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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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Decrease in D. discoideum mitochondrial mRNAs during L. pneumophila infection.(A) D. discoideum cells were infected with L. pneumophila (JR32) at an M.O.I. of 10 and RNA was extracted at the indicated times and analyzed on northern blots. Substantial cell death occurred after 8 hours in this experiment (data not shown). Messenger RNA for the mitochondrial-encoded genes examined displayed two modes of decline; rapid (nad5 and atp6) and gradual (cobA and cox3). As controls, cells were also exposed to avirulent L. pneumophila (icmT mutant) or the food bacteria K. aerogenes. (B) The mRNA levels of these genes were also examined under conditions that kill D. discoideum cells by oxidative stress (hydrogen peroxide), inhibition of protein synthesis (G418, an aminoglycoside), inhibition of DNA synthesis (Etoposide, a topoisomerase II inhibitor).
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pone-0005706-g001: Decrease in D. discoideum mitochondrial mRNAs during L. pneumophila infection.(A) D. discoideum cells were infected with L. pneumophila (JR32) at an M.O.I. of 10 and RNA was extracted at the indicated times and analyzed on northern blots. Substantial cell death occurred after 8 hours in this experiment (data not shown). Messenger RNA for the mitochondrial-encoded genes examined displayed two modes of decline; rapid (nad5 and atp6) and gradual (cobA and cox3). As controls, cells were also exposed to avirulent L. pneumophila (icmT mutant) or the food bacteria K. aerogenes. (B) The mRNA levels of these genes were also examined under conditions that kill D. discoideum cells by oxidative stress (hydrogen peroxide), inhibition of protein synthesis (G418, an aminoglycoside), inhibition of DNA synthesis (Etoposide, a topoisomerase II inhibitor).

Mentions: In the course of carrying out transcriptional profiling of D. discoideum during infection with L. pneumophila we observed a decrease in the steady-state levels of a number of mRNAs that are related to mitochondrial function (data not shown). Given the reported association of L. pneumophila-containing phagosomes with the host cell mitochondria, we examined this finding more closely. We assessed the steady-state mRNA levels of four mitochondria-encoded genes, each of which code for one subunit of each of the four complexes of the electron transport chain [23]. These four genes; nad5 (NADH dehydrogenase subunit), atp6 (ATP synthase, F0 subunit), cytB (cytochrome b), and cox3 (cytochrome c oxidase subunit) are transcribed from the mitochondrial genome as part of four distinct polycistronic primary transcripts [36]. After infection with the virulent L. pneumophila strain JR32, we observed two modes of mRNA decline, nad5 and atp6 levels decreased dramatically within the first 4 h of infection, while cytB and cox3 levels declined more gradually throughout the infection (Figure 1A). Exposure of cells to an avirulent L. pneumophila strain (icmT mutant) or Klebsiella aerogenes (a bacterium commonly used as a laboratory food source for D. discoideum) had no detectable effect on transcript levels. In these experiments, cell viability began to decrease rapidly after 8 h, so the decline in nad5 and atp6 mRNAs appears to precede cell killing.


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

Zhang C, Kuspa A - PLoS ONE (2009)

Decrease in D. discoideum mitochondrial mRNAs during L. pneumophila infection.(A) D. discoideum cells were infected with L. pneumophila (JR32) at an M.O.I. of 10 and RNA was extracted at the indicated times and analyzed on northern blots. Substantial cell death occurred after 8 hours in this experiment (data not shown). Messenger RNA for the mitochondrial-encoded genes examined displayed two modes of decline; rapid (nad5 and atp6) and gradual (cobA and cox3). As controls, cells were also exposed to avirulent L. pneumophila (icmT mutant) or the food bacteria K. aerogenes. (B) The mRNA levels of these genes were also examined under conditions that kill D. discoideum cells by oxidative stress (hydrogen peroxide), inhibition of protein synthesis (G418, an aminoglycoside), inhibition of DNA synthesis (Etoposide, a topoisomerase II inhibitor).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005706-g001: Decrease in D. discoideum mitochondrial mRNAs during L. pneumophila infection.(A) D. discoideum cells were infected with L. pneumophila (JR32) at an M.O.I. of 10 and RNA was extracted at the indicated times and analyzed on northern blots. Substantial cell death occurred after 8 hours in this experiment (data not shown). Messenger RNA for the mitochondrial-encoded genes examined displayed two modes of decline; rapid (nad5 and atp6) and gradual (cobA and cox3). As controls, cells were also exposed to avirulent L. pneumophila (icmT mutant) or the food bacteria K. aerogenes. (B) The mRNA levels of these genes were also examined under conditions that kill D. discoideum cells by oxidative stress (hydrogen peroxide), inhibition of protein synthesis (G418, an aminoglycoside), inhibition of DNA synthesis (Etoposide, a topoisomerase II inhibitor).
Mentions: In the course of carrying out transcriptional profiling of D. discoideum during infection with L. pneumophila we observed a decrease in the steady-state levels of a number of mRNAs that are related to mitochondrial function (data not shown). Given the reported association of L. pneumophila-containing phagosomes with the host cell mitochondria, we examined this finding more closely. We assessed the steady-state mRNA levels of four mitochondria-encoded genes, each of which code for one subunit of each of the four complexes of the electron transport chain [23]. These four genes; nad5 (NADH dehydrogenase subunit), atp6 (ATP synthase, F0 subunit), cytB (cytochrome b), and cox3 (cytochrome c oxidase subunit) are transcribed from the mitochondrial genome as part of four distinct polycistronic primary transcripts [36]. After infection with the virulent L. pneumophila strain JR32, we observed two modes of mRNA decline, nad5 and atp6 levels decreased dramatically within the first 4 h of infection, while cytB and cox3 levels declined more gradually throughout the infection (Figure 1A). Exposure of cells to an avirulent L. pneumophila strain (icmT mutant) or Klebsiella aerogenes (a bacterium commonly used as a laboratory food source for D. discoideum) had no detectable effect on transcript levels. In these experiments, cell viability began to decrease rapidly after 8 h, so the decline in nad5 and atp6 mRNAs appears to precede cell killing.

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