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Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

Zhang C, Kuspa A - PLoS ONE (2009)

Bottom Line: No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways.Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction.The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA) is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial protein synthesis in D. discoideum during the course of infection.

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No LSU rRNA cleavage in Acanthamoeba or human cells during L. pneumophila infection.(A) A. castellanii (ATCC 30234) were infected by L. pneumophila in Ac buffer at 37°C. (B) Human U937 cells (ATCC CRL-1593.2) were infected by L. pneumophila. Total RNA samples were probed on northern blots with a A. castellanii or human mitochondrial LSU rRNA probes. In each experiment, the infection was confirmed by monitoring the internalization of GFP-labeled bacteria and host cell killing (data not shown). Experiments were carried out three times and a representative experiment is shown.
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pone-0005706-g005: No LSU rRNA cleavage in Acanthamoeba or human cells during L. pneumophila infection.(A) A. castellanii (ATCC 30234) were infected by L. pneumophila in Ac buffer at 37°C. (B) Human U937 cells (ATCC CRL-1593.2) were infected by L. pneumophila. Total RNA samples were probed on northern blots with a A. castellanii or human mitochondrial LSU rRNA probes. In each experiment, the infection was confirmed by monitoring the internalization of GFP-labeled bacteria and host cell killing (data not shown). Experiments were carried out three times and a representative experiment is shown.

Mentions: We tested whether LSU rRNA cleavage occurs in other cells infected with L. pneumophila. Acanthamoeba castellanii and human U937 cells are two widely used models of L. pneumophila infection [28], [29]. We infected these cells using established protocols and assayed for host cell viability and mitochondrial LSU rRNA integrity, but we did not observe any cleavage events during either of these infections (Figure 5). This result with human cells is not surprising since the entire structural domain where the primary cleavage site is located in D. discoideum LSU rRNA has been lost in human LSU rRNA [32], [51]. However, in A. castellanii, the major cleavage site and the minor site at base 1878 are conserved, whereas the minor cleavage site at base 832 is within a loop that is unique in D. discoideum [32].


Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

Zhang C, Kuspa A - PLoS ONE (2009)

No LSU rRNA cleavage in Acanthamoeba or human cells during L. pneumophila infection.(A) A. castellanii (ATCC 30234) were infected by L. pneumophila in Ac buffer at 37°C. (B) Human U937 cells (ATCC CRL-1593.2) were infected by L. pneumophila. Total RNA samples were probed on northern blots with a A. castellanii or human mitochondrial LSU rRNA probes. In each experiment, the infection was confirmed by monitoring the internalization of GFP-labeled bacteria and host cell killing (data not shown). Experiments were carried out three times and a representative experiment is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683564&req=5

pone-0005706-g005: No LSU rRNA cleavage in Acanthamoeba or human cells during L. pneumophila infection.(A) A. castellanii (ATCC 30234) were infected by L. pneumophila in Ac buffer at 37°C. (B) Human U937 cells (ATCC CRL-1593.2) were infected by L. pneumophila. Total RNA samples were probed on northern blots with a A. castellanii or human mitochondrial LSU rRNA probes. In each experiment, the infection was confirmed by monitoring the internalization of GFP-labeled bacteria and host cell killing (data not shown). Experiments were carried out three times and a representative experiment is shown.
Mentions: We tested whether LSU rRNA cleavage occurs in other cells infected with L. pneumophila. Acanthamoeba castellanii and human U937 cells are two widely used models of L. pneumophila infection [28], [29]. We infected these cells using established protocols and assayed for host cell viability and mitochondrial LSU rRNA integrity, but we did not observe any cleavage events during either of these infections (Figure 5). This result with human cells is not surprising since the entire structural domain where the primary cleavage site is located in D. discoideum LSU rRNA has been lost in human LSU rRNA [32], [51]. However, in A. castellanii, the major cleavage site and the minor site at base 1878 are conserved, whereas the minor cleavage site at base 832 is within a loop that is unique in D. discoideum [32].

Bottom Line: No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways.Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction.The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA) is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial protein synthesis in D. discoideum during the course of infection.

Show MeSH
Related in: MedlinePlus