Limits...
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.

Show MeSH

Related in: MedlinePlus

Free cholesterol is enriched in A. phagocytophilum inclusions.A. phagocytophilum–infected HL-60 cells (A) and A. phagocytophilum inclusions released from host cells (B) were fixed at 2 d p.i., stained with mouse anti-A. phagocytophilum (green) or normal mouse IgG and filipin (blue), and analyzed by fluorescence microscopy. The experiment shown is representative of at least three independent experiments. Bar, 5 µm. Ap, A. phagocytophilum.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654415&req=5

ppat-1000329-g002: Free cholesterol is enriched in A. phagocytophilum inclusions.A. phagocytophilum–infected HL-60 cells (A) and A. phagocytophilum inclusions released from host cells (B) were fixed at 2 d p.i., stained with mouse anti-A. phagocytophilum (green) or normal mouse IgG and filipin (blue), and analyzed by fluorescence microscopy. The experiment shown is representative of at least three independent experiments. Bar, 5 µm. Ap, A. phagocytophilum.

Mentions: Most of the free (unesterified) cholesterol in eukaryotic cells is located in the plasma membrane [16]. Over-accumulation of free cholesterol in cells can be toxic due to the potential formation of solid crystals [17]. To determine the intracellular distribution of the observed increased cholesterol in A. phagocytophilum–infected HL-60 cells, we used a polyene antibiotic, filipin, which binds specifically to free cholesterol [18]. A specific antibody against A. phagocytophilum was used to localize bacteria by double immunofluorescence microscopy. First, the microscopy analysis clearly showed the overall filipin signal was much stronger in A. phagocytophilum-infected HL-60 cells than that in uninfected HL-60, which supports the data shown in Figure 1 and further suggests the increased total cellular cholesterol might be free cholesterol, but not esterified cholesterol (Figure 2A). Second, most of the filipin signal was confined in A. phagocytophilum–containing vacuoles (“inclusions”) (Figure 2A). Uninfected host cells showed weak filipin signal, which was mostly localized to the plasma membrane and some unknown compartments (assumed to be recycling endocytic compartments [19]). Notably, A. phagocytophilum inclusions outside of host cells also clearly displayed strong filipin signals (Figure 2B), suggesting that the inclusion has intrinsic ability to retain the cholesterol. Recently, it was shown that Chlamydia release from the infected host cells occurs by two mechanisms: lysis and extrusion [20]. How the A. phagocytophilum inclusion became extracellular remains to be studied. Taken together, these results indicate that A. phagocytophilum infection alters host intracellular cholesterol homeostasis and distribution and that free cholesterol is enriched in A. phagocytophilum inclusions.


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

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

Free cholesterol is enriched in A. phagocytophilum inclusions.A. phagocytophilum–infected HL-60 cells (A) and A. phagocytophilum inclusions released from host cells (B) were fixed at 2 d p.i., stained with mouse anti-A. phagocytophilum (green) or normal mouse IgG and filipin (blue), and analyzed by fluorescence microscopy. The experiment shown is representative of at least three independent experiments. Bar, 5 µm. Ap, A. phagocytophilum.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654415&req=5

ppat-1000329-g002: Free cholesterol is enriched in A. phagocytophilum inclusions.A. phagocytophilum–infected HL-60 cells (A) and A. phagocytophilum inclusions released from host cells (B) were fixed at 2 d p.i., stained with mouse anti-A. phagocytophilum (green) or normal mouse IgG and filipin (blue), and analyzed by fluorescence microscopy. The experiment shown is representative of at least three independent experiments. Bar, 5 µm. Ap, A. phagocytophilum.
Mentions: Most of the free (unesterified) cholesterol in eukaryotic cells is located in the plasma membrane [16]. Over-accumulation of free cholesterol in cells can be toxic due to the potential formation of solid crystals [17]. To determine the intracellular distribution of the observed increased cholesterol in A. phagocytophilum–infected HL-60 cells, we used a polyene antibiotic, filipin, which binds specifically to free cholesterol [18]. A specific antibody against A. phagocytophilum was used to localize bacteria by double immunofluorescence microscopy. First, the microscopy analysis clearly showed the overall filipin signal was much stronger in A. phagocytophilum-infected HL-60 cells than that in uninfected HL-60, which supports the data shown in Figure 1 and further suggests the increased total cellular cholesterol might be free cholesterol, but not esterified cholesterol (Figure 2A). Second, most of the filipin signal was confined in A. phagocytophilum–containing vacuoles (“inclusions”) (Figure 2A). Uninfected host cells showed weak filipin signal, which was mostly localized to the plasma membrane and some unknown compartments (assumed to be recycling endocytic compartments [19]). Notably, A. phagocytophilum inclusions outside of host cells also clearly displayed strong filipin signals (Figure 2B), suggesting that the inclusion has intrinsic ability to retain the cholesterol. Recently, it was shown that Chlamydia release from the infected host cells occurs by two mechanisms: lysis and extrusion [20]. How the A. phagocytophilum inclusion became extracellular remains to be studied. Taken together, these results indicate that A. phagocytophilum infection alters host intracellular cholesterol homeostasis and distribution and that free cholesterol is enriched in A. phagocytophilum inclusions.

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