Marek Link

Proteome Analysis of an Attenuated Francisella tularensis dsbA Mutant: Identification of Potential DsbA Substrate Proteins

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

Multimethodological Approach to Identification of Glycoproteins from the Proteome of Francisella tularensis, an Intracellular Microorganism

It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a takeoff platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis.

Amniotic Fluid Cathelicidin in PPROM Pregnancies: From Proteomic Discovery to Assessing Its Potential in Inflammatory Complications Diagnosis

Background Preterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available. Methodology/Principal Findings We performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ∼6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p = 0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA. Conclusions Our multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.

Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning.

Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.

Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography-ion trap mass spectrometry.

Liquid chromatography–ion trap mass spectrometry was used for the detection and structural characterization of metabolites of the anti-obesity drug sibutramine. Metabolites were profiled from incubations of sibutramine in primary cultures of rat hepatocytes. In addition, enantioselectivity of sibutramine metabolism was investigated by carrying out separate incubations with (R)- and (S)-sibutramine. As a result, biotransformation profile for sibutramine with rat hepatocytes is proposed. Nineteen metabolites and several of their isomers formed via demethylation, hydroxylation, dehydrogenation, acetylation, attachment of CO2, and glucuronidation were identified in MS2 and MS3 experiments, though the exact position of the functionality, mostly hydroxylation, could not always be determined from the mass spectrometric information. However, clear enantioselective formation was observed for two hydroxyl derivatives and two glucuronide conjugates, indicating that the hydroxyl/glucuronic acid moiety in those structures is close to the chiral center. Most of the metabolites found in this study are new metabolites of sibutramine, which were not previously reported.

Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

The stereoselective biotransformation of the anti‐obesity drug sibutramine in rat liver microsomes and in primary cultures of rat hepatocytes

Sibutramine is an anti‐obesity drug sold as a racemic mixture under the trademark Meridia or Reductil. With the aim of evaluating the stereoselectivity in phase I of sibutramine biotransformation, the formation of the main metabolites from R‐sibutramine, S‐sibutramine and rac‐sibutramine was studied in rat microsomes and primary cultures of hepatocytes. A novel analytical method for the determination of sibutramine and its phase I metabolites in culture medium and microsomal incubates using isocratic reversed‐phase liquid chromatography with UV detection was developed. Only two metabolites, mono‐desmethylsibutramine (M1) and di‐desmethylsibutramine (M2), were found in the rat microsomes incubated with sibutramine and NADPH. The kinetics of M1 and M2 formation slightly differed depending on the enantiomeric form of the sibutramine used. The stereoselectivity in sibutramine biotransformation was much more evident in primary cultures of rat hepatocytes. While R‐sibutramine incubation led to the formation of M1 and M2 metabolites only, the incubation of S‐sibutramine or rac‐sibutramine (to a lesser extent) resulted in four major metabolites (M1, M2, M3 and M4) and 2 or 3 minor metabolites. On the basis of our results, R‐sibutramine might represent the more advantageous sibutramine enantiomer from the pharmacokinetic standpoint.

Inactivation of Francisella tularensis Gene Encoding Putative ABC Transporter Has a Pleiotropic Effect upon Production of Various Glycoconjugates

Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host-pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines.

Quantitative Proteomics Analysis of Macrophage-Derived Lipid Rafts Reveals Induction of Autophagy Pathway at the Early Time of Francisella tularensis LVS Infection

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.