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Control of parasitophorous vacuole expansion by LYST/Beige restricts the intracellular growth of Leishmania amazonensis.

Wilson J, Huynh C, Kennedy KA, Ward DM, Kaplan J, Aderem A, Andrews NW - PLoS Pathog. (2008)

Bottom Line: In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth.Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs.Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
The intracellular protozoan Leishmania replicates in parasitophorous vacuoles (PV) that share many features with late endosomes/lysosomes. L. amazonensis PVs expand markedly during infections, but the impact of PV size on parasite intracellular survival is still unknown. Here we show that host cells infected with L. amazonensis upregulate transcription of LYST/Beige, which was previously shown to regulate lysosome size. Mutations in LYST/Beige caused further PV expansion and enhanced L. amazonensis replication. In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth. Treatment of LYST/Beige over-expressing cells with vacuolin-1 reversed this phenotype, expanding PVs and promoting parasite growth. The opposite was seen with E-64d, which reduced PV size in LYST-Beige mutant cells and inhibited L. amazonensis replication. Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs. Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival.

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Parasitophorous vacuole size modulates the intracellular growth of Leishmania.(A) Phase contrast images showing that pretreatment with E64d reduces the large vacuoles observed in bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bar = 5 µm. (B) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with E64d. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test). (C) PV reduction with E64d inhibits the intracellular growth of L. amazonensis in bgJ/bgJ fibroblasts. After E64d pretreatment the fibroblasts were exposed to amastigotes for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): * P<0.05 (Student t test). (D) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with 1 µM vacuolin-1. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from non-treated controls (NT): ** P<0.01 (Student t test). (E) Phase contrast images showing that pretreatment with 1 µM vacuolin-1 enlarges the small vacuoles observed in L. amazonensis-infected YAC-bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bars = 10 µm. (F) After vacuolin-1 pretreatment the fibroblasts were infected for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test).
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ppat-1000179-g004: Parasitophorous vacuole size modulates the intracellular growth of Leishmania.(A) Phase contrast images showing that pretreatment with E64d reduces the large vacuoles observed in bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bar = 5 µm. (B) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with E64d. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test). (C) PV reduction with E64d inhibits the intracellular growth of L. amazonensis in bgJ/bgJ fibroblasts. After E64d pretreatment the fibroblasts were exposed to amastigotes for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): * P<0.05 (Student t test). (D) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with 1 µM vacuolin-1. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from non-treated controls (NT): ** P<0.01 (Student t test). (E) Phase contrast images showing that pretreatment with 1 µM vacuolin-1 enlarges the small vacuoles observed in L. amazonensis-infected YAC-bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bars = 10 µm. (F) After vacuolin-1 pretreatment the fibroblasts were infected for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test).

Mentions: The data discussed above suggested that PV expansion facilitates the intracellular survival and growth of Leishmania. To further investigate this possibility, we examined the effects of reducing PV size in bgJ/bgJ fibroblasts and, conversely, increasing the size of PVs in YAC-bgJ/bgJ fibroblasts. Previous studies showed that treatment of bgJ/bgJ cells with the thiol proteinase inhibitor E-64d reduces the size of the enlarged lysosomes normally observed in these cells [21],[22]. A reduction in PV size after pre-treatment with E-64d was evident in bgJ/bgJ fibroblasts (Figure 4A and 4B), and this coincided with a reduction of ∼3 fold in the number of intracellular amastigotes detected after 24 h, when compared to untreated bgJ/bgJ cells (Figure 4C, bgJ/bgJ). In contrast, there was no apparent effect of E-64d on the smaller PVs within the YAC-bgJ/bgJ fibroblasts (Figure 4B, YAC-bgJ/bgJ), and no effect on parasite numbers in these cells (Figure 4C, YAC-bgJ/bgJ).


Control of parasitophorous vacuole expansion by LYST/Beige restricts the intracellular growth of Leishmania amazonensis.

Wilson J, Huynh C, Kennedy KA, Ward DM, Kaplan J, Aderem A, Andrews NW - PLoS Pathog. (2008)

Parasitophorous vacuole size modulates the intracellular growth of Leishmania.(A) Phase contrast images showing that pretreatment with E64d reduces the large vacuoles observed in bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bar = 5 µm. (B) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with E64d. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test). (C) PV reduction with E64d inhibits the intracellular growth of L. amazonensis in bgJ/bgJ fibroblasts. After E64d pretreatment the fibroblasts were exposed to amastigotes for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): * P<0.05 (Student t test). (D) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with 1 µM vacuolin-1. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from non-treated controls (NT): ** P<0.01 (Student t test). (E) Phase contrast images showing that pretreatment with 1 µM vacuolin-1 enlarges the small vacuoles observed in L. amazonensis-infected YAC-bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bars = 10 µm. (F) After vacuolin-1 pretreatment the fibroblasts were infected for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2562527&req=5

ppat-1000179-g004: Parasitophorous vacuole size modulates the intracellular growth of Leishmania.(A) Phase contrast images showing that pretreatment with E64d reduces the large vacuoles observed in bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bar = 5 µm. (B) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with E64d. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test). (C) PV reduction with E64d inhibits the intracellular growth of L. amazonensis in bgJ/bgJ fibroblasts. After E64d pretreatment the fibroblasts were exposed to amastigotes for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): * P<0.05 (Student t test). (D) Analysis of PV size in infected bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with 1 µM vacuolin-1. Fibroblasts were exposed to L. amazonensis amastigotes for 30 min, washed, and further incubated for 24 h followed by microscopic determination of PV size. The values above the symbols represent the mean of 50 independent measurements. Asterisks indicate significant differences from non-treated controls (NT): ** P<0.01 (Student t test). (E) Phase contrast images showing that pretreatment with 1 µM vacuolin-1 enlarges the small vacuoles observed in L. amazonensis-infected YAC-bgJ/bgJ fibroblasts. Arrows point to individual PVs. Bars = 10 µm. (F) After vacuolin-1 pretreatment the fibroblasts were infected for 1 h, washed, and further incubated for 24 h, followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the non-treated controls (NT): ** P<0.01 (Student t test).
Mentions: The data discussed above suggested that PV expansion facilitates the intracellular survival and growth of Leishmania. To further investigate this possibility, we examined the effects of reducing PV size in bgJ/bgJ fibroblasts and, conversely, increasing the size of PVs in YAC-bgJ/bgJ fibroblasts. Previous studies showed that treatment of bgJ/bgJ cells with the thiol proteinase inhibitor E-64d reduces the size of the enlarged lysosomes normally observed in these cells [21],[22]. A reduction in PV size after pre-treatment with E-64d was evident in bgJ/bgJ fibroblasts (Figure 4A and 4B), and this coincided with a reduction of ∼3 fold in the number of intracellular amastigotes detected after 24 h, when compared to untreated bgJ/bgJ cells (Figure 4C, bgJ/bgJ). In contrast, there was no apparent effect of E-64d on the smaller PVs within the YAC-bgJ/bgJ fibroblasts (Figure 4B, YAC-bgJ/bgJ), and no effect on parasite numbers in these cells (Figure 4C, YAC-bgJ/bgJ).

Bottom Line: In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth.Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs.Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
The intracellular protozoan Leishmania replicates in parasitophorous vacuoles (PV) that share many features with late endosomes/lysosomes. L. amazonensis PVs expand markedly during infections, but the impact of PV size on parasite intracellular survival is still unknown. Here we show that host cells infected with L. amazonensis upregulate transcription of LYST/Beige, which was previously shown to regulate lysosome size. Mutations in LYST/Beige caused further PV expansion and enhanced L. amazonensis replication. In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth. Treatment of LYST/Beige over-expressing cells with vacuolin-1 reversed this phenotype, expanding PVs and promoting parasite growth. The opposite was seen with E-64d, which reduced PV size in LYST-Beige mutant cells and inhibited L. amazonensis replication. Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs. Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival.

Show MeSH
Related in: MedlinePlus