| 1. |
- Haider, Susanne, et al.
(författare)
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Raman microspectroscopy reveals long-term extracellular activity of Chlamydiae
- 2010
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Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 77:3, s. 687-700
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Tidskriftsartikel (refereegranskat)abstract
- The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope-labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress-related proteins under these conditions as shown by 2-D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host-free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.
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| 2. |
- Sixt, Barbara Susanne, et al.
(författare)
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Metabolic features of Protochlamydia amoebophila elementary bodies--a link between activity and infectivity in Chlamydiae
- 2013
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 9:8
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Tidskriftsartikel (refereegranskat)abstract
- The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.
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| 3. |
- Sixt, Barbara Susanne, et al.
(författare)
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Proteomic analysis reveals a virtually complete set of proteins for translation and energy generation in elementary bodies of the amoeba symbiont Protochlamydia amoebophila
- 2011
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Ingår i: Proteomics. - : Wiley-VCH Verlagsgesellschaft. - 1615-9853 .- 1615-9861. ; 11:10, s. 1868-1892
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydiae belong to the most successful intracellular bacterial pathogens. They display a complex developmental cycle and an extremely broad host spectrum ranging from vertebrates to protozoa. The family Chlamydiaceae comprises exclusively well-known pathogens of humans and animals, whereas the members of its sister group, the Parachlamydiaceae, naturally occur as symbionts of free-living amoebae. Comparative analysis of these two groups provides valuable insights into chlamydial evolution and mechanisms for microbe-host interaction. Based on the complete genome sequence of the Acanthamoeba spp. symbiont Protochlamydia amoebophila UWE25, we performed the first detailed proteome analysis of the infectious stage of a symbiotic chlamydia. A 2-D reference proteome map was established and the analysis was extensively complemented by shotgun proteomics. In total, 472 proteins were identified, which represent 23.2% of all encoded proteins. These cover a wide range of functional categories, including typical house-keeping proteins, but also putative virulence-associated proteins. A number of proteins that are not encoded in genomes of Chlamydiaceae were observed and the expression of 162 proteins classified as hypothetical or unknown proteins could be demonstrated. Our findings indicate that P. amoebophila exploits its additional genetic repertoire (compared with the Chlamydiaceae), and that its elementary bodies are remarkably well equipped with proteins involved in transcription, translation, and energy generation.
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