In Mycoplasma hominis the OppA-mediated cytoadhesion depends on its ATPase activity

<p>Abstract</p> <p>Background</p> <p>In <it>Mycoplasma homini</it>s, a facultative human pathogen of the human genital tract, OppA, the substrate-binding domain of the oligopeptide permease, is a multifunctional protein involved in nutrition uptake, cytoadhesion and hydrolysis of extracellular ATP.</p> <p>Results</p> <p>To map the function-related protein regions the ATPase activity and adhesive behavior of OppA mutants were analyzed. Mutations of the Walker BA motifs resulted in an inhibition of up to 8% of the OppA ATPase activity, whereas deletion of the N-terminal CS1 or the CS2 region, structural motifs that are conserved in bacterial OppA proteins, reduced ATPase activity to 60% and deletion of CS3, the third conserved region adjacent to the Walker B motif led to a reduction to 42% ATPase activity.</p> <p>Interestingly, adhesion of the OppA mutants to immobilized HeLa cells demonstrated that two distal regions are mainly involved in adherence of OppA: the CS1 region, deletion of which led to 35% of the cytoadhesion, and the Walker BA with the adjacent upstream region CS3, deletion of which led to 25% of the cytoadhesion. The influence of the ATPase activity on the adherence of <it>M. hominis </it>to HeLa cells was confirmed by the use of ATPase inhibitors which reduced mycoplasmal cytoadhesion to 50%.</p> <p>Conclusions</p> <p>These findings suggest that the OppA-mediated cytoadherence of <it>Mycoplasma homini</it>s depends on both, the topology of the neighbouring CS1 and ATPase domain regions and the functionality of the ecto-ATPase activity in addition.</p

Untersuchung elektromagnetisch induzierter Transparenz für das kontinuierliche Vier-Wellen-Mischen

Kontinuierliche, kohärente Strahlung bei der 1S-2P-Übergangsfrequenz des Wasserstoffatoms, kurz Lyman-alpha, wird eine Schlüsselrolle bei Experimenten mit Antiwasserstoffatomen spielen: Sie ist zum Laserkühlen und zur Spektroskopie unumgänglich. Für die Lyman-alpha-Strahlung gibt es seit einigen Jahren eine Reihe gepulster Quellen, deren Effizienz beim Kühlen gefangener Atomen allerdings durch die Repetitionsrate und Bandbreite limitiert ist. Daher wurde vor wenigen Jahren in der Arbeitsgruppe von Prof. Hänsch eine kontinuierlich kohärente Lyman-alpha-Quelle durch Vier-Wellen-Mischen in Quecksilberdampf realisiert. Die vorliegende Arbeit geht der Frage nach, wie durch elektromagnetisch induzierte Transparenz die Konversionseffizienz des Vier-Wellen-Mischens erhöht werden kann. Erstmals wurden in Quecksilberdampf gemischte Zustände induziert und eine Aufspaltung des Ein-Photonenübergangs 6^1S->6^3P und des Zwei-Photonenübergangs 6^1S->7^1S beobachtet. Dazu wurden kontinuierliche Laserlichtquellen für 254 nm und 408 nm aufgebaut, die jeweils in der Frequenz soweit durchgestimmt werden können, daß Spektroskopie an allen natürlichen Quecksilberisotopen vorgenommen werden konnte. Die im Experiment beobachteten Effekte werden durch Rechnungen im Dichtematrixformalismus unter Berücksichtigung von Dopplerverbreiterung und endlicher Wechselwirkungzeit wiedergegeben. Dabei wurden auch Besonderheiten der Zwei-Photonenabsorption im von zwei starken kohärenten Lichtquellen getriebenen Drei-Niveausystem untersucht und im Modell der bekleideten Zustände erklärt. Aus den Ergebnissen der vorliegenden Arbeit lassen sich Anforderungen an eine künftige Apparatur ableiten, welche die Erzeugung von kontinuierlich kohärenter Lyman-alpha-Strahlung mit hoher Konversionseffizienz ermöglichen wird

P80, the HinT interacting membrane protein, is a secreted antigen of Mycoplasma hominis

BACKGROUND: Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT. RESULTS: Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form. We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa. Western blot analysis of recombinant P80 mutants expressed in E. coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro) which is important for signal peptidase I recognition resulted in reduced P80 secretion. All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80. Incubation of M. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions. CONCLUSIONS: Our model for secretion of the P80 protein of M. hominis implies a two-step process. In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence. Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P80 secretion, which is followed by a proteolytic process leading to a helical 74 kDa product. We propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set

HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

BACKGROUND: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. RESULTS: An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase, and Cp266, the HinT protein. CONCLUSION: In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase. Future work will elucidate whether these differing associations indicate that HinT proteins have evolved independently or are indeed two hotspots of a common sphere of action of bacterial HinT proteins

A set of novel multiplex Taqman real-time PCRs for the detection of diarrhoeagenic Escherichia coli and its use in determining the prevalence of EPEC and EAEC in a university hospital

<p>Abstract</p> <p>Background</p> <p>Accurate measurement of the incidence of diarrhoeagenic <it>E. coli </it>in patients with diarrhoea is hindered by the current methods of detection and varies from country to country. In order to improve the diagnosis of diarrhoeagenic <it>E. coli </it>(DEC), we developed a set of multiplex TaqMan real-time PCRs designed to detect the respective pathogens from an overnight stool culture.</p> <p>Methods</p> <p>Over the period Jan. 2006 to Dec. 2006 all stool specimens (n = 1981) received were investigated for EPEC and EAEC.</p> <p>Results</p> <p>Of these, 371 specimens had no growth of <it>Enterobacteriaceae</it>. Of the remaining 1610 specimens 144 (8,9%) were positive for EPEC and 78 (4,8%) positive for EAEC. Among the EPEC positive stool specimens 28 (19,4%) were received from the tropical diseases unit, 49 (34%) from the paediatric dept. and 67 (46,5%) from the remainder of the wards. The EAEC were distributed as follows: 39 (50%) - tropical diseases, 19 (24,4%) -paediatrics and 20 (25,6%) other wards. Proportionately more EAEC and EPEC were found in children less than 3 years of age than other age groups. In only 22,2% of the detected EPEC and 23% of EAEC was the investigation requested by hospital staff.</p> <p>Conclusions</p> <p>This is, to our knowledge, the first study using a multiplex TaqMan PCR for the successful detection of diarrhoeagenic <it>E. coli</it>. In conclusion, due to the high prevalence of DEC detected, investigation of EPEC and EAEC should be recommended as a routine diagnostic test for patients with infectious diarrhoea.</p

Circulating Leukotriene B4 Identifies Respiratory Complications after Trauma

Background. Leukotriene B4 (LTB4), a proinflammatory lipid mediator correlates well with the acute phase of Acute Respiratory Distress Syndrome (ARDS). Therefore, LTB4-levels were investigated to determine whether they might be a useful clinical marker in predicting pulmonary complications (PC) in multiply traumatized patients. Methods: Plasma levels of LTB4 were determined in 100 patients on admission (ED) and for five consecutive days (daily). Twenty healthy volunteers served as control. LTB4-levels were measured by ELISA. Thirty patients developed PC (pneumonia, respiratory failure, acute lung injury (ALI), ARDS, pulmonary embolism) and 70 had no PC (ØPC). Results. LTB4-levels in the PC-group [127.8 pg/mL, IQR: 104–200pg/ml] were significantly higher compared to the ØPC-group on admission [95.6 pg/mL, IQR: 55–143 pg/mL] or control-group [58.4 pg/mL, IQR: 36–108 pg/mL]. LTB4 continuously declined to basal levels from day 1 to 5 without differences between the groups. The cutoff to predict PC was calculated at 109.6 pg/mL (72% specificity, 67% sensitivity). LTB4 was not influenced by overall or chest injury severity, age, gender or massive transfusion. Patients with PC received mechanical ventilation for a significantly longer period of time, and had prolonged intensive care unit and overall hospital stay. Conclusion. High LTB4-levels indicate risk for PC development in multiply traumatized patients

Reexamination In Vitro

Purpose. To introduce additional methods to detect and to quantify single pathogens in the complex biofilm formation on an antibacterial dental material. Materials and Methods. A conventional (ST) and an antibacterial dental composite (B) were manufactured. In vitro: specimens were incubated with a mixture of early colonizers. Bacterial adhesion was analyzed by TaqMan PCR after 8/24 h. In situ: TaqMan PCR and 16S rRNA Next Generation Sequencing (NGS) were performed. Results. In vitro: after 8 h incubation, B was covered by 58.6% of the bacterial amount that was attached to ST. After 24 h, the amount of attached bacteria to ST remained constant on ST only slightly lower on B. In situ: after 8 h the amount of adhering A. viscosus and S. mitis was prominent on ST and reduced on B. NGS revealed that S. sanguinis, S. parasanguinis, and Gemella sanguinis were the mainly attached species with S. sanguinis dominant on ST and S. parasanguinis and G. sanguinis dominant on B. Conclusions. Initial biofilm formation was altered by B. A shift between actinomycetes and streptococci was observed in situ. TaqMan PCR and 16S rRNA NGS revealed comparable results in situ and demonstrated the usefulness of NGS to characterize complex bacterial communities

Perinatal Ureaplasma Exposure Is Associated With Increased Risk of Late Onset Sepsis and Imbalanced Inflammation in Preterm Infants and May Add to Lung Injury

Background: Controversy remains concerning the impact of Ureaplasma on preterm neonatal morbidity.Methods: Prospective single-center study in very low birth weight infants &lt;30 weeks' gestation. Cord blood and initial nasopharyngeal swabs were screened for Ureaplasma parvum and U. urealyticum using culture technique and polymerase chain reaction. Neonatal outcomes were followed until death or discharge. Multi-analyte immunoassay provided cord blood levels of inflammatory markers. Using multivariate regression analyses, perinatal Ureaplasma exposure was evaluated as risk factor for the development of bronchopulmonary dysplasia (BPD), other neonatal morbidities until discharge and systemic inflammation at admission.Results: 40/103 (39%) infants were positive for Ureaplasma in one or both specimens, with U. parvum being the predominant species. While exposure to Ureaplasma alone was not associated with BPD, we found an increased risk of BPD in Ureaplasma-positive infants ventilated ≥5 days (OR 1.64; 95% CI 0.12–22.98; p = 0.009). Presence of Ureaplasma was associated with a 7-fold risk of late onset sepsis (LOS) (95% CI 1.80–27.39; p = 0.014). Moreover, Ureaplasma-positive infants had higher I/T ratios (b 0.39; 95% CI 0.08–0.71; p = 0.014), increased levels of interleukin (IL)-17 (b 0.16; 95% CI 0.02–0.30; p = 0.025) and matrix metalloproteinase 8 (b 0.77; 95% CI 0.10–1.44; p = 0.020), decreased levels of IL-10 (b −0.77; 95% CI −1.58 to −0.01; p = 0.043) and increased ratios of Tumor necrosis factor-α, IL-8, and IL-17 to anti-inflammatory IL-10 (p = 0.003, p = 0.012, p &lt; 0.001).Conclusions: Positive Ureaplasma screening was not associated with BPD. However, exposure contributed to BPD in infants ventilated ≥5 days and conferred an increased risk of LOS and imbalanced inflammatory cytokine responses

Typing of Lymphogranuloma Venereum Chlamydia trachomatis Strains

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe