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Cholesterol-dependent anaplasma phagocytophilum exploits the low-density lipoprotein uptake pathway.

Xiong Q, Lin M, Rikihisa Y - PLoS Pathog. (2009)

Bottom Line: We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes.Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum.These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3' UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal-regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

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LDLR mRNA is stabilized in A. phagocytophilum–infected host cells.Actinomycin D (5 µg/ml) was added to uninfected and A. phagocytophilum–infected HL-60 cells for different periods. Total RNA was isolated, and LDLR mRNA (A) and HMG-CoA reductase mRNA (B) were analyzed by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. The decay rates of LDLR mRNA (A) are significantly different (p<0.01), as tested by two-way ANOVA. (C) Chimeric pLuc/LDLR 3′UTR-2 was transfected into RF/6A cells using FuGene HD reagent, and host cell–free A. phagocytophilum was added to transfected RF/6A cells at 1 day post transfection. Luc mRNA was analyzed on day 2 p.i. by quantitative RT-PCR. Cell samples were normalized by the antibiotic gene zeocin mRNA level. Data are representative of two independent experiments. Ap, A. phagocytophilum. ND, not detectable. **, p<0.01 (unpaired two-tailed t-test).
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ppat-1000329-g007: LDLR mRNA is stabilized in A. phagocytophilum–infected host cells.Actinomycin D (5 µg/ml) was added to uninfected and A. phagocytophilum–infected HL-60 cells for different periods. Total RNA was isolated, and LDLR mRNA (A) and HMG-CoA reductase mRNA (B) were analyzed by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. The decay rates of LDLR mRNA (A) are significantly different (p<0.01), as tested by two-way ANOVA. (C) Chimeric pLuc/LDLR 3′UTR-2 was transfected into RF/6A cells using FuGene HD reagent, and host cell–free A. phagocytophilum was added to transfected RF/6A cells at 1 day post transfection. Luc mRNA was analyzed on day 2 p.i. by quantitative RT-PCR. Cell samples were normalized by the antibiotic gene zeocin mRNA level. Data are representative of two independent experiments. Ap, A. phagocytophilum. ND, not detectable. **, p<0.01 (unpaired two-tailed t-test).

Mentions: LDLR is regulated not only at the transcriptional level but also at the posttranscriptional level via modulation of LDLR mRNA stability [34]. Therefore, we investigated LDLR mRNA stability in A. phagocytophilum–infected HL-60 cells after treatment with actinomycin D, a eukaryotic DNA-dependent RNA polymerase inhibitor. The half-life of LDLR mRNA in infected HL-60 cells was increased by almost 2-fold as compared to uninfected cells (Figure 7A). In contrast, the stability of HMG-CoA reductase mRNA did not change noticeably during A. phagocytophilum infection (Figure 7B). Human LDLR mRNA contains a 2.5-kb 3′UTR [35]. The 3′UTR of LDLR mRNA can be stabilized by phorbol-12-myristate-13-acetate (PMA) and a Chinese herbal compound, berberine in the human hepatic cell line HepG2 [36],[37]. Three AU-rich elements (AREs) are located in the 5′ proximal region of the 3′UTR, which have been shown to be responsible for the stabilization of LDLR mRNA by berberine, but not by PMA [37]. To investigate whether the LDLR 3′UTR containing three AREs is involved in A. phagocytophilum–induced LDLR stabilization, we transfected the luciferase fusion plasmid pLuc/LDLR 3′UTR-2, containing three AREs of LDLR 3′UTR (nt 2,677–3,582) [37], into RF/6A cells and measured the luciferase mRNA levels in A. phagocytophilum–infected and control RF/6A cells. As shown in Figure 7C, luciferase mRNA levels normalized to the antibiotic zeocin resistance gene (plasmid copy number) were significantly increased in A. phagocytophilum–infected cells. Data normalized by G3PDH (host cell number) showed a similar pattern (data not shown). These results indicate that the LDLR 3′UTR containing three AREs may be involved in enhancing LDLR mRNA stability in A. phagocytophilum–infected host cells.


Cholesterol-dependent anaplasma phagocytophilum exploits the low-density lipoprotein uptake pathway.

Xiong Q, Lin M, Rikihisa Y - PLoS Pathog. (2009)

LDLR mRNA is stabilized in A. phagocytophilum–infected host cells.Actinomycin D (5 µg/ml) was added to uninfected and A. phagocytophilum–infected HL-60 cells for different periods. Total RNA was isolated, and LDLR mRNA (A) and HMG-CoA reductase mRNA (B) were analyzed by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. The decay rates of LDLR mRNA (A) are significantly different (p<0.01), as tested by two-way ANOVA. (C) Chimeric pLuc/LDLR 3′UTR-2 was transfected into RF/6A cells using FuGene HD reagent, and host cell–free A. phagocytophilum was added to transfected RF/6A cells at 1 day post transfection. Luc mRNA was analyzed on day 2 p.i. by quantitative RT-PCR. Cell samples were normalized by the antibiotic gene zeocin mRNA level. Data are representative of two independent experiments. Ap, A. phagocytophilum. ND, not detectable. **, p<0.01 (unpaired two-tailed t-test).
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ppat-1000329-g007: LDLR mRNA is stabilized in A. phagocytophilum–infected host cells.Actinomycin D (5 µg/ml) was added to uninfected and A. phagocytophilum–infected HL-60 cells for different periods. Total RNA was isolated, and LDLR mRNA (A) and HMG-CoA reductase mRNA (B) were analyzed by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. The decay rates of LDLR mRNA (A) are significantly different (p<0.01), as tested by two-way ANOVA. (C) Chimeric pLuc/LDLR 3′UTR-2 was transfected into RF/6A cells using FuGene HD reagent, and host cell–free A. phagocytophilum was added to transfected RF/6A cells at 1 day post transfection. Luc mRNA was analyzed on day 2 p.i. by quantitative RT-PCR. Cell samples were normalized by the antibiotic gene zeocin mRNA level. Data are representative of two independent experiments. Ap, A. phagocytophilum. ND, not detectable. **, p<0.01 (unpaired two-tailed t-test).
Mentions: LDLR is regulated not only at the transcriptional level but also at the posttranscriptional level via modulation of LDLR mRNA stability [34]. Therefore, we investigated LDLR mRNA stability in A. phagocytophilum–infected HL-60 cells after treatment with actinomycin D, a eukaryotic DNA-dependent RNA polymerase inhibitor. The half-life of LDLR mRNA in infected HL-60 cells was increased by almost 2-fold as compared to uninfected cells (Figure 7A). In contrast, the stability of HMG-CoA reductase mRNA did not change noticeably during A. phagocytophilum infection (Figure 7B). Human LDLR mRNA contains a 2.5-kb 3′UTR [35]. The 3′UTR of LDLR mRNA can be stabilized by phorbol-12-myristate-13-acetate (PMA) and a Chinese herbal compound, berberine in the human hepatic cell line HepG2 [36],[37]. Three AU-rich elements (AREs) are located in the 5′ proximal region of the 3′UTR, which have been shown to be responsible for the stabilization of LDLR mRNA by berberine, but not by PMA [37]. To investigate whether the LDLR 3′UTR containing three AREs is involved in A. phagocytophilum–induced LDLR stabilization, we transfected the luciferase fusion plasmid pLuc/LDLR 3′UTR-2, containing three AREs of LDLR 3′UTR (nt 2,677–3,582) [37], into RF/6A cells and measured the luciferase mRNA levels in A. phagocytophilum–infected and control RF/6A cells. As shown in Figure 7C, luciferase mRNA levels normalized to the antibiotic zeocin resistance gene (plasmid copy number) were significantly increased in A. phagocytophilum–infected cells. Data normalized by G3PDH (host cell number) showed a similar pattern (data not shown). These results indicate that the LDLR 3′UTR containing three AREs may be involved in enhancing LDLR mRNA stability in A. phagocytophilum–infected host cells.

Bottom Line: We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes.Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum.These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3' UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal-regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

Show MeSH
Related in: MedlinePlus