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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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Location of the cleavage sites on the D. discoideum mitochondrial LSU rRNA structure.(A) Predicted secondary structure of D. discoideum mitochondrial LSU rRNA showing the locations of the cleavage sites (red arrows). For clarity, the rRNA is split between domains III and IV and the thick dashed line indicates the covalent linkage between the two halves. Lines indicate predicted basepairs in the three dimensional structure between bases that widely separated in the primary sequence. The domains are labeled in roman numerals near where their secondary structure emanates from the center. (B) LSU rRNA cleavage sites (red) mapped onto the three-dimensional structure of the Haloarcula marismortui mitochondrial ribosome (pdb 1ffk). The LSU rRNA (grey line), the 5S rRNA (blue line) and ribosomal proteins (yellow) are shown. The rRNA sarcin/ricin loop is indicated (green). The points of contact with the small subunit (purple) in domain IV of the rRNA secondary structure are where essential ribosomal functions are carried out and where the cleavage site at base 1878 is located (red). (C) Predicted structure in B rotated 60 degrees counterclockwise on the vertical axis.
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pone-0005706-g004: Location of the cleavage sites on the D. discoideum mitochondrial LSU rRNA structure.(A) Predicted secondary structure of D. discoideum mitochondrial LSU rRNA showing the locations of the cleavage sites (red arrows). For clarity, the rRNA is split between domains III and IV and the thick dashed line indicates the covalent linkage between the two halves. Lines indicate predicted basepairs in the three dimensional structure between bases that widely separated in the primary sequence. The domains are labeled in roman numerals near where their secondary structure emanates from the center. (B) LSU rRNA cleavage sites (red) mapped onto the three-dimensional structure of the Haloarcula marismortui mitochondrial ribosome (pdb 1ffk). The LSU rRNA (grey line), the 5S rRNA (blue line) and ribosomal proteins (yellow) are shown. The rRNA sarcin/ricin loop is indicated (green). The points of contact with the small subunit (purple) in domain IV of the rRNA secondary structure are where essential ribosomal functions are carried out and where the cleavage site at base 1878 is located (red). (C) Predicted structure in B rotated 60 degrees counterclockwise on the vertical axis.

Mentions: The northern blot data indicates that the LSU rRNA is cleaved into discrete fragments and the unambiguous ends identified by sequencing each of the RACE products strongly suggests that the fragmentation results from cleavage at discrete sites. The major cleavage site mapped between nucleotides 1533 and 1534 (Figure 3A). A minor cleavage site on the 5′ side of the major site mapped between nucleotides 862 and 863 and another minor cleavage site on the 3′ side of the major site mapped between nucleotides 1878 and 1879. Because the eukaryotic mitochondrial ribosomes are thought to have shared a common ancestor with archaebacterial ribosomes, we mapped the cleavage sites onto the predicted secondary structure of the D. discoideum mitochondrial LSU rRNA based on the halobacteria Haloarcula marismortui rRNA structure [33], [50] (Figure 4A). When mapped onto the corresponding regions of the H. marismortui rRNA high-resolution crystal structure, the major cleavage site is predicted to be on the surface of the ribosome (Figure 4B). This is consistent with the expectation that the initial cleavage site is accessible on the intact ribosome similar to the sarcin/ricin loop, a distinct site that is subject to cleavage by those toxins (Figure 4B and 4C). The minor cleavage site at base 1878 is predicted to be in domain IV of the LSU rRNA (Figure 4A), where essential functional contacts are made between the ribosome's large and small subunits (Figure 4B and 4C). The minor cleavage site at base 862 and is predicted to be deep within the ribosomal structure (Figure 4C).


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

Zhang C, Kuspa A - PLoS ONE (2009)

Location of the cleavage sites on the D. discoideum mitochondrial LSU rRNA structure.(A) Predicted secondary structure of D. discoideum mitochondrial LSU rRNA showing the locations of the cleavage sites (red arrows). For clarity, the rRNA is split between domains III and IV and the thick dashed line indicates the covalent linkage between the two halves. Lines indicate predicted basepairs in the three dimensional structure between bases that widely separated in the primary sequence. The domains are labeled in roman numerals near where their secondary structure emanates from the center. (B) LSU rRNA cleavage sites (red) mapped onto the three-dimensional structure of the Haloarcula marismortui mitochondrial ribosome (pdb 1ffk). The LSU rRNA (grey line), the 5S rRNA (blue line) and ribosomal proteins (yellow) are shown. The rRNA sarcin/ricin loop is indicated (green). The points of contact with the small subunit (purple) in domain IV of the rRNA secondary structure are where essential ribosomal functions are carried out and where the cleavage site at base 1878 is located (red). (C) Predicted structure in B rotated 60 degrees counterclockwise on the vertical axis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683564&req=5

pone-0005706-g004: Location of the cleavage sites on the D. discoideum mitochondrial LSU rRNA structure.(A) Predicted secondary structure of D. discoideum mitochondrial LSU rRNA showing the locations of the cleavage sites (red arrows). For clarity, the rRNA is split between domains III and IV and the thick dashed line indicates the covalent linkage between the two halves. Lines indicate predicted basepairs in the three dimensional structure between bases that widely separated in the primary sequence. The domains are labeled in roman numerals near where their secondary structure emanates from the center. (B) LSU rRNA cleavage sites (red) mapped onto the three-dimensional structure of the Haloarcula marismortui mitochondrial ribosome (pdb 1ffk). The LSU rRNA (grey line), the 5S rRNA (blue line) and ribosomal proteins (yellow) are shown. The rRNA sarcin/ricin loop is indicated (green). The points of contact with the small subunit (purple) in domain IV of the rRNA secondary structure are where essential ribosomal functions are carried out and where the cleavage site at base 1878 is located (red). (C) Predicted structure in B rotated 60 degrees counterclockwise on the vertical axis.
Mentions: The northern blot data indicates that the LSU rRNA is cleaved into discrete fragments and the unambiguous ends identified by sequencing each of the RACE products strongly suggests that the fragmentation results from cleavage at discrete sites. The major cleavage site mapped between nucleotides 1533 and 1534 (Figure 3A). A minor cleavage site on the 5′ side of the major site mapped between nucleotides 862 and 863 and another minor cleavage site on the 3′ side of the major site mapped between nucleotides 1878 and 1879. Because the eukaryotic mitochondrial ribosomes are thought to have shared a common ancestor with archaebacterial ribosomes, we mapped the cleavage sites onto the predicted secondary structure of the D. discoideum mitochondrial LSU rRNA based on the halobacteria Haloarcula marismortui rRNA structure [33], [50] (Figure 4A). When mapped onto the corresponding regions of the H. marismortui rRNA high-resolution crystal structure, the major cleavage site is predicted to be on the surface of the ribosome (Figure 4B). This is consistent with the expectation that the initial cleavage site is accessible on the intact ribosome similar to the sarcin/ricin loop, a distinct site that is subject to cleavage by those toxins (Figure 4B and 4C). The minor cleavage site at base 1878 is predicted to be in domain IV of the LSU rRNA (Figure 4A), where essential functional contacts are made between the ribosome's large and small subunits (Figure 4B and 4C). The minor cleavage site at base 862 and is predicted to be deep within the ribosomal structure (Figure 4C).

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