Soluble virulence-associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o-, S9 or A549 cells with recombinant Hla (rHla). In a dose-dependent manner, rHla induced secretion of IL-8 in all three cell types, but IL-6 release only in 16HBE14o- and S9 cells. rHla-mediated secretion of IL-8 and IL-6 was suppressed by pre-incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL-8 release in all three cell types and for IL-6 release in 16HBE14o- and S9 cells. The rHla-mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca(2+)]i, an early response in rHla-treated cells. Inhibitors of calmodulin or calcium/calmodulin-dependent kinase II attenuated rHla-mediated release of IL-8 in 16HBE14o- and A549 cells and of IL-6 in 16HBE14o- cells. This indicates that rHla may mediate simultaneous activation of calmodulin-dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin-dependent signalling did not affect rHla-induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.
Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process. Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process. Staphylococcus aureus is a Gram-positive opportunistic pathogen of humans, but also a commensal of the human body. Specifically, S. aureus is commonly found in the anterior nares of around 30% of the human population (1.Wertheim H.F. Melles D.C. Vos M.C. van Leeuwen W. van Belkum A. Verbrugh H.A. Nouwen J.L. The role of nasal carriage in Staphylococcus aureus infections.Lancet Infect. 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Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management.Clin. Microbiol. Rev. 2015; 28: 603-661Crossref PubMed Scopus (2410) Google Scholar, 4.Breathnach A.S. Nosocomial infections and infection control.Medicine. 2013; 41: 649-653Abstract Full Text Full Text PDF Scopus (26) Google Scholar, 5.Appelbaum P.C. MRSA–the tip of the iceberg.Clin. Microbiol. Infect. 2006; 12: 3-10Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Although S. aureus often acts as an extracellular pathogen, it can evade immune responses and antibiotic therapy by entering human cells. The latter strategy is also used by the bacteria as a mechanism to spread to other tissues and both professional as well as non-professional phagocytic cells are used for internalization (6.Fraunholz M. Sinha B. Intracellular Staphylococcus aureus: Live-in and let die.Front. Cell. Infect. 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After the bacteria have been taken up by the host cells, they will initially be localized in vesicles, which subsequently might fuse with lysosomes or be engulfed by an isolation membrane because of autophagy, and the bacteria inside them may prevail or escape into the cytosol. Although the internalization by host cells is potentially lethal for the bacteria, the survivors will have two options: proliferation or persistence. In the first case, the bacteria replicate intracellularly and subsequently induce lysis of the host cells. The released bacteria search for new host cells to be infected and spread into new tissues (6.Fraunholz M. Sinha B. Intracellular Staphylococcus aureus: Live-in and let die.Front. Cell. Infect. Microbiol. 2012; 2: 43Crossref PubMed Scopus (224) Google Scholar, 7.Garzoni C. Kelley W.L. Staphylococcus aureus: new evidence for intracellular persistence.Trends Microbiol. 2009; 17: 59-65Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 8.Lehar S.M. Pillow T. Xu M. Staben L. Kajihara K.K. Vandlen R. DePalatis L. Raab H. Hazenbos W.L. Hiroshi Morisaki J. Kim J. Park S. Darwish M. Lee B.-C. Hernandez H. Loyet K.M. Lupardus P. Fong R. Yan D. Chalouni C. Luis E. Khalfin Y. Plise E. Cheong J. Lyssikatos J.P. Strandh M. Koefoed K. Andersen P.S. Flygare J.A. Wah Tan M. Brown E.J. Mariathasan S. Novel antibody–antibiotic conjugate eliminates intracellular S. aureus.Nature. 2015; 527: 323-328Crossref PubMed Scopus (506) Google Scholar). In the second case, the persistent bacteria do not multiply, but adapt to the intracellular environment and may survive intracellularly without causing clinical symptoms for extended time periods. This pattern has been linked to relapse of infections or emergence of small colony variants (SCVs) 1 of S. aureus which display reduced metabolic activity (9.Proctor R.A. van Langevelde P. Kristjansson M. Maslow J.N. Arbeit R.D. Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 1995; 20: 95-102Crossref PubMed Scopus (339) Google Scholar, 10.Seifert H. Wisplinghoff H. Schnabel P. von Eiff C. Small colony variants of Staphylococcus aureus and pacemaker-related infection.Emerg. Infect. Dis. 2003; 9: 1316-1318Crossref PubMed Scopus (68) Google Scholar, 11.Tuchscherr L. Bischoff M. Lattar S.M. Noto Llana M. Pförtner H. Niemann S. Geraci J. Van de Vyver H. Fraunholz M.J. Cheung A.L. Herrmann M. Völker U. Sordelli D.O. Peters G. Löffler B. Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections.PLoS Pathog. 2015; 11: e1004870Crossref PubMed Scopus (118) Google Scholar). Despite strain-specific differences in overall virulence, all S. aureus strains, including laboratory strains, can display proliferative and persistent phenotypes. Although this phenomenon has been known (6.Fraunholz M. Sinha B. Intracellular Staphylococcus aureus: Live-in and let die.Front. Cell. Infect. Microbiol. 2012; 2: 43Crossref PubMed Scopus (224) Google Scholar), the actual adaptations either enabling active intracellular proliferation or reduced metabolic activity and persistence are still poorly understood. The precise outcome of the interplay between the bacterium and the host depends on the type of host cell involved and, perhaps most importantly, the physiological states of both parties (12.Strobel M. Pförtner H. Tuchscherr L. Völker U. Schmidt F. Kramko N. Schnittler H.-J. Fraunholz M.J. Löffler B. Peters G. Niemann S. Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.Clin. Microbiol. Infect. 2016; 22: 799-809Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13.Surmann K. Michalik S. Hildebrandt P. Gierok P. Depke M. Brinkmann L. Bernhardt J. Gesell Salazar M. Sun Z. Shteynberg D. Kusebauch U. Moritz R.L. Wollscheid B. Lalk M. Völker U. Schmidt F. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells.Front. Microbiol. 2014; 5: 392Crossref PubMed Scopus (34) Google Scholar). The main challenge in obtaining a detailed understanding of the adaptive behavior of internalized S. aureus lies in the fact that it is essential to study quantitative changes over an extended period, not only in one of the two interacting parties but simultaneously in both. Previous studies have addressed these aspects only partially either by focusing on the internalized bacteria only, or over only short periods of time post infection (p.i.) (11.Tuchscherr L. Bischoff M. Lattar S.M. Noto Llana M. Pförtner H. Niemann S. Geraci J. Van de Vyver H. Fraunholz M.J. Cheung A.L. Herrmann M. Völker U. Sordelli D.O. Peters G. Löffler B. Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections.PLoS Pathog. 2015; 11: e1004870Crossref PubMed Scopus (118) Google Scholar, 12.Strobel M. Pförtner H. Tuchscherr L. Völker U. Schmidt F. Kramko N. Schnittler H.-J. Fraunholz M.J. Löffler B. Peters G. Niemann S. Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.Clin. Microbiol. Infect. 2016; 22: 799-809Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14.Hecker M. Mäder U. Völker U. From the genome sequence via the proteome to cell physiology – Pathoproteomics and pathophysiology of Staphylococcus aureus.Int. J. Med. Microbiol. 2018; 308: 545-557Crossref PubMed Scopus (12) Google Scholar, 15.Kiedrowski M.R. Paharik A.E. Ackermann L.W. Shelton A.U. Singh S.B. Starner T.D. Horswill A.R. Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.Cell. Microbiol. 2016; 18: 720-732Crossref PubMed Scopus (19) Google Scholar, 16.Richter E. Harms M. Ventz K. Nölker R. Fraunholz M.J. Mostertz J. Hochgräfe F. Quantitative proteomics reveals the dynamics of protein phosphorylation in human bronchial epithelial cells during internalization, phagosomal escape, and intracellular replication of Staphylococcus aureus.J. Proteome Res. 2016; 15: 4369-4386Crossref PubMed Scopus (7) Google Scholar, 17.Surmann K. Simon M. Hildebrandt P. Pförtner H. Michalik S. Stentzel S. Steil L. Dhople V.M. Bernhardt J. Schlüter R. Depke M. Gierok P. Lalk M. Bröker B.M. Schmidt F. Völker U. A proteomic perspective of the interplay of Staphylococcus aureus and human alveolar epithelial cells during infection.J. Proteomics. 2015; 128: 203-217Crossref PubMed Scopus (21) Google Scholar). Yet, it is important to get the "complete picture" of such an infection scenario, because the invasion and destruction of lung epithelial cells is representative for some of the most serious staphylococcal diseases possible, especially necrotizing pneumonia. The present study was designed to close the current knowledge gap on the interplay between S. aureus and lung epithelial cells by a time-resolved analysis of both parties over the longest possible period. The limits for such an analysis are set by the amount of material that can be extracted for bacteria- and host cell-specific analyses, and the parameters to be measured. This led us to a proteomics approach, where adaptations of the bronchial epithelial cell line 16HBE14o- and S. aureus were followed up to 4 days p.i. using a data independent acquisition (DIA) method. Importantly, our findings highlight dynamic adaptive changes, in both the host and the internalized pathogen, and describe the active cross-talk between them at different stages of infection. Additionally, we correlate these adaptations with the intracellular localization of the bacteria p.i. and the epithelial cells' response. The observations suggest that, after a period of violent conflict, both parties reach an equilibrium phase where they are apparently at peace and the bacteria have reached a persister status. S. aureus strain HG001 (18.Herbert S. Ziebandt A.-K. Ohlsen K. Schäfer T. Hecker M. Albrecht D. Novick R. Götz F. Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates.Infect. Immun. 2010; 78: 2877-2889Crossref PubMed Scopus (269) Google Scholar) was used to perform all experiments. The bacteria carried plasmid pJL-sar-GFP to constitutively express the green fluorescent protein (GFP; Liese et al., 2013). For the immunostaining protocols, a spa mutant was used to prevent unspecific binding of marker antibodies to protein A. The HG001 Δspa strain was kindly provided by Dr. Jan Pané-Farré, University of Greifswald. Cultivation of bacteria was performed in prokaryotic minimal essential medium (pMEM): 1x MEM without sodium bicarbonate (Invitrogen, Karlsruhe, Germany) supplemented with 1x non-essential amino acids (PAN-Biotech GmbH, Aidenbach, Germany), 4 mm l-glutamine (PAN-Biotech GmbH), 10 mm HEPES (PAN-Biotech GmbH), 2 mm l-alanine, 2 mm l-leucine, 2 mm l-isoleucine, 2 mm l-valine, 2 mm l-aspartate, 2 mm l-glutamate, 2 mm l-serine, 2 mm l-threonine, 2 mm l-cysteine, 2 mm l-proline, 2 mm l-histidine, 2 mm l-phenylalanine and 2 mm l-tryptophan (All from Sigma-Aldrich, Schnelldorf, Germany), adjusted to pH 7.4 and sterilized by filtration. One day before the infection of epithelial cells, bacterial overnight cultures in pMEM supplemented with 0.01% yeast extract (Sigma-Aldrich) and 10 μg/ml erythromycin (Sigma-Aldrich) were prepared by serial dilutions (1×10−6 up to 1×10−10) of a 100 μl glycerol stock of a bacterial culture with an OD600 of 1.2. Incubation was performed at 37 °C and 220 rpm. The following day, the main culture was inoculated from an overnight culture with an OD600 between 0.3 to 0.8. The starting OD600 of the main culture was set to 0.05 and it was incubated for ∼2 h in a shaking water bath at 150 rpm and 37 °C until it reached the mid-exponential phase at an OD600 of ∼0.4 (supplemental Fig. S1). The bacteria were then harvested and used for preparation of the master mix for infection as explained below in the Internalization Experiments" paragraph. The human epithelial cell line 16HBE14o- is a transformed bronchial epithelial cell line originally derived from a 1-year-old heart-lung transplant patient (20.Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (776) Google Scholar). This cell line is known for its ability to form tight junctions and to differentiate. The cells were cultured at 37 °C in 5% CO2 in eukaryotic minimal essential medium (eMEM): 1× MEM (Biochrom AG, Berlin, Germany) supplemented with 10% (v/v) fetal calf serum (FCS; Biochrom AG), 2% (v/v) l-glutamine 200 mm (PAN-Biotech GmbH) and 1% (v/v) nonessential amino acids 100x (PAN-Biotech GmbH). The splitting of cells was carried out every 3 days with 0.25% trypsin-EDTA (Gibco®, Grand Island, NY). After thawing of frozen stocks (in liquid N2) the cells were maintained for 20 additional passages. The cell lines stocks used are not authenticated. Four independent biological replicates of the infection set-up were used for quantification of bacterial and host cell populations, and for mass spectrometry measurements. The number of replicates was selected to ensure that at every time point there were at least three consistent measurements for every protein. The sampling of each independent infection consisted of 8 samples taken over the course of 4 days, including a 0 h sample which is the control condition. In total, 32 samples of the cytosolic proteome of bacteria and 32 samples of the human bronchial epithelial cell proteome were measured. To avoid measuring replicates of the same condition sequentially, the measuring order of each set of samples was determined by assigning a random number between 1 and 32 to each sample (function sample in R version 3.4.4 (21.R Core Team R: A language and environment for statistical computing.R Foundation for Statistical Computing. 2018; Google Scholar)). Additionally, samples for imaging were collected from three independent infection experiments. To determine changes over time in protein abundance, an empirical Bayes moderated F-test was conducted for each protein profile. This test also evaluates the similarity of the replicates. The moderated p values were corrected for multiple testing using Benjamini and Hochberg's multiple testing correction. Internalization experiments were performed essentially as described by Pförtner et al. (22.Pförtner H. Wagner J. Surmann K. Hildebrandt P. Ernst S. Bernhardt J. Schurmann C. Gutjahr M. Depke M. Jehmlich U. Dhople V. Hammer E. Steil L. Völker U. Schmidt F. A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.Methods. 2013; 61: 244-250Crossref PubMed Scopus (25) Google Scholar). Briefly, internalization was performed using a confluent 16HBE14o- cell layer seeded at a density of 1×105 cells/cm2 in 12-well plates, 3 days before infection. The infection was carried out at a multiplicity of infection (MOI) of 25 bacteria per host cell. The master mix for infection was prepared from a mid-exponential (OD600 of 0.4) culture of S. aureus HG001 diluted in eMEM, buffered with 2.9 μl sodium hydrogen carbonate (7.5%, PAN-Biotech GmbH) per ml bacterial culture added. The growth medium over the confluent epithelial layer was replaced with the master mix, and the coculture was incubated for 1 h at 37 °C in 5% CO2. Afterward, the medium was collected (non-adherent sample) and replaced with eMEM medium containing 10 μg 47 ml of lysostaphin (AMBI Products LLC, Lawrence, NY). The medium was replaced every 2 days. For collection of the proteome samples, the culturing medium was aspirated, and the epithelial cell layers were treated for 5 min at 37 °C with UT buffer (8 m urea, 2 m thiourea in MS-grade water; Sigma-Aldrich) to generate samples for analysis by mass spectrometry (MS). If samples were intended for the collection of bacteria, the disruption of epithelial cells was performed for 5 min at 37 °C in 0.05% sodium dodecyl sulfate (SDS; Carl Roth, Karlsruhe, Germany). Samples were collected at 2.5 h, 6.5 h, 24 h, 48 h, 72 h, and 96 h p.i. To monitor changes in the abundance of human and bacterial cells, counting was performed at the times of sample collection. Epithelial cells were counted after staining with trypan blue dye using a Countess® system (Invitrogen). Quantification of intracellular bacteria and infected epithelial cells was performed with a Guava® easyCyte flow cytometer (Merck Millipore, Darmstadt, Germany) by excitation of the GFP with a 488 nm laser and detection at 510–540 nm. After disruption of epithelial cells with 0.05% SDS, two million liberated bacteria were sorted by flow cytometry using a FACSAria IIIu cell sorter (Becton Dickinson Biosciences, Franklin Lakes, NJ) per time point. The recognition of bacteria was carried out by excitation with a 488 nm laser and the emission was detected in the range of 515–545 nm. The bacterial cells were collected on low protein binding filter membranes with a pore size of 0.22 μm (Merck Millipore). These bacteria-containing filters were immediately placed in Eppendorf tubes that were then frozen by transferring them to a −20 °C freezer for the course of the experiment and then kept at −80 °C until use. The bacteria on the filter were lysed by incubation for 30 min at 37 °C with 7.4 μg/ml lysostaphin in 50 mm ammonium bicarbonate (Sigma-Aldrich) (23.Depke M. Michalik S. Rabe A. Surmann K. Brinkmann L. Jehmlich N. Bernhardt J. Hecker M. Wollscheid B. Sun Z. Moritz R.L. Völker U. Schmidt F. A peptide resource for the analysis of Staphylococcus aureus in host pathogen interaction studies.Proteomics. 2015; 15: 3648-3661Crossref PubMed Scopus (17) Google Scholar). Digestion of bacterial proteins on the filter was performed overnight at 37 °C with 0.1% Rapigest SF surfactant (Waters, Eschborn, Germany) and 0.3 μg of trypsin (Promega, Madison, WI). For human proteome analyses, the protein content of samples was quantified using a Bradford assay (Bio-Rad, Hercules, CA). Four μg of protein per sample were prepared for MS measurements by reduction with 2.5 mmol/L dithiothreitol (Thermo Fisher Scientific, Idstein, Germany) for 1 h at 60 °C and alkylation with 10 mmol/L iodoacetamide (Sigma-Aldrich) for 30 min at 37 °C. Then, the samples were digested overnight with trypsin (protein/trypsin 25:1) at 37 °C. The following 16HBE14o- samples were used for the construction of the spectral library of the host: a confluent cell layer cultured in a 10 cm dish for 3 days, an apoptotic cell layer in a 10 cm dish cultured for a week, non-polarized cells cultured for 3 days over Transwells®, and lastly polarized cells cultured for 11 days over Transwells® (Corning, Schnelldorf, Germany). The last two conditions were grown over 12 mm inserts with 0.4 μm pores, and with media volumes of 400 μl on the apical side and 1300 μl on the basal side of the cultures. Furthermore, to expand the host proteome library, published reads (24.Michalik S. Depke M. Murr A. Gesell Salazar M. Kusebauch U. Sun Z. Meyer T.C. Surmann K. Pförtner H. Hildebrandt P. Weiss S. Palma Medina L.M. Gutjahr M. Hammer E. Becher D. Pribyl T. Hammerschmidt S. Deutsch E.W. Bader S.L. Hecker M. Moritz R.L. Mäder U. Völker U. Schmidt F. A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.Sci. Rep. 2017; 7: 9718Crossref PubMed Scopus (35) Google Scholar) of the bronchial epithelium cell line S9 were also used. These cells are immortalized cells isolated from a patient with cystic fibrosis that were transformed with a hybrid virus adeno-12-SV40 (ATCC® number CRL-2778) (25.Zeitlin P.L. Lu L. Rhim J. Cutting G. Stetten G. Kieffer K.A. Craig R. Guggino W.B. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection.Am. J. Respir. Cell Mol. Biol. 1991; 4: 313-319Crossref PubMed Scopus (273) Google Scholar). For the construction of the host proteome spectral library, aliquots of the different samples of whole cell lysates of 16HBE14o- in UT buffer were mixed, and then 25 μg of the extract mixture was fractionated by SDS-PAGE. The gel was partitioned into ten protein-containing pieces that were destained by 15 min washes with ammonium bicarbonate solution (200 mm) in 50% acetonitrile (Mallinckrodt Baker, Inc., Deventer, Netherlands) at 37 °C and 500 rpm. Then, the gel pieces were dehydrated by incubation with acetonitrile at 37 °C and 500 rpm. The supernatant was discarded afterward. Proteins in each gel piece were in-gel digested overnight at 37 °C with 20 μl of trypsin (10 ng/μl) and 30 μl ammonium bicarbonate solution (20 mm). Lastly, the peptides were extracted by addition of 0.1% acetic acid (Carl Roth) and incubation in an ultrasound bath for 30 min. Afterward, the supernatant was collected, 50% acetonitrile with 0.05% acetic acid were added to the gel pieces for another 30 min incubation, and both supernatant fractions were united. Two of the supernatants of the ten SDS-PAGE fractions were mixed to generate five final samples with essentially the same protein quantity, which were then used for further processing and DDA-measurements. The tryptic peptides derived from bacterial or human proteins were concentrated and purified using C18 ZipTip columns (Merck Millipore). All samples were resuspended in a buffer consisting of 2% acetonitrile and 0.1% acetic acid in MS-grade water. Indexed Retention Time (iRT) peptides (Biognosys AG, Schlieren, Switzerland) were added to the samples for feature alignment, peak calibration and signal quantification. The spike in of the samples was carried out according to the manufacturer′s instructions assuring that the injected volumes have one IE (injection equivalent) of iRT peptide mix. The final volume of the samples and the injection volumes were set to 12 μl and 10 μl for S. aureus samples, 20 μl and 5 μl for 16HBE14o- samples, and 20 μl and 4 μl for the spectral library samples, respectively. Tryptic peptides were separated on an Accucore 150-C18 analytical column of 250 mm (25 cm × 75 μm, 2.6 μm C18 particles, 150 Å pore size, Thermo Fischer Scientific, Waltham, MA) using a Dionex Ultimate 3000 nano-LC system (Thermo Fischer Scientific). Peptides were eluted at a constant temperature of 40 °C and a flow rate of 300 nL/min with a 120 min linear gradient (2% to 25%) of buffer (acetonitrile in 0.1% acetic acid). To design a spectral library MS/MS data were recorded on a Q Exactive mass spectrometer (Thermo Fischer Scientific) in data dependent mode (DDA). The MS scans were carried out in a m/z range of 300 to 1650 m/z. Data was acquired with a resolution of 70,000 and an AGC target of 3×106. The top 10 most abundant isotope patterns with charge ≥2 from the survey scan were selected for fragmentation by high energy collisional dissociation (HCD) with a maximal injection time of 120 ms, an isolation window of 3 m/z, and a normalized collision energy of 27.5 eV. Dynamic exclusion was set to 30 s. The MS/MS scans had a resolution of 17,500 and an AGC target of 2×105. MS/MS analyses of samples were performed in data independent mode (DIA) on a Q Exactive Plus mass spectrometer (Thermo Fischer Scientific) following the method described by Bruderer et al. (26.Bruderer R. Bernhardt O.M. Gandhi T. Miladinović S.M. Cheng L.-Y. Messner S. Ehrenberger T. Zanotelli V. Butscheid Y. Escher C. Vitek O. Rinner O. Reiter L. Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.Mol. Cell. Proteomics. 2015; 14: 1400-1410Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). Briefly, the data was acquired in the m/z range from 400 to 1220 m/z, the resolution for MS and MS/MS was 35,000, and the AGC target was 5×106 for MS, and 3×106 for MS/MS. The number of DIA isolation windows was 19 with 2 m/z overlap. For further details to the instrumental setup and the parameters for LC-MS/MS analysis in DDA and DIA mode see supplemental Tables S1 and S2. Time-lapse imaging was carried out with a DeltaVisionRT deconvolution microscope (GE Healthcare Europe GmbH, Freiburg im Breisgau, Germany). To perform the imaging, the actual infection experiment was carried out on a glass bottom 35-mm plate (MatTek, Ashland, MA). After a change of media with lysostaphin, the plate was transferred to the microscope base under incubation conditions. Imaging of the epithelial layer was performed by light microscopy, whereas GFP fluorescent bacteria were observed by excitation with a 490/20 nm mercury vapor lamp and detection of fluorescence at 528/38 nm. Image acquisition was performed every 5 min for the first 48 h, then from 48 h to 72 h and finally from 92 h to 96 h. Picture processing was performed with Fiji (http://fiji.sc/Fiji). Subcellular localization of microtubule-associated protein 1A/1B-light chain (LC3) and lysosomal-associated membrane protein 1 (LAMP-1) by immunofluorescence microscopy was performed using a Leica TCS SP8 Confocal laser scanning microscope (Leica Microsystems B.V., Amsterdam, Netherlands). The cells were seeded over coverslips of 18 mm diameter 3 days before infection as described above. However, in this case a HG001 Δspa mutant was used to avoid aspecific IgG binding. The samples were collected at 0 h, 1 h, 2.5 h, 6.5 h, 24 h, 48 h, 72 h, and 96 h by fixation with 2% para-formaldehyde (PFA, Merck Millipore) for 20 min at room temperature. Preparation of the samples for the actual microscopy was performed simultaneously after conclusion of the experiment. The samples were permeabilized with 0.5% Tween 20 (Sigma-Aldrich) for 30 min at room temperature and then nonspecific binging sites were blocked with 1% bovine serum albumin, 10% FCS in 0.07% Tween 20 for 120 min at room temperature. All antibodies were diluted in blocking solution. Primary rabbit anti-LC3B (Cat. No. 1384; Novus Biologicals, Oxon, England) and mouse CD107a (LAMP-1; Cat. No. 555798; BD, Drachten, Netherlands) antibodies were used at 1:500 and 1:100 dilutions, respe
Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.
ABSTRACT Seemingly simple bacteria mount intricate adaptive responses when exposed to physical stress or nutrient limitation, and the activation of these responses is governed by complex signal transduction networks. Upon entry into the stationary growth phase, the soil bacterium Bacillus subtilis may develop natural competence, form biofilms or stress-resistant cells, or ultimately trigger a cellular differentiation program leading to spore formation. Master regulators, such as Spo0A, ComK, SinR, and SigB, constantly monitor the bacterium’s environment and then determine appropriate adaptive responses. Here, we show that exposure of B. subtilis to visible light and other stresses triggers a general stress response-dependent block in competence development. SigB serves as an “emergency system” to silence inappropriate expression of an alternative developmental program in the face of unfavorable conditions. In particular, we document a stress-dependent molecular mechanism that prevents accumulation of the central competence regulator ComK via expression of a SigB-driven antisense RNA ( as-comK , S365) which is part of a noncontiguous operon. IMPORTANCE Bacillus subtilis exhibits a large number of different specific and general adaptation reactions, which need to be well balanced to sustain survival under largely unfavorable conditions. Under specific conditions, natural competence develops, which enables B. subtilis to actively take up exogenous DNA to integrate it into its own genome. In contrast to this specific adaptation, the general stress response is induced by a variety of exogenous stress and starvation stimuli, providing comprehensive protection and enabling survival of vegetative B. subtilis cells. In the present work, we reveal the molecular basis for the interconnection of these two important responses in the regulatory network. We describe that the master regulator of the general stress response SigB is activated by physiological stress stimuli, including daylight and ethanol stress, leading to the inactivation of the competence master regulator ComK by transcriptional anti-sense regulation, showing a strict hierarchy of adaptational responses under severe stress.
To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP) encoding a continuously expressed green fluorescent protein (GFP). Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed). Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC) standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC-MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]). They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.
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