The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila

Abstract

Background: Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality.

Results: In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii.

Conclusions: The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

Figure 1

Sequence alignment of the putative ClpP from L. pneumophila with other prokaryotic ClpP proteins. Numbers indicate the positions of amino acids in the sequences, and dashes show gaps inserted for an optimal alignment. Identical or similar residues are labeled with asterisks or periods, respectively. The highly conserved catalytic Ser-110, His-135 and Asp-184 are shown as light color. Lla, Lactococcus lactis. Spn, Streptococcus pneumoniae. Bsu, Bacillus subtilis. Sau, Staphylococcus aureus. Lmo, Listeria monocytogenes. Eco, Escherichia coli. Sty, Salmonella enterica serovar typhimurium. Ype, Yersinia pestis. Pfl, Pseudomonas fluorescens. Lpn, Legionella pneumophila. Hpy, Helicobacter pylori. Ara, Agrobacterium radiobacter. Mtu, Mycobacterium tuberculosis.

Figure 2

The growth curves of L. pneumophila wild-type JR32, the LpΔclpP mutant, both harboring the vector pBC(gfp)Pmip, and the complemented strain LpΔclpP-pclpP. Overnight cultures of mid-exponential bacterial cells were diluted into fresh medium and then incubated at (A) 25°C, (B) 30°C, (C) 37°C, and (D) 42°C, respectively. Growth was monitored by OD₆₀₀ at various time points. Points indicate mean values and error bars indicate standard deviations of three experiments.

Figure 3

Impaired stress tolerance of the L. pneumophila LpΔclpP mutant during stationary phase. Overnight cultures of different strains were inoculated into fresh medium and grew to stationary phase (OD₆₀₀ from 3.5 to 4.5), and the cells were then treated with (A) 1 mM H₂O₂ for 30 minutes. * p < 0.05, (B) pH 4.0 citric acid for 30 minutes. * p < 0.01, (C) 57°C heat shock for 20 minutes. * p < 0.05, or (D) 0.3 M KCl for 1 hour. * p < 0.05. The experiments were carried out in triplicate.

Figure 4

Electron microscopy of stationary-phase L. pneumophila cells revealed cell elongation and abnormal division in the LpΔclpP mutant. Cyro-TEM of (A) JR32, (B) LpΔclpP and (C) LpΔclpP-pclpP and SEM of (D) JR32 and (E) LpΔclpP were carried out. Bar for (A), (B) and (C), 0.2 μm; Bar for (D), 2.0 μm; Bar for (E), 1.0 μm. (F) The percentages of normal and abnormal cells under cyro-TEM in the three L. pneumophila strains. Shown are the averages and standard deviations of three independent counts and the number of cells for each count is about 120 (n = 120).

Figure 5

Sodium tolerance of L. pneumophila LpΔclpP mutant was enhanced. (A). Overnight bacterial cultures in mid-exponential phase were inoculated into fresh medium and grew to exponential phase (OD₆₀₀ from 1.0 to 1.5) or stationary phase (OD₆₀₀ from 3.5 to 4.5), then the CFU was determined by plating duplicate samples of JR32 (black bars), LpΔclpP mutant (white bars), and complemented strain (gray bars) on BCYE and BCYE containing 100 mM NaCl. The experiment was carried out in triplicate. * p < 0.01. (B). For direct visualization, different dilutions of stationary-phase JR32 and LpΔclpP cells were also spotted onto plates in triplicate.

Figure 6

The L. pneumophila clpP mutant was impaired in both cytotoxicity against amoebae A. castellanii and growth on amoebae plates. (A) Growth of L. pneumophila LpΔclpP mutant in the amoebae plate test was impaired. L. pneumophila wild-type strain JR32, LpΔclpP mutant, clpP complemented strain or dotA mutant were spotted respectively in tenfold serial dilutions onto BCYE agar plates containing A. castellanii. The plates were incubated at 37°C for 5 days. (B) Cytotoxicity of L. pneumophila against amoebae A. castellanii was quantified by flow cytometry and (C) detected by PI staining 24 h post infection. The infection was performed using the wild-type strain JR32, LpΔclpP mutant, clpP complemented strain or dotA mutant at an MOI of 100. For fluorescence microscopy, amoebae cells in each well of 24-well plate were stained. The data shown are representative of at least two independent experiments.

Figure 7

Intracellular growth of L. pneumophila LpΔclpP mutant in A. castellanii was abolished. A. castellanii cells were seeded onto 24-well plates and infected with L.pneumophila at an MOI of 10. At each time point indicated, amoebae cells were lysed and the CFU was determined by plating dilutions onto BCYE plates. The intracellular growth kinetics of JR32, LpΔclpP mutant, clpP complemented strain, and dotA mutant are shown. The infection assay was carried out in triplicate.