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Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

Bhargava A, Cotton JA, Dixon BR, Gedamu L, Yates RM, Buret AG - PLoS ONE (2015)

Bottom Line: Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited.Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells.Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada; Inflammation Research Network, University of Calgary, Calgary, Alberta, Canada; Host-Parasite Interactions, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

No MeSH data available.


Related in: MedlinePlus

Pre-treatment of Giardia duodenalis trophozoites with E-64d or Ca-074Me inhibits cathepsin cysteine protease activity.G. duodenalis NF trophozoites were treated with E-64d (10uM), Ca074Me (10uM), or vehicle control (DMSO) for 3 hours and then co-incubated with Caco-2 monolayers for 2 (B and C) or 24 (D and E) hours. G. duodenalis trophozoites were collected and sonicated to assess for intra-trophozoite cathepsin cysteine protease activity. Supernatants were collected and assessed for the viability of G. duodenalis trophozoites by examining the ratio of motile: non-motile trophozoites (A). G. duodenalis sonicates were incubated with catB/L fluorogenic substrate ZFR-AMC (B and D) or the catB fluorogenic substrate ZRR-AMC (C and E) (200 μM: 5 min: 37°C: pH 7.2). Proteolytic activity was calculated by determining the change in RFUs over time. *p<0.05 vs Control cells; #p<0.05 vs G. duodenalis NF trophozoites. Data are mean +/- SEM, n = 3.
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pone.0136102.g006: Pre-treatment of Giardia duodenalis trophozoites with E-64d or Ca-074Me inhibits cathepsin cysteine protease activity.G. duodenalis NF trophozoites were treated with E-64d (10uM), Ca074Me (10uM), or vehicle control (DMSO) for 3 hours and then co-incubated with Caco-2 monolayers for 2 (B and C) or 24 (D and E) hours. G. duodenalis trophozoites were collected and sonicated to assess for intra-trophozoite cathepsin cysteine protease activity. Supernatants were collected and assessed for the viability of G. duodenalis trophozoites by examining the ratio of motile: non-motile trophozoites (A). G. duodenalis sonicates were incubated with catB/L fluorogenic substrate ZFR-AMC (B and D) or the catB fluorogenic substrate ZRR-AMC (C and E) (200 μM: 5 min: 37°C: pH 7.2). Proteolytic activity was calculated by determining the change in RFUs over time. *p<0.05 vs Control cells; #p<0.05 vs G. duodenalis NF trophozoites. Data are mean +/- SEM, n = 3.

Mentions: G. duodenalis NF trophozoites were used for the rest of the study to assess whether and how parasite catB/L proteases may affect host IECs. Initial experiments sought to determine whether catB/L activity could be inhibited within G. duodenalis trophozoites via the broad-spectrum clan CA cysteine protease inhibitor E64d (10 μM) or the catB-specific inhibitor Ca-074Me (10 μM). Parasite viability was not affected by 3-hour treatment with E-64d or Ca-074Me; compared against control G. duodenalis trophozoites, no significant difference in the proportion of motile to non-motile trophozoites in groups treated with E64d, Ca-074Me, or vehicle control (DMSO) was observed (Fig 6A). Next, Caco-2 monolayers were co-incubated with G. duodenalis NF trophozoites pretreated with E64d for 2 or 24 hours; this treatment significantly reduced the hydrolysis of ZFR-AMC within G. duodenalis sonicates (Fig 6B) and cell supernatants (Fig 6C), following their 2-hour co-incubation with Caco-2 monolayers. Similarly, hydrolysis of ZFR-AMC was significantly reduced within G duodenalis sonicates (Fig 6D) and cell supernatants (Fig 6E) when parasites were pre-treated with E-64d or Ca-074Me for 3-hours and subsequently co-incubated with Caco-2 monolayers for 24 hours. Collectively, these results demonstrate that G. duodenalis catB and L proteases are sensitive to inhibition using commercial protease inhibitors.


Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

Bhargava A, Cotton JA, Dixon BR, Gedamu L, Yates RM, Buret AG - PLoS ONE (2015)

Pre-treatment of Giardia duodenalis trophozoites with E-64d or Ca-074Me inhibits cathepsin cysteine protease activity.G. duodenalis NF trophozoites were treated with E-64d (10uM), Ca074Me (10uM), or vehicle control (DMSO) for 3 hours and then co-incubated with Caco-2 monolayers for 2 (B and C) or 24 (D and E) hours. G. duodenalis trophozoites were collected and sonicated to assess for intra-trophozoite cathepsin cysteine protease activity. Supernatants were collected and assessed for the viability of G. duodenalis trophozoites by examining the ratio of motile: non-motile trophozoites (A). G. duodenalis sonicates were incubated with catB/L fluorogenic substrate ZFR-AMC (B and D) or the catB fluorogenic substrate ZRR-AMC (C and E) (200 μM: 5 min: 37°C: pH 7.2). Proteolytic activity was calculated by determining the change in RFUs over time. *p<0.05 vs Control cells; #p<0.05 vs G. duodenalis NF trophozoites. Data are mean +/- SEM, n = 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559405&req=5

pone.0136102.g006: Pre-treatment of Giardia duodenalis trophozoites with E-64d or Ca-074Me inhibits cathepsin cysteine protease activity.G. duodenalis NF trophozoites were treated with E-64d (10uM), Ca074Me (10uM), or vehicle control (DMSO) for 3 hours and then co-incubated with Caco-2 monolayers for 2 (B and C) or 24 (D and E) hours. G. duodenalis trophozoites were collected and sonicated to assess for intra-trophozoite cathepsin cysteine protease activity. Supernatants were collected and assessed for the viability of G. duodenalis trophozoites by examining the ratio of motile: non-motile trophozoites (A). G. duodenalis sonicates were incubated with catB/L fluorogenic substrate ZFR-AMC (B and D) or the catB fluorogenic substrate ZRR-AMC (C and E) (200 μM: 5 min: 37°C: pH 7.2). Proteolytic activity was calculated by determining the change in RFUs over time. *p<0.05 vs Control cells; #p<0.05 vs G. duodenalis NF trophozoites. Data are mean +/- SEM, n = 3.
Mentions: G. duodenalis NF trophozoites were used for the rest of the study to assess whether and how parasite catB/L proteases may affect host IECs. Initial experiments sought to determine whether catB/L activity could be inhibited within G. duodenalis trophozoites via the broad-spectrum clan CA cysteine protease inhibitor E64d (10 μM) or the catB-specific inhibitor Ca-074Me (10 μM). Parasite viability was not affected by 3-hour treatment with E-64d or Ca-074Me; compared against control G. duodenalis trophozoites, no significant difference in the proportion of motile to non-motile trophozoites in groups treated with E64d, Ca-074Me, or vehicle control (DMSO) was observed (Fig 6A). Next, Caco-2 monolayers were co-incubated with G. duodenalis NF trophozoites pretreated with E64d for 2 or 24 hours; this treatment significantly reduced the hydrolysis of ZFR-AMC within G. duodenalis sonicates (Fig 6B) and cell supernatants (Fig 6C), following their 2-hour co-incubation with Caco-2 monolayers. Similarly, hydrolysis of ZFR-AMC was significantly reduced within G duodenalis sonicates (Fig 6D) and cell supernatants (Fig 6E) when parasites were pre-treated with E-64d or Ca-074Me for 3-hours and subsequently co-incubated with Caco-2 monolayers for 24 hours. Collectively, these results demonstrate that G. duodenalis catB and L proteases are sensitive to inhibition using commercial protease inhibitors.

Bottom Line: Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited.Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells.Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada; Inflammation Research Network, University of Calgary, Calgary, Alberta, Canada; Host-Parasite Interactions, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

No MeSH data available.


Related in: MedlinePlus