An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency

Abstract

Introduction: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency.

Methods: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei.

Results: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient.

Conclusion: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.

Keywords: Burkholderia mallei; Burkholderia pseudomallei; NBR1; autophagy; intracellular survival; p62.

Figure 1

Autophagy reduced intracellular p62/NBR1 and survival of B. thailandensis in infected cells. J774A.1 cells (3 × 10⁶cells/mL) were first treated with Rapamycin (50 and 100 μM) for 30 min and then infected with B. thailandensis (20 MOI) for 2 h. After infection the cells were washed, pelleted, lysed, and labelled as 0 h. The whole cell lysates were measured for protein concentration and 30 μg protein were used to measure p62 and NBR1 as described in experimental procedures. Infected cells that were incubated for overnight, pelleted, lysed, and labeled as 20 h. Cell lysates 30 ug were used for p62 and NBR1 assay and a part of it plated at different dilutions for bacterial colony count (Table 1). Data presented from two independent experiments. Two‐way ANOVA test was performed at each time point for analyzing the effect of rapamycin and B. thailandensis. For p62, the P‐value at 0 h of rapamycin treatment and effect of concentration are <0.0001 and 0.0091 and at 20 h of rapamycin treatment and effect of concentration are <0.0001 and 0.1057. For NBR1, the P‐value at 0 h of rapamycin treatment and effect of concentration are <0.0029 and 0.5224 at 20 h of rapamycin treatment and effect of concentration are <0.0085 and 0.1977. Asterisk indicates significant difference between the pairwise comparisons (P ≤ 0.0091)

Figure 2

Autophagy reduced intracellular p62/NBR1 and survival of B. pseudomallei in infected HeLa cells. HeLa cells (4 × 10⁶cell/mL) were induced with Rapamycin (100 μM) and infected with BpK96243 (10 MOI) for 20 h. Cells were pelleted and lysed. The whole cell lysates (30 μg) were used to measure p62 and NBR1 at multiple dilutions as described in experimental procedures. Data presented as % change of control of p62/NBR1 from the dose response curve of the combined data (Supplemental Figure S1) from two independent experiments. For statistical significance each treatment and repetition of the experiment was analyzed by the equation as described in the method section. Confidence intervals and standard errors are taken from asymptotic normal methods. For p62, pairwise comparisons shows the following P‐values; BpK96243 versus DMSO (0.0002), BpK96243 versus Rapamycin (<0.0001), BpK96243 versus Rapamycin+ BpK96243 (0.0004), DMSO versus Rapamycin (0.5809), DMSO versus Rapamycin+ BpK96243 (<0.0001), Rapamycin versus Rapamycin+ BpK96243 (<0.0001). For NBR1, pairwise comparisons shows the following P‐values; BpK96243 versus DMSO (<0.0001), BpK96243 versus Rapamycin (<0.0001), BpK96243 versus Rapamycin+ BpK96243 (0.0017), DMSO versus Rapamycin (<0.0001), DMSO versus Rapamycin+ BpK96243 (<0.0001), Rapamycin versus Rapamycin+ BpK96243 (<0.0001). Asterisk indicates significant difference between the pairwise comparisons (P ≤ 0.0017)

Figure 3

Autophagy and bacterial persistence in a mouse glanders model. Aerosol exposure of BALB/c mice at varying concentrations of B. mallei FMH were performed as described in experimental procedures. Bacterial exposure up to 21 days considered as acute phase and post 21‐60 days as considered as chronic phase. Spleens were isolated and measured size of the spleens at 27d (blue [A] and dark red [B]) and at 47d (green [C] and yellow [D]). Spleen from individual mouse was homogenized for purifying spleen cells and lysed for preparing lysates to determine bacterial count after plating and to measure p62 levels as described in section 2. (A–D) Spleens from aerosol exposed mice with bacterial count indicated below, and (E) spleen from normal BALB/c mice. (F) p62 levels in spleen cell lysates of B. mallei exposed to individual mouse at two different time points as indicated above

Figure 4

Autophagy and bacterial persistence in a mouse melioidosis model. Aerosol exposure of BALB/c mice at varying concentrations of B. pseudomallei MSHR305 were performed as described in section 2. Spleens were isolated at day 35 and measured size of the spleens. Spleens from individual mice were homogenized to prepare spleen extracts and cfu determined. p62 levels were measured as described in section 2. (A–D). Spleens of B. pseudomallei with pyogranulomas and persistence of bacteria in chronically infected mice are indicated below; (E) p62 levels in spleen cell extracts from individual mice as shown in A–D (same color)

Figure 5

B. pseudomallei strains Bp406e, Bp1106A, Bp1034a exposure increased p62 in spleens of BALB/c mice. B. pseudomallei strain (a) BP406e; (b) Bp1034a and (c) BP1106a were used to expose/infect BALB/c mice as indicated below. Spleens from individual mice were homogenized to prepare lysates for measureing p62 levels or plated for colony count as described earlier. Data presented as p62 levels in spleen cell lysates from individual mice at different time points. (A) BP406e aerosol infection and p62 (pg) at different concentrations 1.25 to 20 μg of total protein at days (34 to 62) post infection (mice #1B at 34d and at 62d; mice #2B at 34d and 62d); (B) Bp 1034a ip infection and p62 (pg) at 20 and 40 μg of total protein at 3 or 14 days post infection (mice #1B, #2B,#3B at 3d and #1B, #2B at 14d); (C) BP1106a ip infection and p62 (pg) at 20 and 40 μg of total protein at 3 or 14 days post infection (mice #4B, at 3d and 14 and mice #5B, at 3d and14d)

Figure 6

p62 and LC3‐I increased in spleen cells of mice infected with B. mallei or B. pseudomallei. Uninfected, B. mallei FMH, B. pseudomallei strain Bp406, Bp1106, and Bp22 (formerly, Bp KHW) infected spleen cell lysates of BALB/c and C57BL/6 mice were used for detecting p62 and LC3. Samples containing 10 μg of total proteins were separated by SDS‐PAGE gel electrophoresis and transferred to nitrocellulose membranes and then probed with anti‐LC3 or anti‐p62 polyclonal antibody followed by horseradish peroxide‐conjugated secondary antibody (goat anti‐rabbit). A, p62 50 kDa band detected after probing with anti‐p62 antibody in BALB/c mice. B, Immunoblot with anti‐LC3 antibody, the major 18 kDa LC3‐I and less 16 kDa kDa LC3‐II form protein was detected in BALB/c mice. C, Expression of LC3 in B. pseudomallei 22 infected C57BL/6 mice

Figure 7

p62 upregulated in spleen and liver of B. mallei infected mouse. In situ hybridization was performed using synthesized 20 zz probes of mouse SQSTM1 /p62 (Cat# 444221) as described in section 2. Briefly, after deparaffinization and peroxidase blocking, sections were covered with ISH probes and hybridized as described in section 2. (A,B) In comparison with the low level of p62 mRNA (red, A) detected in the uninfected control spleen, high levels of SQSTM1/p62 (red, B) mRNA was detected in the edge region of a pyogranuloma of B. mallei infected spleens. (C,D) Low level of NBR1 (green) was detected in uninfected control liver (C), whereas high levels of NBR1 (green) was detected in the focus of histiocytic infiltrate of liver from a B. mallei infected mouse (D). (E,F) NBR1 was undetectable in the uninfected control spleen (E), but high levels of NBR1 (green) was detected in the spleen of a B. mallei infected mouse (F). (G,H), B. mallei was detected in the tissue sections of spleen after staining with Rabbit anti‐B mallei polyclonal antibody followed by incubation with secondary goat anti‐rabbit Alex Fluor 561 antibodies. G, B. mallei was not detected in an uninfected spleen, (H) high levels of B. mallei (red) was detected in the spleen of a B. mallei infected mouse

Figure 8

Stimulation of autophagy reduced intracellular p62/NBR1 in B. pseudomallei infected human PBMC. PBMCs (3 × 10⁵ cells) were were first treated with Rapamycin (100 μM) for 30 min and then infected with B. pseudomallei BpK96243 (10 MOI) for 20 h. Cells were pelleted and lysed. The whole cell lysates at different concentrations (12.5, 25, and 100 μg of total protein) were used for measuring p62 and NBR1 as described in section 2. Data presented as average from two independent experiments p62/NBR1 present in the total protein as indicated (12.5, 25, and 100 μg of total protein). For statistical significance each treatment and repetition of the experiment was analyzed by the equation relating the target protein concentration to the total protein as Target Protein = α *Total Proteinβ as described in section 2. Two‐way ANOVA test was performed at each time point for analyzing the effect of rapamycin and B. pseudomallei BpK96243. For p62, and NBR1 the P‐value with pairwise comparisons and asterisk indicates significant difference between these pairwise comparisons (P ≤ 0.0084)