sources. C. perfringens strain 13, which is a readily transformable strain,¹⁸⁾ and Bacillus subtilis ISW1214¹⁹⁾ were used in this study. Preparation of a plc gene-knockout mutant of C. perfringens (PLC-KO) has been described in our previous report.¹¹⁾

Purification of α-Toxin

Purification of wild-type or H148G variant α-toxin was performed as described previously.^10,20) Transformation of B. subtilis ISW1214 was performed with recombinant forms of pHY300PLK harboring the structural genes of wild-type or H148G variant α-toxin. The transformants were cultured in Luria–Bertani broth containing 50 µg/mL ampicillin with continuous aeration, and α-toxin secreted into culture medium was purified chromatographically.

Isolation of Bone Marrow Cells

Isolation of bone marrow cells (BMCs) was performed as described previously.¹¹⁾ Femurs and tibias were crushed in phosphate-buffered saline (PBS) supplemented with 2% heat-inactivated fetal bovine serum (FBS; AusGeneX), and filtered through a 40 µm mesh. After hemolysis of red blood cells with ammonium–chloride–potassium lysing buffer (GIBCO, U.S.A.), the number of living cells was counted with trypan blue staining. RPMI 1640 medium was supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin for the culture of BMCs.

Bacterial Culture and Infection

C. perfringens strain 13 and PLC-KO were grown in tryptone glucose yeast (TGY) medium in anaerobic conditions at 37°C as described previously.¹¹⁾ Exponentially growing bacteria were harvested, washed, re-suspended in TGY medium, and 2.5×10⁷ colony-forming units (CFUs) of the bacteria were injected into the left femoral muscle of mice. As an uninfected control, the same amount of TGY medium was injected. BMCs were isolated from the mice after 24 h. For the quantification of CFUs, residual bacteria were serially diluted, plated on brain heart infusion agar plates, and cultured anaerobically at 37°C.

Flow Cytometry Analysis

Fc-receptors on BMCs were blocked with purified rat an anti-mouse CD16/CD32 antibody, and the cells were labeled with antibodies diluted with PBS containing 2% FBS. To label GM1, Alexa Fluor 488-conjugated cholera toxin B subunit (CTB) was diluted with PBS containing 2% FBS, and incubated with the cells for 30 min on ice. FACS Aria II (BD Biosciences, U.S.A.) or a Guava easyCyte (Millipore, U.S.A.) instruments were used to analyze the labeled cells. Data were analyzed by using FlowJo (Tree Star) software.

Immunofluorescence Microscopy

Isolated cells were incubated with Alexa Fluor 488-conjugated CTB diluted with PBS containing 2% FBS for 30 min on ice. After washing, samples were placed onto CELLview glass bottomed dishes (Greiner Bio-One, Austria), and images were captured using a confocal laser-scanning fluorescence microscope (Nikon A1; Nikon Instruments, Japan).

Statistical Analysis

All statistical analyses were performed with Easy R (Saitama Medical Center, Jichi Medical University, Japan).²¹⁾ One-way ANOVA followed by the Tukey’s test was used to evaluate differences among three or more groups. Differences were considered to be significant for values of p<0.05.

RESULTS

Firstly, we quantified the cell surface expression of GM1 in several different lineages from naive C57BL/6 mice using CTB, which is known to specifically bind to GM1.²²⁾ Three distinct populations in steady-state BMCs were identified as GM1-negative (GM1⁻), GM1-intermediate (GM1^int), and GM1-high (GM1^high) (Fig. 1A). Overall, lymphoid lineage cells (CD3e⁺, CD4⁺, CD8a⁺, or B220⁺ cells) had higher cell surface expression of GM1 than myeloid lineage cells (TER119⁺, CD11b⁺, or Gr-1⁺ cells), suggesting that the state of lipid rafts differs in a cell-lineage-dependent manner during hematopoiesis (Figs. 1A, B). In CD11b⁺ or Gr-1⁺ myeloid cells, the main population was GM1^int, but about 10% of cells were identified as GM1^high (Fig. 1A).

Fig. 1. Expression of GM1 Ganglioside Differs in a Cell-Lineage-Dependent Manner

Bone marrow cells from mice (n=3) were labeled with Alexa Fluor 488-CTB and specific antibodies against CD3e, CD4, CD8a, B220, TER119, CD11b, or Gr-1. The cells were analyzed using a FACS Aria II. Fluorescence of Alexa Fluor 488-CTB in each cell population was compared with an unstained control (A), and the mean fluorescence intensity was determined (B). Values are the mean±S.D.

Expression of CD11b and Gr-1 represents stages of neutrophil maturation.^11,23) The CD11b⁺Gr-1^high cell population contains mature neutrophils, the CD11b⁺Gr-1^low cell population represents an intermediate stage of maturation, and the CD11b⁻Gr-1⁺ cell population contains primitive precursor cells. Moreover, c-kit is known to be a marker of immaturity.^24,25) A comparison of GM1 expression levels among four populations at different stages of granulopoiesis revealed that cell surface expression of GM1 decreased gradually in association with neutrophil differentiation (Fig. 2). The morphological characteristics of neutrophils differ depending on their state of maturation. Mature neutrophils have condensed and segmented nuclei, whereas primitive precursor cells, myeloblasts, have a large nucleus and a lower nuclear cytoplasmic ratio. To determine the morphological characteristics of the Gr-1⁺GM1^high cell population, we stained and sorted Gr-1⁺GM1^int and Gr-1⁺GM1^high cells with Giemsa and found that Gr-1⁺GM1^int cells presented a mature morphology, whereas Gr-1⁺GM1^high cells were immature (Fig. 3). Thus, the expression of GM1 correlates with the maturity of neutrophils. In addition, we tested if localization of lipid rafts was altered in association with neutrophil differentiation. In CD11b⁺Gr-1^lowc-kit⁺ cells, GM1 was widely dispersed in the cell membrane, whereas scattered localization of GM1 was seen in CD11b⁺Gr-1^high cells (Fig. 4). Both types of the distribution were observed in the CD11b⁺Gr-1^lowc-kit⁻ cell population. These results suggested not only the amount but also the localization of GM1 in the cell membrane correlates with the maturity of neutrophils. Together, these results suggested that the integrity of lipid rafts should be tightly regulated during granulopoiesis.

Fig. 2. Expression of GM1 Ganglioside Decreases in Association with Neutrophil Differentiation

Bone marrow cells from mice (n=3) were labeled with Alexa Fluor 488-CTB and specific antibodies against CD11b, Gr-1, and c-kit. The cells were analyzed using a FACS Aria II. A comparison of Alexa Fluor 488-CTB fluorescence among four distinct populations: CD11b⁺Gr-1^high, CD11b⁺Gr-1^low, CD11b⁺Gr-1^lowc-kit⁺, and CD11b⁻Gr-1⁺, was performed (A), and the mean fluorescence intensity was determined (B). Values are the mean±S.D. One-way ANOVA was employed to assess statistical significance.

Fig. 3. Morphological Characteristics of the Gr-1⁺GM1^int and Gr-1⁺GM1^high Cell Populations

Bone marrow cells were labeled with Alexa Fluor 488-CTB and a specific antibody against Gr-1. Gr-1⁺GM1^int and Gr-1⁺GM1^high cells were sorted using a FACS Aria II and stained with Giemsa.

Fig. 4. Localization of GM1 Ganglioside Is Altered in Association with Neutrophil Differentiation

Bone marrow cells from mice were labeled with specific antibodies against CD11b, Gr-1, or c-kit. CD11b⁺Gr-1^high, CD11b⁺Gr-1^lowc-kit⁻, and CD11b⁺Gr-1^lowc-kit⁺ cells were sorted using a FACS Aria II. After the cells were incubated with Alexa Fluor 488-CTB, the cells were inspected using a confocal laser scanning microscope.

Next, we tested whether α-toxin affected the state of lipid rafts by quantifying the cell surface expression of GM1. Figure 5A shows that α-toxin treatment dose-dependently increased the expression of GM1 in CD11b⁺ cells, suggesting that α-toxin affected lipid raft integrity in neutrophils. Because a variant α-toxin (H148G) did not affect the expression of GM1, the α-toxin-induced alteration of GM1 expression was dependent on its enzyme activities (Figs. 5B, C). In addition, an increase in GM1 expression was observed in CD11b⁺ BMCs from wild-type C. perfringens-infected mice, and the increase was attenuated in those from a plc-gene knockout mutant C. perfringens (PLC-KO)-infected mice, suggesting that lipid raft integrity is perturbed in neutrophils during infection (Fig. 6).

Fig. 5. α-Toxin Increases Expression of GM1 Ganglioside

A total of 5×10⁶ bone marrow cells were treated with α-toxin for 24 h, and Alexa Fluor 488-CTB fluorescence of CD11b⁺ cell population was compared with that of α-toxin-untreated cells (Control) (n=3 per condition). The cells were treated with the indicated concentration of wild-type α-toxin (A), 100 ng/mL α-toxin (WT) or a variant α-toxin (H148G) (B, C). The mean fluorescence intensity was determined (A, C). Values are the mean±S.D. One-way ANOVA was employed to assess statistical significance.

Fig. 6. Clostridium perfringens Infection Increases Expression of GM1 Ganglioside on Myeloid Cells

Bone marrow cells from mice intramuscularly injected with 2.5×10⁷ colony-forming units of C. perfringens strain 13 (WT) or PLC-KO (KO) were labeled with Alexa Fluor 488-CTB and a specific antibody against CD11b. Fluorescence of Alexa Fluor 488-CTB in the CD11b⁺ cell population was compared with that of an uninfected control (Uninfected) (n=4 per condition). Values are the mean±S.E.M. One-way ANOVA was employed to assess statistical significance.

To clarify whether the perturbation of lipid rafts is responsible for the blockage of neutrophil differentiation, we treated BMCs with an inhibitor of lipid raft formation, MβCD,²⁶⁾ and quantified the CD11b⁺Gr-1^high cell population. As shown in Fig. 7A, the expression of GM1 in CD11b⁺ cells was increased by treatment with MβCD, indicating that the lipid rafts had been perturbed. In this condition, the CD11b⁺Gr-1^high cell population decreased, whereas the CD11b⁺Gr-1^low cell population increased (Fig. 7B). These results indicated that the perturbation of lipid raft integrity could cause the blockage of neutrophil differentiation.

Fig. 7. Perturbation of Lipid Raft Integrity by MβCD Impairs Neutrophil Differentiation

A total of 5×10⁶ bone marrow cells were treated with 2.5 mM MβCD for 24 h, and flow cytometry analysis was performed with a Guava easyCyte. Fluorescence of Alexa Fluor 488-CTB of MβCD-treated cells in CD11b⁺ cell population (MβCD) was compared with that of untreated control (Control) (A). Expression profiling of CD11b and Gr-1 was performed (B).

DISCUSSION

Lipid rafts can act as platforms for signaling molecules involved in the regulation of cell differentiation in many cell types,^13,14) including hematopoietic cells, but the role of lipid rafts in granulopoiesis has not been elucidated. In the present study, the cell surface expression of a lipid raft marker, GM1, differed in a cell-lineage-dependent manner (Fig. 1). In addition, the expression of GM1 decreased gradually in association with neutrophil differentiation (Fig. 2), suggesting that the state of lipid rafts could be altered during granulopoiesis. Previously, Kim et al. identified mutations of a neutral SMase gene, SMPD3, in human acute myeloid leukemias that is characterized by differentiation blockage of immature myeloid cells.²⁷⁾ SMase hydrolyzes cell membrane sphingomyelin to ceramide,²⁸⁾ which is a well-known lipid raft component.²⁹⁾ Ceramide is also known to be a lipid messenger implicated in various cellular responses, including cell growth, differentiation, and apoptosis.³⁰⁾ Taken together, lipid rafts might play an important role in myeloid cell differentiation through ceramide metabolism, and the integrity of lipid rafts should be properly maintained during granulopoiesis.

We reported previously that treatment of BMCs with α-toxin, which is a major virulence factor of C. perfringens,^6,31) caused the blockage of neutrophil differentiation in a PLC and/or SMase activity-dependent manner.¹¹⁾ In addition, we reported that α-toxin induces clustering of GM1 on the cell membrane in a lung adenocarcinoma epithelial cell line, A549 cells.³²⁾ Bacterial SMase is known to hydrolyze cell membrane sphingomyelin to ceramide as described above. Therefore, α-toxin might alter the localization of lipid rafts on the cell surface and/or cause the overproduction of ceramide in neutrophils, leading to the disturbance of lipid raft integrity. The increase in cell surface expression of GM1 by α-toxin treatment supports the notion that α-toxin affects lipid raft integrity (Fig. 5).

Similarly, MβCD, which is known to deplete cholesterol from the cell membrane, increased the expression of GM1 (Fig. 7A). MβCD has been reported to have bilateral effects on cellular cholesterol.^26,33) In detail, MβCD depletes membrane cholesterol in the absence of cholesterol, whereas MβCD-cholesterol complexes are formed in the presence of cholesterol and serve as donors to integrate cholesterol into cell membranes. This means that the effect of MβCD on the cell membrane is dependent on cell culture conditions. In this study, we supplemented the culture medium with FBS, which generally contains cholesterol, so whether the treatment with MβCD quantitatively increased lipid rafts or decreased them was unknown. Irrespective of this, lipid raft integrity would be affected by MβCD treatment because it affected the expression of GM1 (Fig. 7A). Together with the results obtained in α-toxin-treated cells, α-toxin could block neutrophil differentiation by perturbing lipid raft integrity.

In conclusion, C. perfringens α-toxin perturbed the integrity of lipid rafts and this mediated the blockage of neutrophil differentiation. Our data offer a novel insight into the role of α-toxin in host-pathogen interactions.

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Some experiments with the FACS Aria II were performed in the Institute for Genome Research, The University of Tokushima. We thank the members of the Institute for their assistance.

Conflict of Interest

The authors declare no conflict of interest.

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