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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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iNOS inhibition rescues Leishmania replication within small parasitophorous vacuoles.(A) Treatment with iNOS inhibitors does not affect L. amazonensis growth in the enlarged PVs of bgJ/bgJ fibroblasts, but rescues growth in the small PVs of YAC-bgJ/bgJ fibroblasts. After pretreatment with 50 µM L-NMMA or 50 µM L-NAME, the fibroblasts were infected for 1 h, washed and 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 non-treated controls (NT): * P<0.05 (Student t test). (B) iNOS inhibitors do not alter PV size in bgJ/bgJ or YAC-bgJ/bgJ fibroblasts. Cells 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 represent the mean of 50 independent measurements. (C) Phase contrast images of bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with L-NMMA or L-NAME, exposed to L. amazonensis amastigotes for 1 h and fixed after 24 h. Arrows point to individual PVs. Bars = 5 µm.
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ppat-1000179-g005: iNOS inhibition rescues Leishmania replication within small parasitophorous vacuoles.(A) Treatment with iNOS inhibitors does not affect L. amazonensis growth in the enlarged PVs of bgJ/bgJ fibroblasts, but rescues growth in the small PVs of YAC-bgJ/bgJ fibroblasts. After pretreatment with 50 µM L-NMMA or 50 µM L-NAME, the fibroblasts were infected for 1 h, washed and 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 non-treated controls (NT): * P<0.05 (Student t test). (B) iNOS inhibitors do not alter PV size in bgJ/bgJ or YAC-bgJ/bgJ fibroblasts. Cells 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 represent the mean of 50 independent measurements. (C) Phase contrast images of bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with L-NMMA or L-NAME, exposed to L. amazonensis amastigotes for 1 h and fixed after 24 h. Arrows point to individual PVs. Bars = 5 µm.

Mentions: Since L. amazonensis is capable of productively infecting both macrophages and fibroblasts in culture (Figure 2), and PV enlargement enhances the early survival and replication of L. amazonensis in these two cell types (Figures 3 and 4), it is conceivable that PV enlargement protects Leishmania from a microbicidal mechanism common to both macrophages and fibroblasts. Although NO production in response to IFN-gamma and TNF-alpha is much more robust in macrophages, fibroblasts are also capable of mounting this response [1]. To investigate whether NO production plays a role in the enhanced survival of Leishmania in fibroblasts, we quantified intracellular amastigotes in L. amazonensis-infected bgJ/bgJ and YAC-bgJ/bgJ fibroblasts pre-treated with the iNOS inhibitors L-NMMA and L-NAME. These inhibitors were previously shown to promote the intracellular growth of Leishmania in macrophages by blocking NO production [25],[28]. Quantification of the number of intracellular amastigotes after 24 h showed that these inhibitors did not alter the parasite population size in bgJ/bgJ fibroblasts, which exhibit large PVs (Figure 5A). However, amastigote survival and replication was significantly enhanced in YAC-bgJ/bgJ fibroblasts pre-treated with the iNOS inhibitors (Figure 5B), consistent with the observation of several parasites inside each of the small PVs found in these cells (Figure 5D). Importantly, the size of the small PVs induced by L. amazonensis in the YAC-bgJ/bgJ fibroblasts overexpressing LYST/Beige remained unchanged after treatment with the iNOS inhibitors (Figure 5C), suggesting that inhibition of NO production can promote L. amazonensis survival/growth in the absence of PV expansion. Future studies are required to investigate the possibility that LYST expression might also impact the trafficking of iNOS to L. amazonensis-containing PVs.


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)

iNOS inhibition rescues Leishmania replication within small parasitophorous vacuoles.(A) Treatment with iNOS inhibitors does not affect L. amazonensis growth in the enlarged PVs of bgJ/bgJ fibroblasts, but rescues growth in the small PVs of YAC-bgJ/bgJ fibroblasts. After pretreatment with 50 µM L-NMMA or 50 µM L-NAME, the fibroblasts were infected for 1 h, washed and 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 non-treated controls (NT): * P<0.05 (Student t test). (B) iNOS inhibitors do not alter PV size in bgJ/bgJ or YAC-bgJ/bgJ fibroblasts. Cells 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 represent the mean of 50 independent measurements. (C) Phase contrast images of bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with L-NMMA or L-NAME, exposed to L. amazonensis amastigotes for 1 h and fixed after 24 h. Arrows point to individual PVs. Bars = 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2562527&req=5

ppat-1000179-g005: iNOS inhibition rescues Leishmania replication within small parasitophorous vacuoles.(A) Treatment with iNOS inhibitors does not affect L. amazonensis growth in the enlarged PVs of bgJ/bgJ fibroblasts, but rescues growth in the small PVs of YAC-bgJ/bgJ fibroblasts. After pretreatment with 50 µM L-NMMA or 50 µM L-NAME, the fibroblasts were infected for 1 h, washed and 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 non-treated controls (NT): * P<0.05 (Student t test). (B) iNOS inhibitors do not alter PV size in bgJ/bgJ or YAC-bgJ/bgJ fibroblasts. Cells 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 represent the mean of 50 independent measurements. (C) Phase contrast images of bgJ/bgJ or YAC-bgJ/bgJ fibroblasts pretreated or not with L-NMMA or L-NAME, exposed to L. amazonensis amastigotes for 1 h and fixed after 24 h. Arrows point to individual PVs. Bars = 5 µm.
Mentions: Since L. amazonensis is capable of productively infecting both macrophages and fibroblasts in culture (Figure 2), and PV enlargement enhances the early survival and replication of L. amazonensis in these two cell types (Figures 3 and 4), it is conceivable that PV enlargement protects Leishmania from a microbicidal mechanism common to both macrophages and fibroblasts. Although NO production in response to IFN-gamma and TNF-alpha is much more robust in macrophages, fibroblasts are also capable of mounting this response [1]. To investigate whether NO production plays a role in the enhanced survival of Leishmania in fibroblasts, we quantified intracellular amastigotes in L. amazonensis-infected bgJ/bgJ and YAC-bgJ/bgJ fibroblasts pre-treated with the iNOS inhibitors L-NMMA and L-NAME. These inhibitors were previously shown to promote the intracellular growth of Leishmania in macrophages by blocking NO production [25],[28]. Quantification of the number of intracellular amastigotes after 24 h showed that these inhibitors did not alter the parasite population size in bgJ/bgJ fibroblasts, which exhibit large PVs (Figure 5A). However, amastigote survival and replication was significantly enhanced in YAC-bgJ/bgJ fibroblasts pre-treated with the iNOS inhibitors (Figure 5B), consistent with the observation of several parasites inside each of the small PVs found in these cells (Figure 5D). Importantly, the size of the small PVs induced by L. amazonensis in the YAC-bgJ/bgJ fibroblasts overexpressing LYST/Beige remained unchanged after treatment with the iNOS inhibitors (Figure 5C), suggesting that inhibition of NO production can promote L. amazonensis survival/growth in the absence of PV expansion. Future studies are required to investigate the possibility that LYST expression might also impact the trafficking of iNOS to L. amazonensis-containing PVs.

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