Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux

Abstract

Staphylococcus aureus is a pathogen that is the causative agent of several human and veterinary infections and plays a critical role in the clinical and subclinical mastitis of cattle. Autophagy is a conserved pathogen defence mechanism in eukaryotes. Studies have reported that S aureus can subvert autophagy and survive in cells. Staphylococcus aureus survival in cells is an important cause of chronic persistent mastitis infection. However, it is unclear whether S aureus can escape autophagy in innate immune cells. In this study, initiation of autophagy due to the presence of S aureus was detected in bovine macrophages. We observed autophagic vacuoles increased after S aureus infection of bovine macrophages by transmission electron microscopy (TEM). It was also found that S aureus-infected bovine macrophages increased the expression of LC3 at different times(0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours). Data also showed the accumulation of p62 induced by S aureus infection. Application of autophagy regulatory agents showed that the degradation of p62 was blocked in S aureus induced bovine macrophages. In addition, we also found that the accumulation of autophagosomes promotes S aureus to survive in macrophage cells. In conclusion, this study indicates that autophagy occurs in S aureus-infected bovine macrophages but is blocked at a later stage of autophagy. The accumulation of autophagosomes facilitates the survival of S aureus in bovine macrophages. These findings provide new insights into the interaction of S aureus with autophagy in bovine macrophages.

Keywords: Staphylococcus aureus; autophagy; bovine macrophage.

Figure 1

Autophagy in bovine macrophage cells was triggered by Staphylococcus aureus. Bovine macrophages were infected with S aureus for 2 h (MOI = 1:1). Samples were prepared as described in Materials and Methods and examined under transmission electron microscopy (TEM). Scale bar = 2 μm or 600 nm

Figure 2

Enhanced expression of LC3 in Staphylococcus aureus‐stimulated bovine macrophage cells. A, Bovine macrophages were infected with S aureus (MOI = 1:1). Cells were harvested at the indicated times and subjected to Western blot analysis using antibodies. After S aureus infects bovine macrophages, the expression of LC3‐II protein is increased. B, The average number of LC3 puncta per cell is displayed. C, Immunofluorescence assay was used to further observe changes in LC3 expression in S aureus‐infected bovine macrophages. Bovine macrophages were grown on coverslips in 24‐well plates and infected at a MOI = 1:1. Cells were fixed at different time points after infection and incubated with anti‐LC3 antibody to visualize LC3 particles. Scale bar = 10 μm. The results are representative of three independent experiments. Statistical significance was determined by Student's t test (*P < .05; **P < .01)

Figure 3

Degradation of autophagy substrate p62 and autophagosome‐lysosomal fusion were arrested after Staphylococcus aureus infection of bovine macrophages. A, Bovine macrophages were infected with S aureus (MOI = 1:1) at the indicated time points. The expression level of p62 was measured by immunoblotting. Relative p62 expression level was calculated, after being normalized to β‐actin, using ImageJ. B, Cells grown on glass coverslips were transfected with GFP‐RFP‐LC3 plasmid for 24 h and then treated with S aureus MOI = 1:1 for 2 h. Autophagy flow was evaluated by confocal microscopy. The results are representative of three independent experiments. Statistical significance was determined by Student's t test (*P < .05; **P < .01)

Figure 4

Degradation of p62 was blocked after Staphylococcus aureus infection in bovine macrophage cells. A, Effect of early‐autophagy inhibitor (3‐MA) on LC3 and p62. Bovine macrophages were infected with S aureus (MOI = 1:1) for 2 h in the presence or absence of 3‐MA (2.5 mM). Cells were harvested and subjected to Western blot analysis. Relative protein expression levels of LC3 and p62 were calculated after being normalized to β‐actin using ImageJ. B, Effect of late‐autophagy inhibitor (CQ) on LC3 and p62. Bovine macrophages were infected with S aureus (MOI = 1:1) for 2 h in the presence or absence of CQ (40 μM). Cells were harvested and subjected to Western blot analysis. Relative protein expression levels of LC3 and p62 were calculated after being normalized to β‐actin using ImageJ. C, Effect of autophagy promoter (Rap) on LC3 and p62. Bovine macrophages were infected with S aureus (MOI = 1:1) for 2 h in the presence or absence of Rap (2.5 μM). Cells were harvested and subjected to Western blot analysis. Relative protein expression level of LC3 and p62 was calculated after being normalized to β‐actin using ImageJ. D, Immunofluorescence analysis of intracellular LC3 protein levels. Bovine macrophages were infected with S aureus (MOI = 1:1) in the presence or absence of an autophagy regulatory regulator for 2 h and stained with mouse anti‐LC3 monoclonal antibody and FITC‐labelled fluorescent secondary antibody. E, The average number of LC3 puncta per cell is displayed. Scale bar = 10 μm. The results are representative of three independent experiments. Statistical significance was determined by Student's t test. Compared with the control group (*P < .05; **P < .01), compared with the modulator of the autophagy group (#P < .05; ##P < .01)

Figure 5

Blocking autophagic flux facilitates the survival of Staphylococcus aureus within cells. After pretreatment of bovine macrophages with autophagy regulatory agents (2.5 mM 3‐MA, 40 μM CQ, 2.5 μM Rap) for 1 h, bovine macrophages were infected with S aureus (MOI = 1:1) for 2 h. The cells were cultured with 100 μg/mL gentamicin sulphate for 1 h to kill the extracellular bacteria. Cell lysates were collected at 0, 1, 2 and 3 h, and serial dilutions were plated on LB solid medium to determine the amount of CFU/mL. The data represent three different experiments. Data are expressed as mean ± SD from three independent experiments. Compared to the 0 h (*P < .05, **P < .01)