10 μl of each dilution were spotted onto the amoebae-CYET agar plates, and incubated at 37°C for 5 days. Cytotoxicity assay using A. castellanii To determine cytotoxicity, 2.5 × 105 amoebae cells were infected by bacteria at a multiplicity of infection (MOI) of 100. 24 h post infection, propidium iodide (PI) was added to 3 mg ml-1. A. castellanii cells were detached from the wells and 2.5 × 104 infected amoebae per sample were analyzed using a FACSCalibur flow cytometer (Becton Tideglusib solubility dmso Dickinson) with a scatter gate adjusted for

A. castellanii [13]. Excitation was at 458 nm and fluorescence was measured at 495 nm. The data were collected and analyzed using the CELLQUEST software (Becton Dickinson). For fluorescence microscopy, the infected amoebae cells

in each well of 24-well plates were stained with PI, then observed in bright field or by epifluorescence with an inverse microscope (Zeiss SHP099 Axiovert 200 M, EPZ5676 20 × objective). Intracellular growth in A. castellanii For intracellular growth assays, exponentially growing A. castellanii were washed with Ac (A. castellanii) buffer, resuspended in HL5 medium, seeded onto a 24-well plate (2.5 × 105 per well) and were allowed to adhere for 1-2 h. L. pneumophila was grown for 21 h in AYE broth, diluted in HL5 and used to infect amoebae at an MOI of 10. The infection was synchronized by centrifugation at 440 g for 10 min, and the infected amoebae were incubated at 30°C. Thirty minutes post infection, extracellular bacteria were removed by washing 3 times with warm HL5 medium [13]. At the time points indicated, culture supernatant was removed and the amoebae cells were lysed with 0.04% Triton. The supernatant

and the lysates were combined, and serial dilutions were prepared and aliquots were plated on CYE plates for CFU counting [72]. Statistical analysis Basic statistical analyses were performed using Excel, and one-way ANOVA was performed using SPSS followed by a post hoc Student-Newman-Keul’s test. The alignment of amino acid sequences was performed using the online ClustalW2 http://​www.​ebi.​ac.​uk/​Tools/​clustalw2. Acknowledgements We thank Miss Ling-yan Zhu for kindly helping perform the flow cytometry analysis. This work was supported by the National Natural Science Foundation of China (No. 30670106, No. 30970123) and the Guangdong Provincial enough Natural Science Foundation of China (No.06201654) to YJL. References 1. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Sharrar RG, Harris J, Mallison GF, Martin SM, McDade JE, Shepard CC, Brachman PS: Legionnaires’ disease: description of an epidemic of pneumonia. N Engl J Med 1977, 297:1189–1197.PubMedCrossRef 2. Kaufmann AF, McDade JE, Patton CM, Bennett JV, Skaliy P, Feeley JC, Anderson DC, Potter ME, Newhouse VF, Gregg MB, Brachman PS: Pontiac fever: isolation of the etiologic agent ( Legionella pneumophilia ) and demonstration of its mode of transmission. Am J Epidemiol 1981, 114:337–347.PubMed 3.