Characteristics Of Intracellular Growth And Pathogenesis Of Environmental Legionella Pneumophila In Macrophages

Part1Characteristics of Intracellular Growh of Environmental Legionella pneumophilaObjective:Legionella pneumophila, is an intracellular pathogen causing penumonia with high mortality and morbidity. Enviromental L.pneumophila (LPE) is the source of infection through aerosol of contaminated water. In this study, we used LPE isolated from hospital water supply system to determine the bacteria and host interaction during infection.Methods:Using a deriative strain of clinical isolated Leginella pneumophila philadelphia1, namely JR32as a control, we first determined intracellular growth of an environmental strain LPE509in bone marrow derived macrophages from A/J mice (BMDMA/J), human macrophage U937cell line, and the amoebae host Dictyostelium discoideum. Then we carried out genome sequence and β-lactamase translocation assay to evaluate the structural and functional integrity of Dot/Icm type IVB secretion system (T4BSS) in LPE509. Next we used immunofluorescent staining for legionella containing vacuoles, Rabl and LAMP-1to detect the localization of host proteins of BMDMA/J after infection with LPE509, as well as the development of bacteria vacuoles in the host.Results:(1) We found that despite proficient replication in human macrophage U937and the amoebae host Dictyostelium discoideum, the strain LPE509could not grow in BMDMA/J, which are permissive for all examined laboratory strains. The ability of intracellular growth in U937cells was limited by the deletion of dotA.(2) LPE509contained all27protein components of T4BSS. Meanwhile the strain efficently translocated effectors into host cytosol as JR32indicated by β-lactamase tranlocation assay (Ipg1171:76.3±3.9%vs70.5±7.8, P>0.05; lpg0796:56.9±4.9%vs60±6.0%, P>0.05).(3) Immubofluoresent staining showed that after1h,2h and6h of infection with LPE509, the trafficking of host proteins LAMP-1and Rabl of this strain is similar to that of JR32(P>0.05).(4) After14h of infection with LPE509, the percentage of phagosomes that developed into big vacuole is significant lower than that of JR32(18.2±2.0%vs66.0±5.2%, P<0.001).(5) Mice macrophages adhered on the coverslip dramatically reduced after infection of LPE509, the reduction was significantly higher than that of JR32(44.9±14.5%vs9.4±16.3%, P<0.05).(6) Comparing to JR32, infection with LPE509caused much more vacuoles rupture by host cells after5h of infection (60.0±1.7%vs15.5±0.06%, P<0.001).Conclusions:LPE509has host specific intracellular growth deficiency in BMDMA/J, which allow intracellular growth for most studied clinical strains. Intracellular growth limitation of LPE509is probably caused by vacuoles rupture and cell loss during infection. Part2Induction Pyroptosis in Mice Macrophages by Environmental Legeionella pneumophilaObjective:To determine the mechanisms of intracellular growth limitation in mice macrophages of environmental Legionella pneumophila.Methods:Using JR32as a control, we evaluated pore formation on the cell membrane and cell death during infection of LPE509by Hoechst/PI double staining, LDH release assay and TUNEL staining of nucleus. We also assessed the possible release of inflammatory cytokines during infection by detection of HMGB1and IL-1β in culture medium of the mice macrophages. In order to understand the cellular signaling pathway involved during cell death, intracellular growth curve, LDH release assay and glycine protection assay were conducted in caspase-1/11-/-mice macrophages. Finally, to screen the possible bactria effector(s) responsible for cell death during infection of LPE509, EMS mutagenesis was applied to randomly mutate the strain and mice macrophages were used to screen for the mutant(s).Results:(1) Infection of mice macrophages by LPE509caused extensive cell death called pyroptsis in a process that required the Dot/Icm type IV secretion system. Hoechst/PI staining of BMDMA/J after infection with LPE509caused cellular membrane rupture in45%cells, which is much higher than that caused by JR32. LDH release after4h of infection and TUNEL staining after14h of infection represented34%and42%cell death, respectively.(2) Infection of LPE509induced HMGB1and IL-1β release of mice macrophages that was independent of flagellin.(3) LPE509AflaA, a flaA deletion strain of LPE509still caused extensive cell death indicated by Hoechst/PI staining and LDH release. In addition, the deletion of flaA could not rescue the bacteria restriction both in A/J and C57BL/6mice macrophages.(4) The strains LPE509and LPE509△/flαA could not grow in mice macrophages despite the deletion of caspase-1and caspase-11. Infection of these strains in caspase-1/11-/-mice macropahges caused extensive LDH release, which could be rescued by the addition of glycine into the culture medium.(5) Using EMS mutagenesis, we screened a mutant that could grow in the A/J mice macrophages. Genomic sequence and analysis indicated nucleotide substitution or frameshift in hypothetical protein coding genes lvrA, lpg1594, lpg1975, sdeC, legA3and luxN. Conclusion:An environmental L. pneumophila serugroup1strain LPE509can cause extensive inflammatory cell death (pyroptosis) after infection, which in turn restricts its intracellular replication. The pyroptosis caused by LPE509infection is not dependent on either caspase-1or caspase-11.