Staphylococcus Aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line
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H OH metabolites OH Article Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line Philipp Gierok 1, Manuela Harms 2, Karen Methling 1, Falko Hochgräfe 2 and Michael Lalk 1,* 1 Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany; [email protected] (P.G.); [email protected] (K.M.) 2 Competence Center Functional Genomics, Junior Research Group Pathoproteomics, University of Greifswald, 17487 Greifswald, Germany; [email protected] (M.H.); [email protected] (F.H.) * Correspondence: [email protected]; Tel.: +49-3834-86-4867 Academic Editors: Wolfgang Eisenreich and Adelbert Bacher Received: 6 October 2016; Accepted: 2 November 2016; Published: 9 November 2016 Abstract: The Gram positive opportunistic human pathogen Staphylococcus aureus induces a variety of diseases including pneumonia. S. aureus is the second most isolated pathogen in cystic fibrosis patients and accounts for a large proportion of nosocomial pneumonia. Inside the lung, the human airway epithelium is the first line in defence with regard to microbial recognition and clearance as well as regulation of the immune response. The metabolic host response is, however, yet unknown. To address the question of whether the infection alters the metabolome and metabolic activity of airway epithelial cells, we used a metabolomics approach. The nutrition uptake by the human airway epithelial cell line A549 was monitored over time by proton magnetic resonance spectroscopy (1H-NMR) and the intracellular metabolic fingerprints were investigated by gas chromatography and high performance liquid chromatography (GC-MS) and (HPLC-MS). To test the metabolic activity of the host cells, glutamine analogues and labelled precursors were applied after the infection. We found that A549 cells restrict uptake of essential nutrients from the medium after S. aureus infection. Moreover, the infection led to a shutdown of the purine and pyrimidine synthesis in the A549 host cell, whereas other metabolic routes such as the hexosamine biosynthesis pathway remained active. In summary, our data show that the infection with S. aureus negatively affects growth, alters the metabolic composition and specifically impacts the de novo nucleotide biosynthesis in this human airway epithelial cell model. Keywords: host cell metabolism; infection; metabolomics; Staphylococcus aureus 1. Introduction The Gram positive bacterium Staphylococcus aureus is not only a permanent commensal of about 20% of the world population but also an opportunistic pathogen [1,2]. Infections can result in diverse clinical manifestations such as soft local tissue infections, endocarditis, sepsis and also pneumonia [2,3]. S. aureus has been described earlier as an extracellular pathogen that exhibits its pathogenicity with the secretion of virulence factors [4]. Within the last two decades, however, S. aureus has also been recognized as an invasive pathogen with an intracellular lifestyle [5,6]. It was shown by proteomic and transcriptomic studies that intracellular S. aureus undergoes changes in expression of metabolic genes, nutrient transporters and virulence factors to adapt to the intracellular environment [7,8]. To prevent colonization in the human lung, the respiratory epithelium maintains an effective antimicrobial environment. This is accomplished by various antimicrobial strategies such as forming a physical barrier, mucociliary clearance, and production of antimicrobial peptides, surfactant proteins, Metabolites 2016, 6, 41; doi:10.3390/metabo6040041 www.mdpi.com/journal/metabolites Metabolites 2016, 6, 41 2 of 21 Metabolites 2016, 6, 41 2 of 21 physical barrier, mucociliary clearance, and production of antimicrobial peptides, surfactant proteins, complement, chemokines, and cytokines [9–11]. [9–11]. Many Many of of these defence mechanisms are activated by staphylococcal virulence factors [12] [12] but so far only little is known about the consequences on the host cell metabolism. metabolism. Recently, Recently, we we described described the the effect effect of ofstaphylococcal staphylococcal alpha alpha toxin toxin (Hla) (Hla) on glycolysis on glycolysis and andglutaminolysis glutaminolysis of human of human airway airway epithelial epithelial cells cells [13]. [13 Although]. Although this this study study shows shows that that the the hosthost metabolism is affected by the action of single virulencevirulence factors, the complex process of infection might influenceinfluence the host cell metabolismmetabolism very differently.differently. DuringDuring thethe invasioninvasion processprocess S. aureus adherenceadherence proteins such as fibronectinfibronectin binding proteins bind toto hosthost cellcell structuresstructures suchsuch asas αα55ββ11 integrinintegrin via fibronectinfibronectin and induce a zipper-typezipper-type uptakeuptake [[14].14]. The uptake activates the rearrangement of the cytoskeleton [15] [15] and several regulators that are also involved in metabolism such as thethe PI3K-AktPI3K-Akt pathway [[16–18].16–18]. Moreover, cellular processes that are directly coupled to the host metabolism such as autophagy [[19]19] andand apoptosis apoptosis [20 [20]] are are affected affected by S.by aureus. S. aureus.In between In between this complex this complex interplay interplay of cellular of processescellular processes and signalling and signalling events metabolites events metabolite serve as signals serve molecules, as signal precursors molecules, for precursors antimicrobial for effectorantimicrobial molecules effector and molecules also fuel and primary also fuel anabolic primary and anabolic catabolic and pathways. catabolic From pathways. the view From of the intracellularview of the intracellular pathogen the pathogen host cell the metabolome host cell me representstabolome a sourcerepresents of nutrients a source [21of]. nutrients Interestingly, [21]. onlyInterestingly, adapted only bacteria adapted are able bacteria to grow are inable this to environmentgrow in this environment [22]. Therefore, [22]. alterations Therefore, in alterations the host cell in metabolitethe host cell composition metabolite composition also affect the also intracellular affect the intracellular pathogen. pathogen. In this work the host cell metabolome of A549 human airway epithelial cells was examined and the effect of the infection with S. aureus was elucidated on the intracellular and extracellular level. level. We observedobserved inin infectedinfected A549A549 cellscells aa stronglystrongly reducedreduced uptakeuptake ofof nutrients,nutrients, especially of essential amino acids.acids. MoreoverMoreover the the analysis analysis of theof the intracellular intracellular metabolic metabolic profiles profiles in a time in dependenta time dependent manner showedmanner dynamicshowed dynamic changes inchanges the content in the of freecontent amino of acidsfree amino and certain acids nucleotides.and certain Furthermore,nucleotides. weFurthermore, elucidated we that elucidated the de novo that synthesisthe de novo of purinesynthesi ands of pyrimidine purine and nucleotidespyrimidine nucleotides is shut down is aftershut infectiondown after by infection using metabolic by using inhibitors metabolic and inhibitors a metabolic and a labelling metabolic approach. labelling approach. 2. Results Results 2.1. A549 Cells Enter Growth Arrest after Exposure to S. aureus After the infection, A549 cells were incubated for 72 h and the cell number and the amount of intracellular S. aureus cells was monitored. We We replaced the medium every 24 h to prevent nutrient limitation and to reduce the amount of dead cells sincesince about 25% of the population died within the firstfirst 24 h after infection.infection. BetweenBetween 2424 hh andand 4848h hthe the cell cell number number remained remained stable, stable, whereas whereas between between 48 48 h andh and 72 72 h weh we observed observed an increasean increase of 38% of (Figure38% (Fig1A).ure Simultaneously, 1A). Simultaneously, we observed we observed a constant a constant decline indecline the amount in the amount of intracellular of intracellular bacteria bacteria over 72 hover (Figure 72 h1 B).(Figure The cell1B). numberThe cell ofnumber control of cells control doubled cells withindoubled 24 within h after 24 which h after maximum which maximum confluence confluence were reached. were Thesereached. data These indicate data that indicate the exposure that the ofexposure A549 cells of A549 to S. cells aureus to S.led aureus to cell led death to cell in death a proportion in a proportion of cells of and cells to aand temporary to a temporary growth growth arrest. Sincearrest. most Since dying most hostdying cells host were cells probably were probably infected, infected, this would this would also explain also explain the strong the strong decline decline of the S.of aureusthe S. aureuscell number cell number within within the cell the culture. cell culture. Figure 1. Cell numbers of A549 cells and S. aureus during infection. (A) Growth of infected (grey) and Figure 1. Cell numbers of A549 cells and S. aureus during infection. (A) Growth of infected (grey) and control A549 cells (black) is displayed in percentages with 100% being the initial cell number. Data control A549 cells (black) is displayed in percentages with 100% being the initial cell number. Data are are presented as mean with standard deviation (n = 5). (B) Total amount of colony