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Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

Hayashi T, Nakamichi M, Naitou H, Ohashi N, Imai Y, Miyake M - PLoS ONE (2010)

Bottom Line: Furthermore, 9 up-regulated proteins of unknown function were found.Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs).Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.

ABSTRACT
Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

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Cya reporter assay.(a) Lpg1851 and Lpg2874 are translocated into macrophages via the Icm/Dot secretion apparatus. (b) Lpg0634 and Lpg0901 are not involved in translocation of RalF into macrophages. U937 cells (3×105 cells/well) were infected with wild type JR32 (MOI = 50) or LELA3118 (dotA−) (MOI = 100) producing the indicated proteins fused with Cya or MN101 (lpg0634−) or MN102 (lpg0901−) producing RalF fused with Cya (MOI = 50) for 1 h. After infection, the cells were lysed and the cAMP levels were measured as described in experimental procedure. Asterisks indicate statistically significant differences (p<0.005, by Student t test) between JR32 and LELA3118 expressing the indicated proteins. Sharp indicates statistically significant differences (# p<0.05, compared with JR32 pCya-RalF by ANOVA followed by Dunnett's test). The experiments were performed in triplicate, and the data are shown as means±S.D. The data are representative of one (Cya-Lpg0901, Cya-RalF) or three (Cya-Lpg1851 and Cya-Lpg2874) independent experiments. NS: not significant.
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pone-0011718-g006: Cya reporter assay.(a) Lpg1851 and Lpg2874 are translocated into macrophages via the Icm/Dot secretion apparatus. (b) Lpg0634 and Lpg0901 are not involved in translocation of RalF into macrophages. U937 cells (3×105 cells/well) were infected with wild type JR32 (MOI = 50) or LELA3118 (dotA−) (MOI = 100) producing the indicated proteins fused with Cya or MN101 (lpg0634−) or MN102 (lpg0901−) producing RalF fused with Cya (MOI = 50) for 1 h. After infection, the cells were lysed and the cAMP levels were measured as described in experimental procedure. Asterisks indicate statistically significant differences (p<0.005, by Student t test) between JR32 and LELA3118 expressing the indicated proteins. Sharp indicates statistically significant differences (# p<0.05, compared with JR32 pCya-RalF by ANOVA followed by Dunnett's test). The experiments were performed in triplicate, and the data are shown as means±S.D. The data are representative of one (Cya-Lpg0901, Cya-RalF) or three (Cya-Lpg1851 and Cya-Lpg2874) independent experiments. NS: not significant.

Mentions: We constructed forms of each of 3 uncharacterized proteins (Lpg0901, Lpg1851 and Lpg2874) fused with Cya, expressed in L. pneumophila (JR32 and LELA3118). Then, U937 cells were infected with L. pneumophila producing these Cya-fused proteins. After 1 h, levels of cAMP on infection with the wild type JR32 strain producing Cya-Lpg1851 and Cya-Lpg2874 were over 100000 fmol/well (Fig. 6). The level of cAMP resembled that on infection with the JR32 strain producing a Cya-fused form of RalF, an effector protein previously shown to be translocated into host cells, as a positive control [38]. In contrast, when U937 cells were infected with the dotA mutant LELA3118 strain producing any Cya-fused proteins, the levels of cAMP were similar to that on infection with JR32 producing only Cya or uninfected U937 as a negative control. However, cAMP on infection with JR32 producing Cya-fused Lpg0901 was similar in level to that on infection with LELA3118 producing any Cya-fused proteins (Fig. 6A). Therefore, this result clearly showed that Lpg1851 and Lpg2874 are translocated into U937 cells via the Icm/Dot secretion apparatus. Regarding the translocation of Lpg1851, it has recently been reported elsewhere [15]. As mentioned above, we showed in this study that the inactivation of the genes encoding these 2 proteins had no influence on the intracellular growth of L. pneumophila in U937 macrophage-like cells and A. polyphaga (Fig. 4). However, these results are not surprising, because effectors may have redundant or synergistic functions [8].


Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

Hayashi T, Nakamichi M, Naitou H, Ohashi N, Imai Y, Miyake M - PLoS ONE (2010)

Cya reporter assay.(a) Lpg1851 and Lpg2874 are translocated into macrophages via the Icm/Dot secretion apparatus. (b) Lpg0634 and Lpg0901 are not involved in translocation of RalF into macrophages. U937 cells (3×105 cells/well) were infected with wild type JR32 (MOI = 50) or LELA3118 (dotA−) (MOI = 100) producing the indicated proteins fused with Cya or MN101 (lpg0634−) or MN102 (lpg0901−) producing RalF fused with Cya (MOI = 50) for 1 h. After infection, the cells were lysed and the cAMP levels were measured as described in experimental procedure. Asterisks indicate statistically significant differences (p<0.005, by Student t test) between JR32 and LELA3118 expressing the indicated proteins. Sharp indicates statistically significant differences (# p<0.05, compared with JR32 pCya-RalF by ANOVA followed by Dunnett's test). The experiments were performed in triplicate, and the data are shown as means±S.D. The data are representative of one (Cya-Lpg0901, Cya-RalF) or three (Cya-Lpg1851 and Cya-Lpg2874) independent experiments. NS: not significant.
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pone-0011718-g006: Cya reporter assay.(a) Lpg1851 and Lpg2874 are translocated into macrophages via the Icm/Dot secretion apparatus. (b) Lpg0634 and Lpg0901 are not involved in translocation of RalF into macrophages. U937 cells (3×105 cells/well) were infected with wild type JR32 (MOI = 50) or LELA3118 (dotA−) (MOI = 100) producing the indicated proteins fused with Cya or MN101 (lpg0634−) or MN102 (lpg0901−) producing RalF fused with Cya (MOI = 50) for 1 h. After infection, the cells were lysed and the cAMP levels were measured as described in experimental procedure. Asterisks indicate statistically significant differences (p<0.005, by Student t test) between JR32 and LELA3118 expressing the indicated proteins. Sharp indicates statistically significant differences (# p<0.05, compared with JR32 pCya-RalF by ANOVA followed by Dunnett's test). The experiments were performed in triplicate, and the data are shown as means±S.D. The data are representative of one (Cya-Lpg0901, Cya-RalF) or three (Cya-Lpg1851 and Cya-Lpg2874) independent experiments. NS: not significant.
Mentions: We constructed forms of each of 3 uncharacterized proteins (Lpg0901, Lpg1851 and Lpg2874) fused with Cya, expressed in L. pneumophila (JR32 and LELA3118). Then, U937 cells were infected with L. pneumophila producing these Cya-fused proteins. After 1 h, levels of cAMP on infection with the wild type JR32 strain producing Cya-Lpg1851 and Cya-Lpg2874 were over 100000 fmol/well (Fig. 6). The level of cAMP resembled that on infection with the JR32 strain producing a Cya-fused form of RalF, an effector protein previously shown to be translocated into host cells, as a positive control [38]. In contrast, when U937 cells were infected with the dotA mutant LELA3118 strain producing any Cya-fused proteins, the levels of cAMP were similar to that on infection with JR32 producing only Cya or uninfected U937 as a negative control. However, cAMP on infection with JR32 producing Cya-fused Lpg0901 was similar in level to that on infection with LELA3118 producing any Cya-fused proteins (Fig. 6A). Therefore, this result clearly showed that Lpg1851 and Lpg2874 are translocated into U937 cells via the Icm/Dot secretion apparatus. Regarding the translocation of Lpg1851, it has recently been reported elsewhere [15]. As mentioned above, we showed in this study that the inactivation of the genes encoding these 2 proteins had no influence on the intracellular growth of L. pneumophila in U937 macrophage-like cells and A. polyphaga (Fig. 4). However, these results are not surprising, because effectors may have redundant or synergistic functions [8].

Bottom Line: Furthermore, 9 up-regulated proteins of unknown function were found.Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs).Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.

ABSTRACT
Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

Show MeSH
Related in: MedlinePlus