Limits...
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.

Show MeSH

Related in: MedlinePlus

Identified proteins in a schematic overview of metabolic pathways of L. pneumophila.Metabolic pathways of L. pneumophila related to our results were constructed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Some of the identified enzymes are involved in Glycolysis, the TCA cycle, the Synthesis and degradation of ketone bodies, and PHB biogenesis. Eleven identified enzymes are shown in underlined boldface and the reaction steps that they catalyze are shown by black arrows. The numbers in parentheses correspond to the spot numbers in 2D-DIGE gel images of Fig. 1 and the list of identified proteins in Table S4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2908689&req=5

pone-0011718-g002: Identified proteins in a schematic overview of metabolic pathways of L. pneumophila.Metabolic pathways of L. pneumophila related to our results were constructed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Some of the identified enzymes are involved in Glycolysis, the TCA cycle, the Synthesis and degradation of ketone bodies, and PHB biogenesis. Eleven identified enzymes are shown in underlined boldface and the reaction steps that they catalyze are shown by black arrows. The numbers in parentheses correspond to the spot numbers in 2D-DIGE gel images of Fig. 1 and the list of identified proteins in Table S4.

Mentions: Approximately 60% of the identified proteins were defined as enzymes. Most of the identified enzymes were up-regulated at the post-exponential phase, and categorized as involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Moreover, the identified enzymes were sorted according to KEGG pathway maps of L. pneumophila Philadelphia-1 available at http://www.genome.jp/kegg/[41]. (Fig. 2). The data showed that enzymes related to glycolysis (pyrubate kinase II (YP_094190) and malate dehydrogenase (YP_096964)), the TCA cycle (2-oxoglutarate ferredoxin oxidoreductase beta subunit (YP_094982)), ketone biogenesis (HMG-CoA lyase (YP_095856) and acetoacectate decarboxylase (ADC) (YP_094708)) and poly-3-hydroxybutyrate (PHB) biogenesis (acetoacetyl CoA reductase (YP_095092), acetyoacetyl CoA reductase (YP_094601, YP_094602), 3-hydroxyisobutyryl Coenzyme A hydrolase (YP_094904)) were up-regulated at the post-exponential phase. This is the first report that the expression of enzymes related to carbohydrate or lipid metabolism in Legionella species are up-regulated at a post-exponential phase. L. pneumophila is thought to utilize only amino acids as carbon and energy sources [42], but a study of the whole-genome sequence study reported that this bacterium has genes encoding enzymes involved in carbohydrate metabolism, lipid metabolism, and the TCA cycle [43], [44]. Moreover, it is known that L. pneumophila utilizes PHB as an energy source for survival in nutrient-poor environments such as tap water [45]. Therefore, L. pneumophila may utilize sugars and lipids as energy sources at the post-exponential phase or within host cells in which amino acids are scarce.


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)

Identified proteins in a schematic overview of metabolic pathways of L. pneumophila.Metabolic pathways of L. pneumophila related to our results were constructed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Some of the identified enzymes are involved in Glycolysis, the TCA cycle, the Synthesis and degradation of ketone bodies, and PHB biogenesis. Eleven identified enzymes are shown in underlined boldface and the reaction steps that they catalyze are shown by black arrows. The numbers in parentheses correspond to the spot numbers in 2D-DIGE gel images of Fig. 1 and the list of identified proteins in Table S4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908689&req=5

pone-0011718-g002: Identified proteins in a schematic overview of metabolic pathways of L. pneumophila.Metabolic pathways of L. pneumophila related to our results were constructed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Some of the identified enzymes are involved in Glycolysis, the TCA cycle, the Synthesis and degradation of ketone bodies, and PHB biogenesis. Eleven identified enzymes are shown in underlined boldface and the reaction steps that they catalyze are shown by black arrows. The numbers in parentheses correspond to the spot numbers in 2D-DIGE gel images of Fig. 1 and the list of identified proteins in Table S4.
Mentions: Approximately 60% of the identified proteins were defined as enzymes. Most of the identified enzymes were up-regulated at the post-exponential phase, and categorized as involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Moreover, the identified enzymes were sorted according to KEGG pathway maps of L. pneumophila Philadelphia-1 available at http://www.genome.jp/kegg/[41]. (Fig. 2). The data showed that enzymes related to glycolysis (pyrubate kinase II (YP_094190) and malate dehydrogenase (YP_096964)), the TCA cycle (2-oxoglutarate ferredoxin oxidoreductase beta subunit (YP_094982)), ketone biogenesis (HMG-CoA lyase (YP_095856) and acetoacectate decarboxylase (ADC) (YP_094708)) and poly-3-hydroxybutyrate (PHB) biogenesis (acetoacetyl CoA reductase (YP_095092), acetyoacetyl CoA reductase (YP_094601, YP_094602), 3-hydroxyisobutyryl Coenzyme A hydrolase (YP_094904)) were up-regulated at the post-exponential phase. This is the first report that the expression of enzymes related to carbohydrate or lipid metabolism in Legionella species are up-regulated at a post-exponential phase. L. pneumophila is thought to utilize only amino acids as carbon and energy sources [42], but a study of the whole-genome sequence study reported that this bacterium has genes encoding enzymes involved in carbohydrate metabolism, lipid metabolism, and the TCA cycle [43], [44]. Moreover, it is known that L. pneumophila utilizes PHB as an energy source for survival in nutrient-poor environments such as tap water [45]. Therefore, L. pneumophila may utilize sugars and lipids as energy sources at the post-exponential phase or within host cells in which amino acids are scarce.

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