Transcriptional Down-Regulation and rRNA Cleavage in Dictyostelium discoideum Mitochondria during Legionella pneumophila Infection

Chenyu Zhang,Adam Kuspa,Malcolm James Horsburgh

doi:10.1371/journal.pone.0005706

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.

Highlights

Legionella pneumophila is an intracellular gram-negative bacterium that causes Legionnaires’ disease, a severe form of pneumonia [1,2,3]
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
Exposure of cells to an avirulent L. pneumophila strain or Klebsiella aerogenes had no detectable effect on transcript levels

Summary

Introduction

Legionella pneumophila is an intracellular gram-negative bacterium that causes Legionnaires’ disease, a severe form of pneumonia [1,2,3]. The human disease is associated with inhalation of contaminated aerosols, usually emanating from air conditioning units, where amoebal species have been found to contain the pathogen [4,5,6]. The bacterium attacks the human alveolar macrophage cells when inhaled. As the infection progresses ER membrane surrounds the vacuole and the bacterium starts to multiply, eventually lysing the host cell. L. pneumophila can mediate its own release without lysing the host cell [11]. None of these cellular hallmarks is found in hosts that have engulfed dead bacteria or avirulent strains of the bacteria [7]

Methods

Results

Conclusion