BACKGROUND: Rickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation. RESULTS: Three hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera. CONCLUSION: Our proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.
- MeSH
- chromatografie kapalinová MeSH
- králíci MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- proteiny vnější bakteriální membrány chemie imunologie metabolismus MeSH
- proteomika metody MeSH
- protilátky bakteriální krev MeSH
- Rickettsia akari imunologie izolace a purifikace metabolismus MeSH
- sekundární struktura proteinů MeSH
- skvrnité horečky diagnóza imunologie MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The regulation of the pyrimidine biosynthetic pathway by pyrimidines was investigated in the biological control agent Pseudomonas aureofaciens ATCC 17418. Using succinate as a carbon source, orotic acid or uracil supplementation had a repressive effect in ATCC 17418 cells on dihydroorotate dehydrogenase or orotidine 5'- monophosphate decarboxylase activity but only orotic acid supplementation appeared to repress the level of orotate phosphoribosyltransferase activity. In glucose-grown ATCC 17418 cells, orotic acid supplementation appeared to repress the level of phosphoribosyltransferase or decarboxylase while uracil supplementation depressed the dihydroorotase, dehydrogenase, and decarboxylase activities. The pyrimidine auxotrophic mutant strain GW-2, isolated from ATCC 17418 using chemical mutagenesis and resistance to 5-fluoroorotic acid, was found to be deficient for orotidine 5'-monophosphate decarboxylase activity. Pyrimidine limitation of the succinate-grown mutant strain cells resulted in only a slight derepression of transcarbamoylase activity while pyrimidine limitation of glucose-grown mutant cells caused a derepression of the four active pyrimidine biosynthetic enzyme activities relative to their activities in the mutant cells grown with excess uracil. The control of the known regulatory enzyme aspartate transcarbamoylase was examined in P. aureofaciens ATCC 17418. Transcarbamoylase activity was shown to be inhibited by pyrophosphate, ATP, UTP, and ADP. It was concluded that the pyrimidine biosynthetic pathway in P. aureofaciens ATCC 17418 was subject to regulation at the transcriptional level and at the level of aspartate transcarbamoylase activity, which could be valuable in comprehending its nucleic acid metabolism as well as its taxonomic assignment to the Pseudomonas chlororaphis homology group.
- MeSH
- aspartátkarbamoyltransferasa metabolismus MeSH
- bakteriální proteiny metabolismus genetika MeSH
- biosyntetické dráhy MeSH
- dihydroorotátdehydrogenasa MeSH
- glukosa metabolismus MeSH
- kyselina jantarová metabolismus MeSH
- kyselina orotová metabolismus MeSH
- orotátfosforibosyltransferasa metabolismus MeSH
- orotidin-5'-fosfátdekarboxylasa metabolismus genetika MeSH
- oxidoreduktasy působící na CH-CH vazby metabolismus MeSH
- Pseudomonas * metabolismus genetika enzymologie MeSH
- pyrimidiny * biosyntéza MeSH
- regulace genové exprese u bakterií * MeSH
- uracil metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.
- MeSH
- Escherichia coli metabolismus MeSH
- hmotnostní spektrometrie MeSH
- molekulární chaperony genetika metabolismus MeSH
- peptidylprolylisomerasa genetika metabolismus MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.
- MeSH
- fylogeneze MeSH
- genetická variace MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- proteinové domény MeSH
- proteiny vnější bakteriální membrány chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- Spirochaetales klasifikace genetika růst a vývoj izolace a purifikace MeSH
- syfilis mikrobiologie MeSH
- Treponema pallidum klasifikace genetika růst a vývoj izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1-) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1- strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.
- MeSH
- bakteriální pouzdra genetika MeSH
- baktericidní aktivita krve * MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Escherichia coli chemie izolace a purifikace fyziologie MeSH
- infekce močového ústrojí mikrobiologie MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- lidé MeSH
- mikrobiální viabilita MeSH
- proteiny vnější bakteriální membrány analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
