Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

The Krüppel-like transcription factor (KLF) BCL11B is characterized by a wide tissue distribution and crucial functions in key developmental and cellular processes, as well as in various pathologies including cancer and HIV infection. Although the basics of BCL11B activity and relevant interactions with other proteins have been uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but has an independent, previously unidentified, nuclear localization signal (NLS), which is located distantly from the zinc finger domains and fulfills the essential criteria of being an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutation causes aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin-α. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13-14-KR+K++ ('+' indicates amino acids with similar chemical properties), which can be found in all BCL11B orthologs among vertebrates and in the closely related protein BCL11A.

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.

Single-Cell and Time-Resolved Profiling of Intracellular Salmonella Metabolism in Primary Human Cells.

The intracellular pathogen Salmonella enterica has evolved an array of traits for propagation and invasion of the intestinal layers. It remains largely elusive how Salmonella adjusts its metabolic states to survive inside immune host cells. In this study, single-cell Raman biotechnology combined with deuterium isotope probing (Raman-DIP) have been applied to reveal metabolic changes of the typhoidal Salmonella Typhi Ty2, the nontyphoidal Salmonella Typhimurium LT2, and a clinical isolate Typhimurium D23580. By initially labeling the Salmonella strains with deuterium, we employed reverse labeling to track their metabolic changes in the time-course infection of THP-1 cell line, human monocyte-derived dendritic cells (MoDCs) and macrophages (Mf). We found that, in comparison with a noninvasive serovar, the invasive Salmonella strains Ty2 and D23580 have downregulated metabolic activity inside human macrophages and dendritic cells and used lipids as alternative carbon source, perhaps a strategy to escape from the host immune response. Proteomic analysis using high sensitivity mass spectrometry validated the findings of Raman-DIP analysis.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions.

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

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.

A peptide resource for the analysis of Staphylococcus aureus in host-pathogen interaction studies.

Staphylococcus aureus is an opportunistic human pathogen, which can cause life-threatening disease. Proteome analyses of the bacterium can provide new insights into its pathophysiology and important facets of metabolic adaptation and, thus, aid the recognition of targets for intervention. However, the value of such proteome studies increases with their comprehensiveness. We present an MS-driven, proteome-wide characterization of the strain S. aureus HG001. Combining 144 high precision proteomic data sets, we identified 19 109 peptides from 2088 distinct S. aureus HG001 proteins, which account for 72% of the predicted ORFs. Peptides were further characterized concerning pI, GRAVY, and detectability scores in order to understand the low peptide coverage of 8.7% (19 109 out of 220 245 theoretical peptides). The high quality peptide-centric spectra have been organized into a comprehensive peptide fragmentation library (SpectraST) and used for identification of S. aureus-typic peptides in highly complex host-pathogen interaction experiments, which significantly improved the number of identified S. aureus proteins compared to a MASCOT search. This effort now allows the elucidation of crucial pathophysiological questions in S. aureus-specific host-pathogen interaction studies through comprehensive proteome analysis. The S. aureus-specific spectra resource developed here also represents an important spectral repository for SRM or for data-independent acquisition MS approaches. All MS data have been deposited in the ProteomeXchange with identifier PXD000702 (http://proteomecentral.proteomexchange.org/dataset/PXD000702).

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells - an in vivo proteomics perspective.

Staphylococcus aureus is a versatile pathogen that can be a commensal but also cause a wide range of different infections. This broad disease spectrum is a reflection of the complex regulation of a large collection of virulence factors that together with metabolic fitness allow adaptation to different niches. The alternative sigma factor SigB is one of the global regulators mediating this adaptation. However, even if SigB contributes to expression of many virulence factors its importance for successful infection greatly varies with the strain and the infection setting analyzed. We have recently established a proteomics workflow that combines high efficiency cell sorting with sensitive mass spectrometry and allows monitoring of global proteome adaptations with roughly one million bacterial cells. Thus, we can now approach the adaptation of pathogens to the intracellular milieu. In the current study this proteomics workflow was used in conjunction with qRT-PCR and confocal fluorescence microscopy to comparatively analyze the adaptation of the S. aureus wild type strain HG001 and its isogenic sigB mutant to the intracellular milieu of human S9 bronchial epithelial cells. The study revealed fast and transient activation of SigB following internalization by human host cells and the requirement of SigB for intracellular growth. Loss of SigB triggered proteome changes reflecting the different residual growth rates of wild type and sigB mutant, respectively, the resistance to methicillin, adaptation to oxidative stress and protein quality control mechanisms.

Labeling of the pathogenic bacterium Staphylococcus aureus with gold or ferric oxide-core nanoparticles highlights new capabilities for investigation of host-pathogen interactions.

Throughout the world, infections caused by bacteria such as Staphylococcus aureus are a major cause of morbidity and mortality. In order to gain some understanding of the complicated physiological link between host and pathogen, modern techniques such as confocal microscopy and sophisticated OMICs technologies are suitable. However, labeling of pathogens such as S. aureus with green fluorescent protein, for example, or the generation of a reliable antibody, which are prerequisites for the application of reproducible isolation techniques, does not always succeed. Here, we present a universal approach for monitoring pathogen traffic after internalization into host cells by fluorescence microscopy and for isolation of bacteria from host-pathogen interaction assays using gold or ferric oxide-core, poly(vinyl alcohol) coated, and fluorescence-labeled nanoparticles (NP). The incubation of S. aureus HG001 with those NP had only minor effects on the bacterial growth in vitro. Quantitative proteome analysis after 24 h of NP incubation revealed that presence of NP provoked only marginal changes in the proteome pattern. The method presented enabled us to investigate the behavior of S. aureus HG001 during infection of S9 human epithelial cells by means of fluorescence microscopy and proteomics using magnetic separation or cell sorting.

A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.

The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 106 cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

Analysis of human TAAR8 and murine Taar8b mediated signaling pathways and expression profile.

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates G(s) signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of G(s) and/or G(i/o) signaling. Activation of G-proteins G(q/11) and G(12/13) was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G(i/o) signaling activity, a so far unknown signaling pathway for TAARs.

Defining the structure of the general stress regulon of Bacillus subtilis using targeted microarray analysis and random forest classification.

The structure of the SigB-dependent general stress regulon of Bacillus subtilis has previously been characterized by proteomics approaches as well as DNA array-based expression studies. However, comparing the SigB targets published in three previous major transcriptional profiling studies it is obvious that although each of them identified well above 100 target genes, only 67 were identified in all three studies. These substantial differences can likely be attributed to the different strains, growth conditions, microarray platforms and experimental setups used in the studies. In order to gain a better understanding of the structure of this important regulon, a targeted DNA microarray analysis covering most of the known SigB-inducing conditions was performed, and the changes in expression kinetics of 252 potential members of the SigB regulon and appropriate control genes were recorded. Transcriptional data for the B. subtilis wild-type strain 168 and its isogenic sigB mutant BSM29 were analysed using random forest, a machine learning algorithm, by incorporating the knowledge from previous studies. This analysis revealed a strictly SigB-dependent expression pattern for 166 genes following ethanol, butanol, osmotic and oxidative stress, low-temperature growth and heat shock, as well as limitation of oxygen or glucose. Kinetic analysis of the data for the wild-type strain identified 30 additional members of the SigB regulon, which were also subject to control by additional transcriptional regulators, thus displaying atypical SigB-independent induction patterns in the mutant strain under some of the conditions tested. For 19 of these 30 SigB regulon members, published reports support control by secondary regulators along with SigB. Thus, this microarray-based study assigns a total of 196 genes to the SigB-dependent general stress regulon of B. subtilis.

The alternative sigma factor B modulates virulence gene expression in a murine Staphylococcus aureus infection model but does not influence kidney gene expression pattern of the host.

Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Kidney tissues of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant were analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild-type strain, but not in its isogenic sigB mutant (p<0.0001). Despite a clear-cut difference in the SigB-dependent transcription pattern of virulence genes (clfA, aur, and hla), the host reaction to infection (either wild type or sigB mutant) was almost identical. Despite its significant activity in vivo, loss of SigB did neither have an effect on the outcome of infection nor on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

Proteome analysis of NRF2 inhibition in melanoma reveals CD44 up-regulation and increased apoptosis resistance upon vemurafenib treatment.

Malignant melanoma is the deadliest form of skin cancer and NRF2 has been proposed as a main regulator of tumor cell malignancy. Still the mechanisms how NRF2 is contributing to melanoma progression are incompletely understood. Here we analyzed the effects of either NRF2 induction or depletion, and we also quantified changes on the whole cell proteome level. Our results showed that inhibition of NRF2 leads to a loss of reactive oxygen species protection, but at the same time to an induction of an epithelial mesenchymal transition (EMT) phenotype and an up-regulation of the stem cell marker CD44. Additionally, cells devoid of NRF2 showed increased cell viability after treatment with a MYC and a BRAF inhibitor. Importantly, survival upon vemurafenib treatment was dependent on CD44 expression. Finally, analysis of archival melanoma patient samples confirmed a vice versa relationship of NRF2 and CD44 expression. In summary, we recorded changes in the proteome after NRF2 modulation in melanoma cells. Surprisingly, we identified that NRF2 inhibition lead to induction of an EMT phenotype and an increase in survival of cells after apoptosis induction. Therefore, we propose that it is important for future therapies targeting NRF2 to consider blocking EMT promoting pathways in order to achieve efficient tumor therapy.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

Early-Stage Staphylococcus aureus Bloodstream Infection Causes Changes in the Concentrations of Lipoproteins and Acute-Phase Proteins and Is Associated with Low Antibody Titers against Bacterial Virulence Factors.

Systemic and quantitative investigations of human plasma proteins (proteomics) and Staphylococcus aureus-specific antibodies (immunoproteomics) provide complementary information and hold promise for the discovery of biomarkers in Staphylococcus aureus bloodstream infection (SABSI). Usually, data-dependent acquisition (DDA) is used for proteome analysis of serum or plasma, but data-independent acquisition (DIA) is more comprehensive and reproducible. In this prospective cohort study, we aimed to identify biomarkers associated with the early stages of SABSI using a serum DIA proteomic and immunoproteomic approach. Sera from 49 SABSI patients and 43 noninfected controls were analyzed. In total, 608 human serum proteins were identified with DIA. A total of 386 proteins could be quantified, of which 9 proteins, mainly belonging to acute-phase proteins, were significantly increased, while 7 high-density lipoproteins were lower in SABSI. In SABSI, total anti-S. aureus serum IgG was reduced compared with controls as shown by immunoproteomic quantification of IgG binding to 143 S. aureus antigens. IgG binding to 48 of these anti-S. aureus proteins was significantly lower in SABSI, while anti-Ecb IgG was the only one increased in SABSI. Serum IgG binding to autoinducing peptide MsrB, FadB, EsxA, Pbp2, FadB, SspB, or SodA was very low in SABSI. This marker panel discriminated early SABSI from controls with 95% sensitivity and 100% specificity according to random forest prediction. This holds promise for patient stratification according to their risk of S. aureus infection, underlines the protective function of the adaptive immune system, and encourages further efforts in the development of a vaccine against S. aureus IMPORTANCE S. aureus sepsis has a high complication and mortality rate. Given the limited therapeutic possibilities, effective prevention strategies, e.g., a vaccine, or the early identification of high-risk patients would be important but are not available. Our study showed an acute-phase response in patients with S. aureus bloodstream infection and evidence that lipoproteins are downregulated in plasma. Using immunoproteomics, stratification of patients appears to be achievable, since at the early stages of systemic S. aureus infection patients had low preexisting anti-S. aureus antibody levels. This strengthens the notion that a robust immune memory for S. aureus protects against infections with the pathogen.

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.

Hypermetabolic syndrome as a consequence of repeated psychological stress in mice.

Stress is a powerful modulator of neuroendocrine, behavioral, and immunological functions. After 4.5-d repeated combined acoustic and restraint stress as a murine model of chronic psychological stress, severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with the severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turnover, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure, changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice. Thus, in our murine model, repeated stress caused severe metabolic dysregulations, leading to a drastic reduction of the individual's energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer.

In vivo Proteomics Approaches for the Analysis of Bacterial Adaptation Reactions in Host-Pathogen Settings.

Proteome profiling of bacteria internalized by host cells is still a challenging task, due to low amounts of bacterial proteins in host-pathogen settings and the high amounts of contaminating host proteins. Here, we describe a workflow for the enrichment of intracellular bacteria by fluorescence activated cell sorting which in combination with highly sensitive LC-MS/MS allows monitoring of about 1200 proteins from 2 to 4 × 106 internalized bacterial cells as starting material.

Characterization of human and Staphylococcus aureus proteins in respiratory mucosa by in vivo- and immunoproteomics.

Staphylococcus aureus is a Gram-positive opportunistic bacterium which can be found as a commensal in the nares of about 50% of the human population. Besides asymptomatic carriage, S. aureus has also been found to colonize nasal polyps, a subform of chronic rhinosinusitis, in 60 to 100% of cases, and even reside intracellularly in nasal polyp tissue. The aim of this study was to shed light on the behavior of S. aureus in the human airways by analyzing S. aureus-specific proteins in nasal polyp tissue from patients with chronic rhinosinusitis and to characterize the immunogenic potential of the identified (mainly secreted) proteins. As a result, in total >600 S. aureus proteins were identified by high resolution mass spectrometry or multiple reaction monitoring. Of those roughly 180 are typically localized in the membrane, surface exposed or secreted. For 115 S. aureus proteins, partially also detected in vivo by mass spectrometry, IgA- and IgG-specific antibody signals were profiled. Strong antibody signals were predominantly found for surface expose or secreted proteins. SIGNIFICANCE: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue. We discovered bacterial proteins involved in invasion of tissue, virulence, bacterial signal transduction or acquisition of nutrients. Some of the detected superantigens and Spls are known to provoke secretion of a broad spectrum of cytokines. Therefore, our manuscript contains new information about the invasion of S. aureus in nasal polyp tissue and its protein-specific immunogenicity.