MassSpecPreppy-An end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT-2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD: ± 7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD: ± 9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT-2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection-ready samples for proteomics research.

SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus.

BACKGROUND: The Bacillus cereus Sigma B (SigB) dependent general stress response is activated via the two-component RsbKY system, which involves a phosphate transfer from RsbK to RsbY. It has been hypothesized that the Hpr-like phosphocarrier protein (Bc1009) encoded by bc1009 in the SigB gene cluster may play a role in this transfer, thereby acting as a regulator of SigB activation. Alternatively, Bc1009 may be involved in the activation of a subset of SigB regulon members. RESULTS: We first investigated the potential role of bc1009 to act as a SigB regulator but ruled out this possibility as the deletion of bc1009 did not affect the expression of sigB and other SigB gene cluster members. The SigB-dependent functions of Bc1009 were further examined in B. cereus ATCC14579 via comparative proteome profiling (backed up by transcriptomics) of wt, Δbc1009 and ΔsigB deletion mutants under heat stress at 42 °C. This revealed 284 proteins displaying SigB-dependent alterations in protein expression levels in heat-stressed cells, including a subgroup of 138 proteins for which alterations were also Bc1009-dependent. Next to proteins with roles in stress defense, newly identified SigB and Bc1009-dependent proteins have roles in cell motility, signal transduction, transcription, cell wall biogenesis, and amino acid transport and metabolism. Analysis of lethal stress survival at 50 °C after pre-adaptation at 42 °C showed intermediate survival efficacy of Δbc1009 cells, highest survival of wt, and lowest survival of ΔsigB cells, respectively. Additional comparative proteome analysis of non-stressed wt and mutant cells at 30 °C revealed 96 proteins with SigB and Bc1009-dependent differences in levels: 51 were also identified under heat stress, and 45 showed significant differential expression at 30 °C. This includes proteins with roles in carbohydrate/ion transport and metabolism. Overlapping functions at 30 °C and 42 °C included proteins involved in motility, and ΔsigB and Δbc1009 cells showed reduced motility compared to wt cells in swimming assays at both temperatures. CONCLUSION: Our results extend the B. cereus SigB regulon to > 300 members, with a novel role of SigB-dependent Bc1009 in the activation of a subregulon of  > 180 members, conceivably via interactions with other transcriptional regulatory networks.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

Riociguat attenuates the changes in left ventricular proteome and microRNA profile after experimental aortic stenosis in mice.

BACKGROUND AND PURPOSE: Development and progression of heart failure involve endothelial and myocardial dysfunction as well as a dysregulation of the NO-sGC-cGMP signalling pathway. Recently, we reported that the sGC stimulator riociguat has beneficial effects on cardiac remodelling and progression of heart failure in response to chronic pressure overload. Here, we examined if these beneficial effects of riociguat were also reflected in alterations of the myocardial proteome and microRNA profiles. EXPERIMENTAL APPROACH: Male C57BL/6N mice underwent transverse aortic constriction (TAC) and sham-operated mice served as controls. TAC and sham animals were randomised and treated with either riociguat or vehicle for 5 weeks, starting 3 weeks after surgery, when cardiac hypertrophy was established. Afterwards, we performed mass spectrometric proteome analyses and microRNA sequencing of proteins and RNAs, respectively, isolated from left ventricles (LVs). KEY RESULTS: TAC-induced changes of the LV proteome were significantly reduced by treatment with riociguat. Bioinformatics analyses revealed that riociguat improved TAC-induced cardiovascular disease-related pathways, metabolism and energy production, for example, reversed alterations in the levels of myosin heavy chain 7, cardiac phospholamban and ankyrin repeat domain-containing protein 1. Riociguat also attenuated TAC-induced changes of microRNA levels in the LV. CONCLUSION AND IMPLICATIONS: The sGC stimulator riociguat exerted beneficial effects on cardiac structure and function during pressure overload, which was accompanied by a reversal of TAC-induced changes of the cardiac proteome and microRNA profile. Our data support the potential of riociguat as a novel therapeutic agent for heart failure.

The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis.

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host's immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein. IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

Cytokine-Mediated Alterations of Human Cardiac Fibroblast's Secretome.

Fibroblasts contribute to approximately 20% of the non-cardiomyocytic cells in the heart. They play important roles in the myocardial adaption to stretch, inflammation, and other pathophysiological conditions. Fibroblasts are a major source of extracellular matrix (ECM) proteins whose production is regulated by cytokines, such as TNF-α or TGF-ß. The resulting myocardial fibrosis is a hallmark of pathological remodeling in dilated cardiomyopathy (DCM). Therefore, in the present study, the secretome and corresponding transcriptome of human cardiac fibroblasts from patients with DCM was investigated under normal conditions and after TNF-α or TGF-ß stimulation. Secreted proteins were quantified via mass spectrometry and expression of genes coding for secreted proteins was analyzed via Affymetrix Transcriptome Profiling. Thus, we provide comprehensive proteome and transcriptome data on the human cardiac fibroblast's secretome. In the secretome of quiescent fibroblasts, 58% of the protein amount belonged to the ECM fraction. Interestingly, cytokines were responsible for 5% of the total protein amount in the secretome and up to 10% in the corresponding transcriptome. Furthermore, cytokine gene expression and secretion were upregulated upon TNF-α stimulation, while collagen secretion levels were elevated after TGF-ß treatment. These results suggest that myocardial fibroblasts contribute to pro-fibrotic and to inflammatory processes in response to extracellular stimuli.

Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis.

BACKGROUND: Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. RESULTS: We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5'/3'UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. CONCLUSIONS: Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation.

AMPK/SIRT1 signaling through p38MAPK mediates Interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells.

Neuroendocrine Prostate Cancer (NEPC) is an aggressive form of androgen independent prostate cancer (AIPC), correlated with therapeutic resistance. Interleukin (IL)-6 promotes proliferation and neuroendocrine differentiation (NED) of androgen dependent LNCaP cells. We treated LNCaP cells with IL-6 and observed for in vitro NED of cells and also expression of NE markers ßIII tubulin, neuron-specific enolase (NSE) and chromogranin A (ChA). Here we investigated the proteins and/or pathways involved in NED of LNCaP cells induced by IL-6 and characterized their role in NED of PCa cells. We found that the altered proteins modulated AMPK signaling pathway in NE cells. Remarkably, IL-6 induces NED of LNCaP cells through activation of AMPK and SIRT1 and also both of these are co-regulated while playing a predominant role in NED of LNCaP cells. Of the few requirements of AMPK-SIRT1 activation, increased eNOS is essential for NED by elevating Nitric oxide (NO) levels. Pleiotropic effects of NO ultimately regulate p38MAPK in IL-6 induced NED. Hence, IL-6 induced AMPK-SIRT1 activation eventually transfers its activation signals through p38MAPK for advancing NED of LNCaP cells. Moreover, inactivation of p38MAPK with specific inhibitor (SB203580) attenuated IL-6 induced NED of LNCaP cells. Therefore, IL-6 promotes NED of PCa cells via AMPK/SIRT1/p38MAPK signaling. Finally, targeting AMPK-SIRT1 or p38MAPK in androgen independent PC3 cells with neuroendocrine features reversed their neuroendocrine characteristics. Taken together these novel findings reveal that targeting p38MAPK mitigated NED of PCa cells, and thus it can be a favorable target to overcome progression of NEPC.

Sputum Proteome Signatures of Mechanically Ventilated Intensive Care Unit Patients Distinguish Samples with or without Anti-pneumococcal Activity.

Mechanically ventilated patients are at risk of contracting pneumonia. Therefore, these patients often receive prophylactic systemic antimicrobial therapy. Intriguingly however, a previous study showed that antimicrobial activity in bronchoalveolar aspirates (here referred to as "sputa") from ventilated patients was only partially explained by antibiotic therapy. Here we report that sputa from these patients presented distinct proteome signatures depending on the presence or absence of antimicrobial activity. Moreover, we show that the same distinction applied to antibodies against Streptococcus pneumoniae, which is a major causative agent of pneumonia. Specifically, the investigated sputa that inhibited growth of S. pneumoniae, while containing subinhibitory levels of the antibiotic cefotaxime, presented elevated levels of proteins implicated in innate immune defenses, including complement and apolipoprotein-associated proteins. In contrast, S. pneumoniae-inhibiting sputa with relatively high cefotaxime concentrations or noninhibiting sputa contained higher levels of proteins involved in inflammatory responses, such as neutrophil elastase-associated proteins. In an immunoproteomics analysis, 18 out of 55 S. pneumoniae antigens tested showed significantly increased levels of IgGs in inhibiting sputa. Hence, proteomics and immunoproteomics revealed elevated levels of antimicrobial host proteins or S. pneumoniae antigen-specific IgGs in pneumococcal growth-inhibiting sputa, thus explaining their anti-pneumococcal activity.IMPORTANCE Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. This suggests the presence of potentially protective antimicrobial agents in their lung environment. Our present study shows for the first time that bronchoalveolar aspirates, "sputa," of ventilated patients in a Dutch intensive care unit were characterized by three distinct groups of proteome abundance signatures that can explain their anti-pneumococcal activity. Importantly, this anti-pneumococcal sputum activity was related either to elevated levels of antimicrobial host proteins or to antibiotics and S. pneumoniae-specific antibodies. Further, the sputum composition of some patients changed over time. Therefore, we conclude that our study may provide a novel tool to measure changes that are indicative of infection-related conditions in the lungs of mechanically ventilated patients.

Analysis of DCM associated protein alterations of human right and left ventricles.

Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and impaired myocardial function. Endomyocardial biopsies (EMB) enable immunohistochemical and molecular characterization of this disease. However, knowledge about specific molecular patterns and their relation to cardiac function in both ventricles is rare. Therefore, we performed a mass spectrometric analysis of 28 paired EMBs of left (LV) and right ventricles (RV) of patients with DCM or suspected myocarditis allowing quantitative profiling of 743 proteins. We analysed associations between protein abundance of LV and RV as well as the echocardiographic parameters LVEF, TAPSE, LVEDDI, and RVEDDI by linear regression models. Overall, more LV than RV proteins were associated with LV parameters or with RVEDDI. Most LV and RV proteins increasing in level with impairing of LVEF were annotated to structural components of cardiac tissue. Additionally, a high proportion of LV proteins with metabolic functions decreased in level with decreasing LVEF. Results were validated with LV heart sections of a genetic murine heart failure model. The study shows, that remodelling and systolic dysfunction in DCM is mirrored by distinct alterations in protein composition of both ventricles. Loss of LV systolic function is reflected predominantly by alterations in proteins assigned to metabolic functions in the LV whereas structural remodelling was more obvious in the RV. Alterations related to intermediate filaments were seen in both ventricles and highlight such proteins as early indicators of LV loss of function. SIGNIFICANCE: The present study report protein sets in the RV and the LV being associated with ventricular function and remodelling in DCM. Protein abundances in the LV and the RV emphasize and expand current knowledge on pathophysiological changes in heart failure and DCM. While RV and LV EMBs do not differ concerning diagnostic assessment of inflammatory status and virus persistence, additional information reflecting disease severity associated protein alterations can be gained by EMB protein profiling. RV and LV protein data provided complementary information. The protein pattern of the LV reflects metabolic changes and an impaired energy production, which is associated with the degree of LV systolic dysfunction and remodelling and may yield important information about the disease status in DCM. On the other hand, at this disease stage of DCM with still preserved RV function, RV alterations in structural proteins may reflect myocardial compensatory protective mechanisms for maintenance of structure and cellular function. The study highlight particular proteins being of interest as heart failure biomarkers in both ventricles which seem to reflect the severity of the disease. Further comparative studies between different HF aetiologies have to evaluate those proteins as markers specific for DCM.

From Proteomics to Personalized Medicine: The Importance of Isoflavone Dose and Estrogen Receptor Status in Breast Cancer Cells.

Continuing efforts are directed towards finding alternative breast cancer chemotherapeutics, with improved safety and efficacy profiles. Soy isoflavones represent promising agents but, despite extensive research, limited information exists regarding their impact on the breast cancer cell proteome. The purpose of this study was to compare the proteomic profiles of MCF-7 (estrogen responsive) and MDA-MB-231 (estrogen non-responsive) breast cancer cells exposed to different concentrations of genistein, daidzein, and a soy seed extract, using a high throughput LC-UDMSE protein profiling approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the dual activity of soy isoflavones on MCF-7 cells and the inhibitory effect on MDA-MB-231 cells. Proteome profiling of paramagnetic beads prepared peptides by nano-LC UDMSE and pathway enrichment analysis revealed that isoflavones affected distinct molecular pathways in MCF-7 and MDA-MB-231 cells, such as tyrosine kinases signaling pathway, cytoskeleton organization, lipid and phospholipid catabolism, extracellular matrix degradation and mRNA splicing. Also, in MCF-7 cells, low and high isoflavone doses induced different changes of the proteome, including cell cycle alterations. Therefore, the expression of estrogen receptors and the isoflavone dose are determinant factors for the molecular impact of isoflavones and must be taken into account when considering adjuvant breast cancer therapy towards personalized medicine.

Impact of Storage Conditions on the Breast Milk Peptidome.

Human donor milk (HDM) provides appropriate nutrition and offers protective functions in preterm infants. The aim of the study is to examine the impact of different storage conditions on the stability of the human breast milk peptidome. HDM was directly frozen at -80 °C or stored at -20 °C (120 h), 4 °C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiled by mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered 3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storage temperature and time and varied for different peptides. The highest impact was observed when samples were stored at RT. Smaller but significant effects were still observed in samples stored at 4 °C, while samples showed highest similarity to those immediately frozen at -80 °C when stored at -20 °C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombin and other proteases cleaving proteins at lysine/arginine. The results point to an ongoing protein degradation/peptide production by milk-derived proteases. They underline the need for immediate freezing of HDM at -20 °C or -80 °C to prevent degradation of peptides and enable reproducible investigation of prospectively collected samples.

Hfq modulates global protein pattern and stress response in Bordetella pertussis.

B. pertussis is the etiological agent of whooping cough, a highly contagious respiratory disease which remains uncontrolled worldwide. Understanding how this pathogen responds to the environmental changes and adapts to different niches found inside the host might contribute to gain insight into bacterial pathogenesis. Comparative analyses of previous transcriptomic and proteomic data suggested that post-transcriptional regulatory mechanisms modulate B. pertussis virulence in response to iron availability. Iron scarcity represents one of the major stresses faced by bacterial pathogens inside the host. In this study, we used gel-free nanoLC-MS/MS-based proteomics to investigate whether Hfq, a highly conserved post-transcriptional regulatory protein, is involved in B. pertussis adaptation to low iron environment. To this end, we compared the protein profiles of wild type B. pertussis and its isogenic hfq deletion mutant strain under iron-replete and iron-depleted conditions. Almost of 33% of the proteins identified under iron starvation was found to be Hfq-dependent. Among them, proteins involved in oxidative stress tolerance and virulence factors that play a key role in the early steps of host colonization and bacterial persistence inside the host cells. Altogether these results suggest that Hfq shapes the infective phenotype of B. pertussis. SIGNIFICANCE: In the last years, it became evident that post-transcriptional regulation of gene expression in ba cteria plays a central role in host-pathogen interactions. Hfq is a bacterial protein that regulates gene expression at post-transcriptional level found pivotal in the establishment of successful infections. In this study, we investigated the role of Hfq in Bordetella pertussis response to iron starvation, one of the main stresses imposed by the host. The data demonstrate that Hfq regulates the abundance of a significant number of B. pertussis proteins in response to iron starvation. Among them, virulence factors and proteins involved in oxidative stress tolerance, key players in host colonization and intracellular bacterial survival. Altogether, our results suggest a relevant role of Hfq in B. pertussis adaptation to the different niches found inside the host eventually granting bacterial pathogenesis.

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli.

In the absence of sugars, C4-dicarboxylates (C4DC) like fumarate represent important substrates for growth of Escherichia coli. Aerobically, C4DCs are oxidized to CO2 whereas anaerobically, C4DCs are used for fumarate respiration. In order to determine the impact of fumarate under aerobic and anaerobic conditions, proteomes of E. coli W3110 grown aerobically or anaerobically with fumarate and/or the non-C4DC substrate glycerol were comparatively profiled by nanoLC-MS/MS. Membrane enrichment allowed sensitive detection of membrane proteins. A total of 1657 proteins of which 646 and 374 were assigned to the cytosol or membrane, respectively, were covered. Presence of fumarate triggered changes (≥ 2fold) to the levels of 211 and 76 proteins under aerobic and anaerobic growth, respectively. The fumarate induced changes included proteins encoded by genes regulated by the C4DC two-component system DcuS-DcuR (DctA, DcuB, FumB, FrdABC proteins) catalyzing uptake and initial catabolic steps. Many of the proteins displaying altered levels are not part of the DcuS-DcuR regulon, including proteins of citric acid cycle and associated pathways (aerobic), proteins involved in motility and chemotaxis (anaerobic), and oxidative stress. Their genes are mostly preceded by cAMP receptor protein (CRP) sites, some by DcuR-like sites. Testing of selected genes confirmed regulation by CRP and DcuS-DcuR. SIGNIFICANCE: Global protein profiling of the soluble and the membrane fraction provides a comprehensive view on the protein pattern of E. coli grown aerobically and anaerobically with or without fumarate. The results disclose during aerobic growth besides the known impact of the C4-dicarboxylates (C4DC) on carbon utilization and citric acid cycle major adaptations in amino acid metabolism. In contrast, protein alterations in the presence of fumarate under anaerobic conditions point to enhanced motility and chemotaxis. Only proteins (transporters, initial metabolic steps) feeding external C4DCs to the central pathways were regulated by the C4DC two-component system DcuS-DcuR, whereas other protein levels were controlled in an indirect manner by CRP triggered catabolite control and other mechanisms. Consequently, metabolic and transcriptional regulation by C4DCs is apparently effected by a network of the DcuS-DcuR system with important contribution by catabolite control.

Shotgun proteomic analysis of Bordetella parapertussis provides insights into the physiological response to iron starvation and potential new virulence determinants absent in Bordetella pertussis.

Bordetella parapertussis is one of the pathogens that cause whooping cough. Even though its incidence has been rising in the last decades, this species remained poorly investigated. This study reports the first extensive proteome analysis of this bacterium. In an attempt to gain some insight into the infective phenotype, we evaluated the response of B. parapertussis to iron starvation, a critical stress the bacteria face during infection. Among other relevant findings, we observed that the adaptation to this condition involves significant changes in the abundance of two important virulence factors of this pathogen, namely, adenylate cyclase and the O-antigen. We further used the proteomic data to search for B. parapertussis proteins that are absent or classified as pseudogenes in the genome of Bordetella pertussis to unravel differences between both whooping cough causative agents. Among them, we identified proteins involved in stress resistance and virulence determinants that might help to explain the differences in the pathogenesis of these species and the lack of cross-protection of current acellular vaccines. Altogether, these results contribute to a better understanding of B. parapertussis biology and pathogenesis. SIGNIFICANCE: Whooping cough is a reemerging disease caused by both Bordetella pertussis and Bordetella parapertussis. Current vaccines fail to induce protection against B parapertussis and the incidence of this species has been rising over the years. The proteomic analysis of this study provided relevant insights into potential virulence determinants of this poorly-studied pathogen. It further identified proteins produced by B. parapertussis not present in B. pertussis, which might help to explain both the differences on their respective infectious process and the current vaccine failure. Altogether, the results of this study contribute to the better understanding of B. parapertussis pathogenesis and the eventual design of improved preventive strategies against whooping cough.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

Role of Platelet Size Revisited-Function and Protein Composition of Large and Small Platelets.

Epidemiological studies found an association between increased platelet size and the risk for thrombotic complications, but functional differences of large and small platelets remain poorly understood due to a lack of standardized protocols separating platelets with different size. We designed a protocol to separate large and small platelets from 15 mL whole blood. Separated large and small platelet fractions differed in mean platelet volume: 12.1 fl (10.3-13.8 fl) versus 7.7 fl (6.8-9.5 fl, p < 0.01), and forward scatter mean fluorescence intensity: 24.75 (19.9-30.9) versus 16.85 (11.3-20.6; p < 0.01). Similar fold differences were observed in cell diameter and plateletcrit. Large platelets express 30 to 50% more glycoprotein (GP) Ia, GPIb, GPIIIa, GPVI and P2Y12 on their membranes compared with small ones. Single large platelets covered a 50% larger area on a collagen surface. Adhesion to collagen was faster in large platelets compared with small ones indicating enhanced outside-in signal transduction in large platelets via collagen receptors. In contrast, integrin activation was more pronounced in small platelets after ADP stimulation. Proteome analysis revealed that 80 of the 894 proteins quantified differed in abundance: ADP-ribosylation factor 1/3, guanosine triphosphate-binding protein SAR1a, Voltage-dependent anion-selective channel protein 3 and guanylate cyclase soluble sub-unit α-3 were higher abundant in large, whereas immunoglobulins, haptoglobin, hemopexin, α-1-antitrypsin, serotransferrin and vitronectin were more abundant in small platelets. We conclude that some functions and the protein composition of large and small platelets differ, which cannot only be explained by the size difference. Our data suggest different functional roles of large and small platelets.

Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization.

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

Analysis of Staphylococcus aureus proteins secreted inside infected human epithelial cells.

Staphylococcus aureus, an opportunistic pathogen is able to invade into and persist inside non-professional phagocytic cells. To do so, this bacterium possesses a wide range of secreted virulence factors which enable attachment to the host as well as intracellular survival. Hence, a monitoring of virulence factors specifically produced upon internalization might reveal targets for prevention or therapy of S. aureus infections. However, previous proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors. Therefore, we used density gradient centrifugation and mass spectrometry to identify S. aureus HG001 proteins which were secreted into compartments of infected human bronchial epithelial S9 cells. Because shotgun mass spectrometry revealed only few bacterial proteins amongst 1905 host proteins, we used highly sensitive and selective single reaction monitoring mass spectrometry as an alternative approach and quantified 37 bacterial proteins within the S. aureus containing host cell compartment 2.5 h and 6.5 h post infection. Among them were secreted bacterial virulence factors like lipases, pore forming toxins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to their low abundance. S. aureus adapted its proteome to improve its response to oxidative and cell wall stress occurring inside the host, but also, increased the amounts of some adhesins and pore-forming toxins, required for attachment and host cell lysis.

Plasma protein absolute quantification by nano-LC Q-TOF UDMSE for clinical biomarker verification.

BACKGROUND AND AIMS: Proteome-based biomarker studies are targeting proteins that could serve as diagnostic, prognosis, and prediction molecules. In the clinical routine, immunoassays are currently used for the absolute quantification of such biomarkers, with the major limitation that only one molecule can be targeted per assay. The aim of our study was to test a mass spectrometry based absolute quantification method for the verification of plasma protein sets which might serve as reliable biomarker panels for the clinical practice. METHODS: Six EDTA plasma samples were analyzed after tryptic digestion using a high throughput data independent acquisition nano-LC Q-TOF UDMSE proteomics approach. Synthetic Escherichia coli standard peptides were spiked in each sample for the absolute quantification. Data analysis was performed using ProgenesisQI v2.0 software (Waters Corporation). RESULTS: Our method ensured absolute quantification of 242 non redundant plasma proteins in a single run analysis. The dynamic range covered was 105. 86% were represented by classical plasma proteins. The overall median coefficient of variation was 0.36, while a set of 63 proteins was found to be highly stable. Absolute protein concentrations strongly correlated with values reviewed in the literature. CONCLUSIONS: Nano-LC Q-TOF UDMSE proteomic analysis can be used for a simple and rapid determination of absolute amounts of plasma proteins. A large number of plasma proteins could be analyzed, while a wide dynamic range was covered with low coefficient of variation at protein level. The method proved to be a reliable tool for the quantification of protein panel for biomarker verification in the clinical practice.