Influence of Impaired Lipoprotein Biogenesis on ... - ACS Publications

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Influence of Impaired Lipoprotein Biogenesis on Surface and Exoproteome of Streptococcus pneumoniae Thomas Pribyl,† Martin Moche,‡,∥ Annette Dreisbach,§,∥ Jetta J.E. Bijlsma,§,∥ Malek Saleh,† Mohammed R. Abdullah,† Michael Hecker,‡ Jan Maarten van Dijl,§ Dörte Becher,*,‡ and Sven Hammerschmidt*,† †

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, Greifswald D-17487, Germany ‡ Department of Microbial Physiology, Institute for Microbiology, Ernst Moritz Arndt University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15, Greifswald D-17487, Germany § Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands S Supporting Information *

ABSTRACT: Surface proteins are important for the fitness and virulence of the Gram-positive pathogen Streptococcus pneumoniae. They are crucial for interaction of the pathogen with its human host during infection. Therefore, the analysis of the pneumococcal surface proteome is an important task that requires powerful tools. In this study, two different methods, an optimized biotinylation approach and shaving with trypsin beads, were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. The identification of nearly 95% of the predicted lipoproteins and 75% of the predicted sortase substrates reflects the high coverage of the two classical surface protein classes accomplished in this study. Furthermore, the biotinylation approach was applied to study the impact of an impaired lipoprotein maturation pathway on the cell envelope proteome and exoproteome. Loss of the lipoprotein diacylglyceryl transferase Lgt leads to striking changes in the lipoprotein distribution. Many lipoproteins disappear from the surface proteome and accumulate in the exoproteome. Further insights into lipoprotein processing in pneumococci are provided by immunoblot analyses of bacterial lysates and corresponding supernatant fractions. Taken together, the first comprehensive overview of the pneumococcal surface and exoproteome is presented, and a model for lipoprotein processing in S. pneumoniae is proposed. KEYWORDS: S. pneumoniae, surface proteome, shaving with trypsin beads, biotinylation approach, exoproteome, lipoprotein processing, lipoprotein maturation mutations, lipoprotein distribution

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INTRODUCTION Streptococcus pneumoniae, also known as pneumococcus, is a Gram-positive facultative pathogen that colonizes the human respiratory tract of healthy individuals as a harmless commensal. However, this bacterium is also responsible for severe local infections like otitis media and sinusitis and lifethreatening invasive diseases such as pneumonia, septicaemia, and meningitis.1 Pneumococci produce a variety of colonization and virulence factors, including the polysaccharide capsule, the toxin pneumolysin, and surface proteins, which are considered to be important for bacterial fitness and virulence. Surfaceexposed proteins are involved in nutrient uptake, mediate bacterial adhesion to host cells, and contribute to colonization, invasive infections and immune evasion.2−5 Studying the pneumococcal surface proteome under different physiological or in vivo relevant conditions will improve our understanding © 2014 American Chemical Society

of the interaction between the pathogen and its host during the infection process. The pneumococcal cell surface is decorated by different classes of proteins, which are distinguished according to their anchoring mechanism. There are membrane-anchored proteins with extracellular domains, LPxTG cell wall-anchored proteins, choline-binding proteins (CBPs), lipid-anchored proteins, secreted proteins, and nonclassical surface proteins.2 Proteins containing the LPxTG-motif are covalently attached to the peptidoglycan by a sortase (SrtA) and are typical for Grampositive bacteria,6,7 whereas CBPs are anchored to the cell wall via noncovalent attachment to phosphorylcholine and represent a class of surface proteins specific to pneumococci.8 NonReceived: July 25, 2013 Published: January 3, 2014 650

dx.doi.org/10.1021/pr400768v | J. Proteome Res. 2014, 13, 650−667

Journal of Proteome Research

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many pneumococcal lipoproteins are substrate-binding proteins of ABC-type transport systems and therefore crucial for bacterial fitness.44,45 In the present study, an optimized biotinylation approach and a highly specific shaving approach with immobilized trypsin were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. A newly developed protocol preventing autolysis of pneumococci during incubation was highly beneficial for both methods and made the presented comprehensive overview of the surface proteome and exoproteome of S. pneumoniae possible. Because the virulence of the pneumococcal Δlgt mutant is attenuated,43,46 we hypothesized that this phenotype might be related to the absence or improper anchoring of lipoproteins on the pneumococcal surface. Therefore, the biotinylation approach was applied to study the impact of mutations in the lipoprotein maturation pathway on the cell envelope proteome and exoproteome with the focus on global changes in the lipoprotein distribution. To gain a deeper insight into lipoprotein processing in S. pneumoniae, nature and localization of some lipoproteins and their precursors were studied by immunoblot analysis.

classical surface proteins lack a typical signal sequence normally required for the translocation across the membrane and a classical anchoring motif,9,10 while secreted proteins attached to the pneumococcal surface just lack an anchoring motif. It can be assumed that the real composition of the surface proteome differs substantially from the predicted one because nonclassical anchorless surface proteins9−11 such as the pneumococcal adherence and virulence factor A (PavA)12,13 or the glycolytic enzymes enolase14 and glyceraldehyde-3phosphate dehydrogenase (GAPDH) 15 have also been identified on the pneumococcal cell surface. Consequently, the development of powerful experimental approaches is needed for a detailed characterization of the pneumococcal surface proteome and a better understanding of the implication of surface factors in pneumococcal pathogenesis. Making surface-exposed proteins of bacteria accessible to proteome analyses has been a challenging task. Several methods have been established to study the surface proteome of other Gram-positive bacteria. 16,17 A strategy based on serial solubilization of surface proteins by salt and detergents in high concentrations has been performed by Schaumburg et al.18 for Listeria monocytogenes. An enzymatic extraction using lysostaphin has been employed to release envelope-associated proteins of Staphylococcus aureus.19,20 In proteolytic ‘shaving’ approaches, intact bacteria were treated with trypsin or immobilized trypsin and released surface protein peptides were identified by LC−MS/MS.17,21−24 In addition, biotinylation approaches with subsequent enrichment of envelopeassociated proteins were applied to characterize the staphylococcal surface proteome.19,25 In a proteomic approach for the identification of cell-wall-associated proteins of S. pneumoniae, protein extracts obtained by mutanolysin digestion or at high pH were analyzed using gel-based and gel-free mass spectrometric strategies.26 Moreover, a gel-based shotgun proteomics approach in combination with sodium carbonate precipitation and ultracentrifugation was applied for the analysis of pneumococcal membrane proteins.27 Ultracentrifugation and a sophisticated protein extraction procedure were combined with multiple separation methods to maximize the identification of membrane proteins.28 Furthermore, an immunoproteomic approach was applied to the secretome of pneumococci,29 and recently shaving of pneumococcal surface proteins with soluble trypsin was reported.30 A major proportion of pneumococcal surface proteins are the lipoproteins, which are synthesized in the cytoplasm as precursors containing an N-terminal signal peptide and a conserved lipobox-motif (LVI)(ASTVI)(GAS)C.31−34 After translocation across the membrane the lipoprotein precursors are subject to lipoprotein maturation leading to the covalent binding of the lipoproteins to the outer leaflet of the phospholipid bilayer. The initial attachment of a diacylglyceryl residue to the thiol group of the conserved cysteine in the lipobox is catalyzed by the lipoprotein diacylglyceryl transferase Lgt. The signal peptide is cleaved off by the lipoprotein-specific signal peptidase Lsp, and the modified cysteine residue remains at the mature lipoprotein as the new N-terminus.31 Lipoproteins are characterized by a broad functional diversity. They play important roles in processes such as signal transduction, adhesion, nutrient uptake, antibiotic resistance, oxidative stress resistance, and protein folding.31,35−39 Because of their diverse biological activities, lipoproteins also contribute to pneumococcal pathogenesis by facilitating colonization, invasion, or immune evasion and modulation.40−43 Importantly,

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EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in Table 1. Pneumococci were grown on Columbia blood agar (Oxoid, Basingstoke, U.K.) or in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY; Roth, Karlsruhe, Germany) at 37 °C and 5% CO2. E. coli was grown in Lysogeny broth (LB) or on LB agar supplemented with the appropriate antibiotics: ampicillin (100 μg mL−1), erythromycin (250 μg mL−1), or spectinomycin (100 μg mL−1). Transformation of E. coli with plasmid DNA was carried out with CaCl2-treated competent cells according to standard procedures. For proteome analyses, S. pneumoniae strains were cultured overnight on Columbia blood agar with the appropriate antibiotics as previously mentioned and then transferred into THY media with a starting OD600 of 0.05 to 0.08. Growth of this liquid preculture at 37 °C without shaking was monitored until an OD600 of 0.5 to 0.6 was reached. An appropriate volume of the culture was used to inoculate the main culture in prewarmed THY media with a starting OD600 of ∼0.08. Bacteria were harvested in midexponential growth phase at an OD600 of 0.5 for the biotinylation approach. For the shaving experiments, harvesting was conducted at an OD600 of 0.25. Molecular Techniques and Generation of S. pneumoniae Mutants

Pneumococcal genomic DNA was purified using a standard phenol/chloroform extraction method described previously.47 DNA amplification was performed by PCR using Taq DNA Polymerase (New England Biolabs, Ipswich, MA). PCR reactions (50 μL) were subjected to 30 cycles of denaturation at 94 °C for 10 s, primer annealing at 55 °C for 30 s, and elongation at 72 °C as needed. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System and plasmids were extracted with the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI). DNA sequencing was carried out by Qiagen (Hilden, Germany) and primers were synthesized by Eurofins MWG (Ebersberg, Germany). S. pneumoniae strain D39Δcps48,49 used in this study carries a deletion of the ’capsular polysaccharide 651


Journal of Proteome Research Bethesda Research Laboratories, Gaithersburg, MD

Promega, Madison, WI this study

Δ(lac)U169, endA1, gyrA46, hsdR17, Φ80Δ(lacZ)M15, recA1, relA1, supE44, thi-1

cloning vector for PCR products, Apr pGEM-T derivative vector with the lgt gene region interrupted by the ermB resistance gene cassette for mutagenesis (1940 bp); fragment amplified by PCR using genomic DNA of strain R6Δlgt as template pGEM-T derivative vector with the lsp gene region interrupted by the Spec resistance cassette (aad9) for mutagenesis (2230 bp) pGEM556

Ap, ampicillin; Km, kanamycin; Erm, erythromycin; Spec, spectinomycin; r, resistant.

Assessment of Pneumococcal Autolysis during Growth

Bacteria were harvested at different time points during growth in THY media. After centrifugation the bacterial sediment and the supernatant were treated as described previously.23 Protein amounts were not normalized. Autolysis was analyzed by immunoblot using a specific polyclonal rabbit anti-TrxA antibody generated by a standard immunization procedure with TrxA protein from Staphylococcus aureus as described previously38,52 and a specific polyclonal rabbit anti-Ply (pneumolysin) antiserum, which was a kind gift of Tim Mitchell (Birmingham, U.K.). Screening for Experimental Conditions with Reduced Pneumococcal Autolysis

Bacteria were harvested at an OD600 of 0.5, washed twice, and incubated for 1 h at room temperature in different buffer systems with or without supplements (PBS, pH 7.4, pure or containing 10 mM glucose, 1 M arabinose, 27% (w/v) sucrose, 40% (w/v) sucrose, 1% (w/v) choline chloride, or 10 mM EDTA; PBS, pH 9.5; citrate buffer, pH 7.5, pure or containing either 27% (w/v) sucrose, 40% (w/v) sucrose, or 1% (w/v) choline chloride). After centrifugation, the resulting supernatant and the bacterial sediment were treated as previously described.23 In brief, proteins in the supernatant were precipitated overnight at 4 °C using trichloroacetic acid (TCA) at a final concentration of 10% (v/v). After

a

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46

S. pneumoniae Strains PN137 PN111 PN220 PN222 PN231 E. coli Strains DH5α plasmids pGEM-T Easy pGEM552

synthesis’ (cps) genome region located between the loci dexB (spd_0311) and aliA (spd_0334), which leads to the capsule knockout. Pneumococcal mutants in D39Δcps were generated by insertion deletion mutagenesis. For the construction of mutants deficient in the lgt gene (spd_1243) a 1940 bpfragment containing the lgt gene region interrupted by the ermB-gene was amplified by PCR using primers lgt1fw (5′GCCGTGCAGCTACCAGTCG-3′) and lgt7rev (5′-CATCGATGACACGACCAAGC-3′) as well as genomic DNA of strain R6Δlgt46 as template and cloned into pGEM-T Easy (Promega). The mutant R6Δlgt was kindly provided by Regine Landmann (Basel, Switzerland). For the construction of the Δlsp mutants the lsp (spd_0819) gene region of D39Δcps (1506 bp) was amplified by PCR using primers lsp1fwd (5′CGGCCTTTTCAGAGCGCTATCC-3′) and lsp4rev (5′CCTTGAGTTCTTGGGCAAGTGC-3′) and cloned into pGEM-T Easy. An internal part of lsp was then deleted in this construct by an inverse PCR with primers lsp2rev (5′ATCGATCATGACTTCTACGAAGAGAGTC-3′) and lsp3fw (5′-ATCGATGTGGCAGATAGCTATC-3′) incorporating ClaI-restriction endonuclease sites (underlined). The spectinomycin resistance gene aad950 was ligated with the PCR product. S. pneumoniae strains were transformed either with the generated plasmid constructs or linear DNA fragments amplified by PCR in the presence of competence-stimulating peptide-1 as described previously.51 Pneumococcal mutants were cultivated in THY with the appropriate antibiotics: erythromycin (2.5 or 5 μg mL−1), kanamycin (200 μg mL−1), and/or spectinomycin (50 μg mL−1). Gene knockouts of pneumococcal transformants were verified by PCR using template DNA isolated by heat lysis (96 °C for 8 min). In addition, growth behavior and stability of the pneumococcal knockout mutants were tested. The stability was verified by cultivating the mutants twice in THY without antibiotic pressure before spreading identical culture volumes on Columbia blood agar plates with and without the appropriate antibiotics.

this study

this study this study this study

R6Δlgt::Ermr (Δspr1269), serotype 2 (R6 is a nonencapsulated derivative of D39) D39Δcps::Kmr, serotype 2 D39Δcps::KmrΔlgt::Ermr (Δspd_1243) D39Δcps::KmrΔlgt::ErmrΔlsp::Specr (Δspd_1243Δspd_0819) D39Δcps::KmrΔlsp::Specr (Δspd_0819)

source or reference characteristic(s)a strain or plasmid

Table 1. Bacterial Strains and Plasmids Used in This Study

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centrifugation (20 000g; 20 min; 4 °C) the precipitate was washed once with ice cold acetone before the proteins were resuspended in 1× SDS gel loading buffer. The bacterial sediment was resuspended in PBS, pH 7.4, and cells were disrupted using the Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France; three cycles of 30 s at 6800 rpm with breaks of 30 s at 4 °C). For removing cell debris and glass beads, the sample was centrifuged and the crude extract was transferred into a fresh tube and mixed with SDS gel loading buffer. Finally, the distribution of proteins between bacterial sediment and supernatant was analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie brilliant blue staining.

four times with PBS, pH 7.4 containing 1% (v/v) NP-40 and 6% (w/v) CHAPS as well as two times with PBS, pH 7.4 containing 1% (v/v) NP-40, and 1% (w/v) SDS. Biotinylated proteins were eluted by adding 1 mL of reductive elution buffer (H2O containing 5% (v/v) β-mercaptoethanol). Proteins in the eluate were precipitated with acetone at −20 °C overnight. After centrifugation at 10 000g for 30 min biotinylated proteins were redissolved in 15 μL of 6 M urea, 2 M thiourea, and 10 μL of SDS gel loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/ v) SDS, 20% (w/v) glycerol, 50 mM dithiothreitol (DTT) and 5% (v/v) β-mercaptoethanol) was added. A control reaction treated in the same way except for the addition of biotinylation reagent sulfo-NHS-SS-biotin was used to verify biotin tagging of proteins in the sample.

Preparation of the Surface Proteome Using the Biotinylation Approach

Preparation of the Surface Proteome Using the Shaving Approach

Pneumococci were harvested at 4000g for 10 min at room temperature. The culture supernatant was subjected to mass spectrometry for the analysis of the exoproteome (see Preparation of the Exoproteome section). The bacterial sediment was washed once with resuspension buffer (PBS, pH 7.4; 1% (w/v) choline chloride; 1× complete protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany), and a wet weight of 0.1 g bacteria was resuspended in 0.5 mL of resuspension buffer. Labeling of surface-exposed proteins was basically carried out as described by Hempel and colleagues.25 In brief, to biotinylate surface proteins, we added 50 μL of fresh 1% (w/v) sulfo-NHS-SS-biotin solution in PBS, pH 7.4 (Thermo Scientific/Pierce, Rockford, IL) to the suspension, leading to a final concentration of ∼1.5 mM sulfo-NHS-SSbiotin in the sample. Biotinylation was performed on a rotator at room temperature for 1 h. For quenching the reaction and removing excess of biotinylating reagent, bacteria were centrifuged (8000g; 1 min; room temperature), followed by three washing steps with resuspension buffer containing 0.5 M glycine. The final bacterial sediment was resuspended in 0.5 mL of resuspension buffer containing 5% (w/v) freshly dissolved iodoacetamide (IAA). Then, bacteria were lysed by sonication with four pulses of 15 s and breaks of 30 s while the sample was kept on ice (max. power for small tip; 60% amplitude; 50% cycle). The lysate was transferred into thick-walled 1 mL tubes and an ultracentrifugation step in a Beckman Coulter Optima Max-XP Ultracentrifuge (Beckman Coulter, Brea, CA) was performed using the TLA-120.2 rotor (100 000g; 1 h; 4 °C). The supernatant containing soluble proteins was discarded. Cell debris and membranes were resuspended in 0.5 mL of resuspension buffer containing 5% (w/v) IAA and a detergent mix composed of 4% (w/v) CHAPS and 4% (w/v) ASB-14 (Sigma-Aldrich, St. Louis, MO) for supporting the solubilization of envelope-associated proteins. The suspension was mixed using the Precellys 24 homogenizer (Bertin Technologies; three cycles of 30 s at 6 800 rpm with breaks of 30 s) and 0.5 g glass beads with a diameter of 0.5 mm to optimize protein extraction. After an incubation on ice for 20 min, the sample was centrifuged (15 800g; 25 min; 4 °C) and solubilized biotinylated surface proteins in the supernatant were purified by NeutrAvidin agarose affinity-chromatography (Thermo Scientific/Pierce). A volume of 150 μL of NeutrAvidin agarose per sample was equilibrated two times with PBS, pH 7.4 containing 1% (v/v) NP-40 (Sigma-Aldrich) and incubated with the protein extract for 1 h by gentle shaking on ice. Subsequently, agarose beads were sedimented by centrifugation (80g, 1 min, 4 °C) and the supernatant was discarded. The resin was washed

Shaving was performed essentially as described by Dreisbach and colleagues.23 In brief, aliquots of 3 mL were harvested from growing cultures at an OD600 of 0.25 by centrifugation (9500g; 5 min). Bacteria were washed twice with shaving buffer (PBS, pH 7.4 supplemented with 1% (w/v) choline chloride and 20 mM azide). After removal of the storage buffer, 20 μL of immobilized trypsin (Thermo Scientific/Pierce) was activated by adding 100 μL of 50 mM ammonium bicarbonate. Subsequently, the immobilized trypsin was sedimented by centrifugation (380g; 2 min), dissolved in 50 μL of shaving buffer, and added to the bacteria. The samples were incubated at 37 °C and after 45 min centrifuged (9500g; 5 min) to separate bacteria and agarose beads with immobilized trypsin from the supernatant. The supernatant was centrifuged, and thereafter proteins in the supernatant were reduced by adding 10 mM DTT (30 min) and alkylated with 10 mM IAA (30 min in the dark). After adding 20 ng trypsin (Promega), the samples were incubated overnight at 37 °C. Afterward, samples were acidified with 0.1% (v/v) trifluoroacetic acid (TFA) and purified with ZipTips (Merck Millipore, Billerica, MA). The tips were stepwise equilibrated with 30 μL of acetonitrile (ACN), 30 μL of 80% (v/v) ACN 0.1% (v/v) TFA, 30 μL of 50% (v/v) ACN 0.1% (v/v) TFA, 30 μL of 30% (v/v) ACN 0.1% (v/v) TFA, and finally 30 μL of 0.1% (v/v) TFA. Peptides were bound to the ZipTip resin by pipetting 10 μL of the sample ten times up and down. After washing the resin with 50 μL of 0.1% (v/v) TFA, the peptides were eluted with 20 μL of 50% (v/v) ACN 0.1% (v/v) TFA and 20 μL of 80% (v/v) ACN 0.1% (v/v) TFA. The eluates from two purifications were pooled and concentrated using a vacuum centrifuge. As a control, pneumococci were exposed to identical conditions, and the procedure was performed in the same way except for the addition of immobilized trypsin. Preparation of the Exoproteome

The extracellular protein fraction was basically prepared as previously described.53 In brief, proteins in 100 mL of culture supernatant were precipitated overnight at 4 °C using TCA (Roth) at a final concentration of 10% (v/v). After centrifugation (1 h; 4 °C; 10 000g), the precipitate was washed with ice-cold 70% (v/v) ethanol and transferred to a small tube. Washing was repeated five times with centrifugations in between (10 000g; 4 °C; 7 min). The final protein precipitate was dried overnight and then dissolved thoroughly in 0.3 mL of 8 M urea, 2 M thiourea. Shaking the sample at room temperature for 1 h was followed by centrifugation (10 000g; room temperature; 10 min). Proteins in the supernatant were 653



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diameter × 100 mm; Waters) by a binary gradient of buffers A and B (0.1% (v/v) acetic acid in acetonitrile) over a period of 80 min with a flow rate of 400 nL min−1. For MS/MS analysis, a full survey scan in the Orbitrap (m/z 300−2000) with a resolution of 30.000 was followed by MS/MS experiments of the five most abundant precursor ions acquired in the LTQ via CID. Precursors were dynamically excluded for 30 s, and unassigned charge states as well as singly charged ions were rejected. Real-time recalibration was implemented in the FTanalyzer (lock mass 445.120025) to avoid mass errors.

precipitated with four volumes of acetone, leading to a final concentration of 80% (v/v). The sample was incubated on a rotator at room temperature for 2 h and centrifuged at 10 000g for 15 min. The protein precipitate was washed twice with 96% (v/v) ethanol, once with 70% (v/v) ethanol with centrifugations in between (10 000g; room temperature; 7 min), and dried overnight. Finally, proteins were dissolved in 80 μL of 8 M urea, 2 M thiourea and incubated at room temperature for 30 min. The protein content was determined with RotiNanoquant (Roth), and 30 μg protein was subjected to 1-D gel electrophoresis-based liquid chromatography−mass spectrometry (1D-GeLC−MS).

Database Search

For protein identification, resulting raw data were searched for full tryptic peptides against a protein database of S. pneumoniae strain D39 using Sorcerer-SEQUEST (Sage-N Research, Milpitas, CA). The database consisted of all protein sequences of strain D39 downloaded from the bacterial genomes section of the National Center for Biotechnology Information Web site (NCBI, Bethesda, MD) (http://www.ncbi.nlm.nih.gov/ genome/proteins/176?project_id=58581). Common contaminants and a decoy database consisting of the reversed sequences of all entries were included in the database to enable estimation of the false discovery rate. Altogether the database contained 3912 sequences. The search parameters specified trypsin cutting after lysine and arginine residues and no blocking amino acids allowing two missed cleavage sites. Mass error tolerance was set to 10 ppm for precursor ions and to 1 amu for fragment ions. Only b- and y-ion series were included, considering oxidation of methionine and carbamidomethylation of cysteine as variable modifications with a maximum of three modifications per peptide. Peptide search matches were filtered by SEQUEST scores [DeltaCn = 0.1, XCorr (+2) = 2.2, XCorr (+3) = 3.3, XCorr (+4) = 3.7]. A protein was considered as identified if at least two unique peptides were detected. Using these parameters, the false discovery rate was below 1%. If not specified otherwise, proteins were identified in at least one of the three biological replicates.

Immunoblot Analysis of Lipoprotein Processing and Localization

Pneumococci were cultured in THY media and harvested at an OD600 of 0.5. culture supernatant and bacterial sediment were separated by centrifugation. Proteins in the culture supernatant were precipitated using TCA, as described in the Preparation of the Exoproteome section. Finally, the proteins were dissolved in 1× SDS gel loading buffer and the sample was incubated at 96 °C for 5 min and analyzed by immunoblot analysis. The bacterial sediment was washed once in PBS (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Roth). Finally, bacteria were resuspended in 1× SDS gel loading buffer and lysed by incubation at 96 °C for 10 min, and the sample was subjected to immunoblot analysis. Different sample volumes were loaded onto 15% SDS−PAGE gels for the immunoblot analysis of the individual lipoproteins. The ratio within the bacterial lysates and within the supernatants remained unchanged, whereas the ratio between the two sample groups had to be adapted. Specific polyclonal mouse antisera raised against MetQ and PsaA as well as specific polyclonal rabbit antisera generated against PpmA and SlrA were used. Analysis of Protein Extracts by GeLC−MS

Proteins were separated by 1D-SDS-PAGE.54 After staining with colloidal Coomassie Brilliant Blue G-250, the entire protein separation lane was cut into 10 equal gel slices after distinct bands, and an in-gel digestion of the proteins was performed with trypsin. Gel slices were destained and equilibrated twice in 50% (v/v) ACN containing 200 mM NH4HCO3 for 20 min. Subsequently, gel slices were dried in a SpeedVac (Eppendorf, Hamburg, Germany) for 30 min and covered with trypsin solution (2 μg mL−1; Promega). After complete rehydration of the gel, trypsin solution in excess was removed and the digest was performed at 37 °C for 16 h. For peptide extraction, gel pieces were covered with 100 μL of water and incubated in an ultrasonic bath for 15 min. Peptide solutions (100 μL) of every fraction were concentrated in a SpeedVac to a final volume of 30 μL and subjected to LC−MS analysis. Equal volumes between fractions of the different samples were separated by liquid chromatography and measured online by ESI-mass spectrometry as previously described.25 In brief, LC−MS/MS analysis was performed using a nanoACQUITY UPLC system (Waters, Milford, MA) coupled to an LTQ Orbitrap classic mass spectrometer (Thermo Scientific, Waltham, MA). Peptides were loaded onto a trap column (Symmetry C18, 5 μm, 180 μm inner diameter ×20 mm; Waters) at a flow rate of 10 μL min−1, and the column was washed for 3 min with 99% buffer A (0.1% (v/ v) acetic acid in water). Elution was performed onto an analytical column (BEH130 C18, 1.7 μm, 100 μm inner

Spectral Counting and Statistical Analysis

A relative quantification of the identified proteins was carried out using spectral counting. The normalized spectral abundance factor (NSAF) was calculated according to Zybailov et al.55 using the unweighted spectrum counts obtained from Scaffold 3.4.9. Thereby zero values were replaced by a spectral count of 0.16 as previously described.55 The spectral counts for each protein (SpC) were divided by the molecular weight of the protein as a measure of protein length (L), and a 100% normalization was done to accommodate run differences. The ratios of the NSAFs were calculated (mutant versus parental strain D39Δcps) and transformed into log2 ratios to allow data visualization on a linear scale. Heatmaps were generated using the Multi Experiment Viewer (MeV; TM4Microarray Software Suite).56 To verify the significance of the semiquantitative data, we applied a t test using MeV.56 The p values were based on all permutations. Adjusted Bonferroni correction was enabled to avoid alpha accumulation errors. Hence, adjusted p values are shown. Subcellular Localization Prediction

To predict the localization of identified proteins, we considered different tools like the PSORTdb 3.0 database,57 the Surface Localization Extracellular Proteins pipeline SLEP (http:// bl210.caspur.it/slep/) and the LocateP database58 (http:// 654



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Table 2. Predicted Classical Pneumococcal Surface Proteins in S. pneumoniae Strain D39 locus Choline-Binding Proteins SPD_0126 SPD_0345 SPD_0357 SPD_0579 SPD_0821 SPD_0853 SPD_1248 SPD_1403 SPD_1737 SPD_1965 SPD_2017 SPD_2028 Sortase Substrates SPD_0063 SPD_0080 SPD_0250 SPD_0287 SPD_0335 SPD_0444 SPD_0558 SPD_0562 SPD_0577 SPD_1018 SPD_1321 SPD_1376 SPD_1504 SPD_1617 SPD_1753 SPD_1789 Lipoproteins SPD_0090 SPD_0109 SPD_0150 SPD_0151 SPD_0179 SPD_0184 SPD_0313a SPD_0334a SPD_0540 SPD_0549 SPD_0572 SPD_0652 SPD_0672 SPD_0739 SPD_0792 SPD_0868 SPD_0886 SPD_0888 SPD_0915 SPD_1038 SPD_1170 SPD_1226 SPD_1232 SPD_1328 SPD_1357 SPD_1463 SPD_1495 SPD_1502 SPD_1585 SPD_1609

protein name

description/molecular function

PspA CbpC CbpF CbpL CbpE LytB CbpM LytC LytA PcpA PspC (SpsA, CbpA) CbpD

pneumococcal surface protein A choline-binding protein C choline-binding protein F choline-binding protein L choline-binding protein E endo-beta-N-acetylglucosaminidase choline-binding protein M 1,4-beta-N-acetylmuramidase autolysin/N-acetylmuramoyl-L-alanine amidase choline-binding protein pneumococcal surface protein C choline-binding protein D

StrH PavB PulA HysA

beta-N-acetylhexosaminidase pneumococcal adherence and virulence factor B alkaline amylopullulanase hyaluronidase cell wall surface anchor family protein endo-beta-N-acetylglucosaminidase cell wall-associated serine protease beta-galactosidase zinc metalloprotease IgA-specific metalloendopeptidase cell wall surface anchor family protein G5 domain-containing protein neuraminidase A; sialidase A plasmin and fibronectin-binding protein A subtilisin-like serine protease cell wall surface anchor family protein

PrtA BgaA ZmpB

NanA PfbA NisP

GshT MetQ

AliA DacB TlpA (Etrx1) LivJ SlrA TmpC (PnrA) PpmA (PrsA) Etrx2 AdcAII (Lmb) PiuA PhtA AppA GlnH AatB AliB PsaA

multiple sugar ABC transporter substrate-binding protein polar amino acid ABC transporter substrate-binding protein glutathione/polar amino acid ABC transporter substrate-binding protein D-methionine ABC transporter substrate-binding protein hypothetical protein hypothetical protein hypothetical protein oligopeptide/peptide/nickel ABC transporter substrate-binding protein glutamine/polar amino acid ABC transporter substrate-binding protein L,D-carboxypeptidase thioredoxin-like protein; extracellular thioredoxin family protein branched-chain amino acid ABC transporter substrate-binding protein streptococcal lipoprotein rotamase A; cyclophilin-type peptidyl-prolyl cis−trans isomerase membrane lipoprotein; purine nucleoside receptor A hypothetical protein; membrane-associated lipoprotein putative proteinase maturation protein A; foldase protein extracellular thioredoxin family protein zinc ABC transporter substrate-binding protein; adhesion lipoprotein iron-compound ABC transporter substrate-binding protein pneumococcal histidine triad protein A oligopeptide/peptide/nickel ABC transporter substrate-binding protein glutamine/polar amino acid ABC transporter substrate-binding protein phosphate ABC transporter substrate-binding protein; PstS-like protein polar amino acid transport system substrate-binding protein oligopeptide ABC transporter substrate-binding protein manganese/iron/zinc/copper ABC transporter substrate-binding protein; adhesion lipoprotein multiple sugar ABC transporter substrate-binding protein multiple sugar ABC transporter substrate-binding protein sugar ABC transporter substrate-binding protein iron(III) transport system substrate-binding protein 655



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Table 2. continued locus Lipoproteins SPD_1652 SPD_1671 SPD_1677 SPD_1910 SPD_1934 SPD_1997 SPD_2025 a

protein name PiaA (FatB) AmiA RafE (MsmE) PstS MalX AdcA

description/molecular function iron-compound ABC transporter substrate-binding protein oligopeptide/nickel ABC transporter substrate-binding protein raffinose/stachyose/multiple sugar ABC transporter substrate-binding protein phosphate ABC transporter substrate-binding protein maltose/maltodextrin ABC transporter substrate-binding protein zinc ABC transporter substrate-binding lipoprotein nitrate/sulfonate/bicarbonate ABC transporter substrate-binding protein

Not present in D39Δcps.

and intracellular proteins.66 Experimental conditions with reduced pneumococcal autolysis had to be identified to avoid unspecific biotin-labeling or trypsin shaving of non-surfaceassociated proteins. Therefore, autolysis of strain D39Δcps was assessed during growth in THY media (Figure 1A). Autolysis was negligible in the early- and mid-exponential growth phases. In contrast, in the late-exponential growth phase, a dramatic increase in autolysis was detected by immunoblot analysis using antibodies recognizing specifically the cytoplasmic proteins thioredoxin (TrxA) and pneumolysin (Figure 1A). Therefore, pneumococci were grown to an OD600 of 0.25 for the trypsin

www.cmbi.ru.nl/locatep-db/cgi-bin/locatepdb.py). LocateP was selected for the analysis, and the data set of strain D39 was further validated by comparison with the localization prediction results of the CBS Prediction Servers (http://www. cbs.dtu.dk/services/). The TMHMM 2.0 algorithm59 was used for the prediction of transmembrane domains (TMDs), and signal sequences were estimated using the software tool SignalP 4.0.60,61 When a predicted signal peptide and cleavage site overlapped with a presumable TMD, the TMD was considered to be a false-prediction. Proteins were considered to be lipidanchored if projected by LipoP 1.0.62 Lipoprotein predictions were compared with DOLOP33 and Augur63 results. Proteins exhibiting an LPxTG-motif were regarded as sortase substrates covalently bound to peptidoglycan.64 Further cell-wallassociated proteins were defined according to previous findings,2 and CBPs were identified via the presence of typical choline-binding domains consisting of characteristic repeat motifs.8 The lgt (spd_1243) and lsp (spd_0819) loci of strain D39 and their upstream and downstream flanking sequences were retrieved from the available genome sequence on the KEGGWeb site (http://www.genome.jp/kegg/). Generation and Visualization of Tertiary Structure Models

Tertiary protein structure models were obtained from the Protein Homology/analogY Recognition Engine (Phyre2).65 Protein domains and identified peptides were highlighted in the structure models using PyMOL (http://www.pymol.org).

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Figure 1. Assessment of autolysis during growth and screening for experimental conditions with reduced pneumococcal autolysis. (A) Assessment of autolysis during growth in THY media. Autolysis of strain D39Δcps was assessed as described in the Experimental Procedures. In brief, pneumococci were cultured in THY media and harvested at the following time points: OD600 of 0.07, 0.3, 0.5, and 1.0. After centrifugation, the distribution of pneumolysin (Ply) and the cytoplasmic marker protein thioredoxin (TrxA) between the bacterial sediment and the supernatant was monitored by immunoblot analysis using a specific rabbit anti-Ply antiserum and a specific polyclonal rabbit anti-TrxA antibody, respectively. (B) Evaluation of buffers for the shaving and biotinylation experiments. Buffer screening was performed with strain D39Δcps as described in the Experimental Procedures. The following buffer systems were checked: PBS, pH 7.4 (1); PBS, pH 7.4, 10 mM glucose (2); PBS, pH 7.4, 1 M arabinose (3); PBS, pH 7.4, 27% (w/v) sucrose (4); PBS, pH 7.4, 40% (w/v) sucrose (5); PBS, pH 7.4, 1% (w/v) choline chloride (6); citrate buffer, pH 7.5 (7); citrate buffer, pH 7.5, 27% (w/v) sucrose (8); citrate buffer, pH 7.5, 40% (w/v) sucrose (9); citrate buffer, pH 7.5, 1% (w/v) choline chloride (10); PBS, pH 7.4, 10 mM EDTA (11); and PBS, pH 9.5 (12). After incubation for 1 h and centrifugation, the supernatant (S) and the pneumococcal cell extract after cell disruption (CE) were separated in an SDS-PAGE, followed by Coomassie staining. The PageRuler Protein Ladder (Thermo Scientific/ Fermentas, Vilnius, Lithuania) was used as molecular weight standard (M).

RESULTS

Predicted Classical Surface Proteins of S. pneumoniae D39Δcps

In a bioinformatic analysis of the genome of S. pneumoniae D39, a total number of 1914 open reading frames coding for proteins were identified. In addition to the approximately 400 membrane proteins, more than 100 surface-associated or secreted proteins are predicted. All of the latter ones contain a signal peptide, and the majority is surface-associated and carries a classical anchoring motif. For strain D39, 12 CBPs, 16 LPxTG proteins, and 37 lipoproteins are predicted based on a validated LocateP58 data set (Table 2). The genes spd_0313 encoding a small hypothetical lipoprotein and spd_0334 encoding AliA, the binding protein of an oligopeptide ABC transporter, are not present in full length in the genome of S. pneumoniae strain D39Δcps48,49 due to the capsule knockout. Consequently, only 35 lipoproteins are predicted for strain D39Δcps (Table 2). Pneumococcal Autolysis Is Prevented by Choline

Autolysis is a process typical for pneumococci. It is mediated by cell-wall-degrading hydrolases and leads to the release of DNA 656



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Figure 2. Characterization of the S. pneumoniae D39Δcps surface proteome using the biotinylation approach. (A) Proteins identified in at least one of the three biological replicates. Identified proteins are classified according to their predicted localization and anchoring mechanism based on a validated LocateP58 data set for strain D39. (See data analysis in Experimental Procedures and Table 2 for details.) (B) Proportion of protein classes in the surface proteome as estimated by the proportion of normalized spectral abundance factors (NSAFs) per protein class. NSAFs of all proteins belonging to the same protein class were summarized and expressed in percentage terms. (C) Identification frequency of proteins. The numbers of proteins detected in all three (3/3), two (2/3), or only one (1/3) biological replicate are shown.

Table 3. Identified Surface and Exoproteomes of the Lipoprotein Maturation Mutants in Comparison to S. pneumoniae D39Δcps subproteome S. pneumoniae strain Surface Proteomea D39Δcps D39ΔcpsΔlgt D39ΔcpsΔlsp D39ΔcpsΔlgtΔlsp Exoproteome D39Δcps D39ΔcpsΔlgt D39ΔcpsΔlsp D39ΔcpsΔlgtΔlsp a

cytoplasmic proteins

lipoproteins

LPxTG-anchored proteins

choline-binding proteins

membrane-anchored proteins

multipass transmembrane proteins

secreted proteins

210 287 319 345

33 27 32 32

12 13 11 12

2 4 4 4

31 34 34 34

23 27 28 32

11 11 12 11

458 374 439 398

25 30 22 27

11 12 12 12

10 10 10 8

21 18 20 17

8 7 8 8

15 13 14 13

Identified using the biotinylation approach.

shaving approach and to an OD600 of 0.5 for labeling with sulfoNHS-SS-biotin. In addition, pneumococcal autolysis was evaluated in several buffer systems with various additives (Figure 1B). Pneumococci showed reduced autolysis in PBS buffer, pH 7.4, compared with citrate buffer, pH 7.5. In shaving experiments with staphylococci, Dreisbach et al.23 used 40% (w/v) sucrose to stabilize the bacteria. In other experiments with staphylococci, 27% (w/ v) sucrose19 and 1 M arabinose24 were shown to be beneficial. However, these sugars had no stabilizing effect on pneumococci (Figure 1B). In addition, higher pH values or EDTA, which are known to inhibit the activity of the autolysins,67 did not reduce autolysis. In contrast, autolysis was significantly reduced, when pneumococci were incubated in PBS buffer supplemented with 1% (w/v) choline chloride (Figure 1B, marked lanes). Therefore, this buffer system was used for both protein labeling with sulfo-NHS-SS-biotin and surface protein shaving with immobilized trypsin. The presence of choline chloride is known

to cause the release of CBPs including autolysin from the pneumococcal surface.68 High Coverage of the Surface Proteome Is Achieved Using the Biotinylation Approach

The biotinylation approach was designed for enrichment and maximum coverage of surface proteins. In total, 112 proteins predicted to be surface-associated were detected in surface proteome preparations of strain D39Δcps (Figure 2A) corresponding to 35% of the identified proteins. Moreover, 210 proteins with a predicted cytoplasmic localization were identified (Table 3; Supplementary Table 1 in the Supporting Information). Because the hypothetical lipoprotein SPD_0313 and AliA are encoded by genes of the deleted cps gene cluster, the identified 33 lipoproteins of strain D39Δcps represent 95% of the predicted lipoproteins. Only the hypothetical lipoprotein SPD_0184 and the multiple sugar ABC transporter substratebinding protein SPD_1502 were not detected. The 12 identified sortase substrates correspond to 75% of the predicted proteins. These results reflect the high coverage of the two 657



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classical surface protein classes achieved in this study. In addition, 31 N-terminally membrane-anchored proteins and 23 proteins possessing multiple transmembrane regions were identified. Furthermore, 11 secreted proteins representing 33% of this protein class were identified on the pneumococcal cell surface. In contrast, CBPs were eluted from the surface due to the presence of choline in the incubation buffer, and thus only 2 of the 12 CBPs could be identified (Table 3). To estimate the proportion of pneumococcal surface proteins in the preparations, we classified proteins according to their predicted localization. NSAFs derived from proteins of the same class were summarized and expressed in percentage terms. The results revealed a remarkably high abundance of lipoproteins in the sample (Figure 2B). Almost 50% of the NSAFs originated from only 33 different lipoproteins, whereas 210 predicted cytoplasmic proteins contributed to 34% of the NSAFs (Figure 2A, B). Accordingly, the proportion of predicted surface proteins was ∼66%. These results demonstrate the potential of the biotinylation approach to efficiently enrich proteins associated with the pneumococcal cell surface. The high reproducibility and reliability of this method are indicated by the number of proteins identified in the individual biological replicates. With 200 out of 322 proteins and a ratio of 62%, the majority of the proteins was detected in all three biological replicates (Figure 2C). The ratio is even higher if only the predicted surface-associated proteins are considered. More than 80% of the 112 identified proteins were detected in all three replicates.

Figure 3. Protein identification on the surface of S. pneumoniae D39Δcps using biotinylation and shaving approaches. A Venn graph with the proteins identified by both methods and by either the biotinylation or shaving approaches is shown. For the shaving approach, the number of proteins identified in at least three out of the four replicates is shown. Proteins were considered as identified by trypsin shaving if the same peptide was detected in three replicates and was not found in the control. Numbers of identified lipoproteins and LPxTG-anchored proteins (sortase substrates) as two prominent classes of classical surface proteins are depicted in detail. In the right section of the graph, the total numbers of identified lipoproteins and sortase substrates are shown in comparison with the predicted proteins. Bright-colored column sections indicate identified proteins (lower number), whereas bright-colored and pale-colored column sections represent the theoretical number of proteins (upper number). *For strain D39, a total number of 37 lipoproteins is predicted. Two of these are encoded in the “capsular polysaccharide synthesis” (cps) genome region and are therefore not present in D39Δcps. (See Table 2 for details.)

Surface-Exposed Protein Domains Are Identified by Shaving with Immobilized Trypsin

The shaving approach with immobilized trypsin was applied to identify exposed domains of pneumococcal surface proteins. In total, peptides belonging to 101 proteins were identified using the shaving approach (Supplementary Table 2 in the Supporting Information). 60 of these proteins were also detected by biotin labeling (Figure 3). Interesting proteins from this overlap fraction reproducibly identified by both methods were selected, and the results were evaluated in more detail. Tertiary structure models of these proteins were generated, and available crystal structures were identified using Phyre2.65 Finally, peptides identified by shaving with trypsin beads were mapped to the structure models with high confidence level. Using the shaving approach, the glutathionebinding protein GshT was identified with a single surfaceexposed peptide derived from the central region of the primary structure of the lipoprotein (Figure 4A; Supplementary Table 3 in the Supporting Information), whereas high protein sequence coverage was obtained for the biotinylated GshT (Supplementary Table 4 in the Supporting Information). Visualization of this peptide in the tertiary structure of GshT indicates that the peptide belongs to a surface-exposed part of the substratebinding domain (Figure 4A). The maltose/maltodextrinbinding protein MalX proved to be another lipoprotein with an apparently well-exposed domain on the pneumococcal cell surface. MalX was identified with a single surface-exposed peptide (T75GDALGGLDK84) derived from the N-terminal region of the substrate-binding domain by shaving with trypsin beads (Figure 4A; Supplementary Table 3 in the Supporting Information), whereas the biotinylation results show the typical high protein sequence coverage achieved for lipoproteins in this approach (Supplementary Table 4 in the Supporting Information). A single surface-exposed peptide was also

identified for the sortase SrtA. In the tertiary structure model of the protein, the peptide E57KLEENQDTEGNFDFDSVK75 is located in a surface-exposed domain in close proximity to the catalytically active domain responsible for covalent binding of substrate proteins to the peptidoglycan (Figure 4A; Supplementary Table 3 in the Supporting Information). The cytoplasmic pyruvate oxidase SpxB was not expected on the pneumococcal surface but was frequently identified by the biotinylation approach and shaving with trypsin beads as well (Figure 4A; Supplementary Tables 3 and 4 in the Supporting Information). Three peptides located in the second half of the primary sequence were reproducibly identified in the shaving samples, suggesting that SpxB is indeed surface-exposed. Anchoring and orientation of this protein on the pneumococcal cell surface cannot be predicted, but the thiamine pyrophosphate binding domain of SpxB, beginning in the middle of the protein sequence and ending close to its C-terminus, seems to form a surface-exposed domain well-accessible to immobilized trypsin. The cytoplasmic and nonclassical surface protein enolase14 was also identified by the shaving method. The peptide Y59GGLGTQK66 close to the N-terminus was reproducibly identified in the shaving experiments. According to the tertiary structure, this peptide is located in an exposed part of the N-terminal protein domain (Figure 4A; Supplementary Table 3 in the Supporting Information). Mapping of the peptide to the octameric structure of pneumococcal enolase (PDB ID: 1W6T)69 confirmed trypsin accessibility on the enzymatic surface (Figure 4B). Remarkably, nearly the entire enolase protein was covered in the biotinylation experiments (Figure 4A; Supplementary Table 4 in the Supporting Information). 658



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Figure 4. continued primary structure starting with the N-terminus on the left. Tertiary structure models were created using Phyre2.65 Transmembrane regions as predicted by Topcons95 (http://topcons.cbr.su.se/), signal peptides and lipoboxes as identified by SignalP60 and LipoP,62 respectively, and functional domains as predicted by Pfam96 (http://pfam.sanger.ac.uk/ ) are shown. (B) Surface exposure of the identified enolase peptide in the octameric structure of the pneumococcal enzyme. The peptide depicted in magenta is shown in the front and side views of the pneumococcal enolase octamer (PDB ID: 1W6T).

Exoproteome of S. pneumoniae D39Δcps

In addition to the surface proteome, the exoproteome of strain D39Δcps was analyzed. In total, 548 pneumococcal proteins were identified in the culture supernatant (Supplementary Table 5 in the Supporting Information). The vast majority of these proteins was predicted to be localized in the cytoplasm. However, the exoproteome also contained 15 secreted proteins, 10 CBPs, 11 sortase substrates, 25 lipoproteins, 21 membraneanchored proteins, and 8 multitransmembrane proteins (Table 3), indicating shedding of surface proteins and release of cytoplasmic proteins into the growth medium. Approximately 45% of the proteins predicted to be secreted by S. pneumoniae D39 could be identified. In vitro Fitness of the Lipoprotein Maturation Mutants

To study the impact of mutations in the lipoprotein maturation pathway on the distribution of the lipoproteome and on lipoprotein processing in S. pneumoniae, we generated mutants deficient in the diacylglyceryl transferase Lgt, the lipoproteinspecific signal peptidase Lsp, or in both enzymes. In growth experiments, the loss of function of Lsp delayed growth in THY, while the deficiency of Lgt did not impair growth, and hence the behavior was comparable to the parental strain D39Δcps (Supplementary Figure 1S in the Supporting Information). The deficiency of both Lsp and Lgt resulted in a striking growth inhibition of the mutant compared with the parental strain D39Δcps. In addition to a decreased growth rate, the final optical density was also reduced, demonstrating the importance of lipoprotein maturation for bacterial fitness and the burden caused by the loss of functions. Distribution of Lipoproteins in the Δlgt Mutant

The surface proteome and exoproteome of the D39ΔcpsΔlgt mutant deficient in Lgt, which is usually responsible for anchoring the lipoproteins to the membrane, were characterized employing the biotinylation approach and precipitation of proteins in the culture supernatant, respectively (Table 3; Supplementary Tables 6 and 7 in the Supporting Information). Despite impaired anchoring, the vast majority of lipoproteins were still identified in the surface proteome preparations of the Δlgt mutant (Supplementary Table 6 in the Supporting Information). In comparison with the parental strain, only the extracellular thioredoxin family proteins TlpA,38 also referred to as Etrx1, and Etrx2,39 as well as the ABC transporter substrate-binding proteins SPD_0540, SPD_1585, PiaA, and RafE were not detectable in the surface proteome of the Δlgt mutant. Semiquantitative analysis of changes in the distribution of the lipoproteome based on spectral counting revealed decreased surface-associated amounts for most of the lipoproteins and accumulation of lipoproteins in the culture supernatant of the Δlgt mutant, indicating their release from the surface (Figure 5;

Figure 4. Surface-exposed protein domains identified by shaving with immobilized trypsin. (A) Peptides identified by the shaving approach highlighted in the tertiary protein structures. Peptides identified by shaving are highlighted in magenta in the tertiary structure model of the corresponding proteins. N-termini cleaved during protein processing are displayed in yellow, and lipoboxes and predicted transmembrane regions are highlighted in orange. Predicted Pfam domains are colored in the 3D model as in the primary sequence. All other parts of the protein are colored in gray. Peptides identified by the biotinylation approach or the shaving approach in the surface proteome of S. pneumoniae D39Δcps are shown schematically in the 659



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Figure 5. Changes in the distribution of lipoproteins between bacterial cell surface and exoproteome of S. pneumoniae D39Δcps and its isogenic lipoprotein maturation mutants. A semiquantitative analysis of the lipoproteome distribution between surface (S) and exoproteomes (E) of the Δlgt-, Δlsp-, and ΔlgtΔlsp mutants compared with D39Δcps is shown. Surface proteomes were identified using the biotinylation approach. The data in Supplementary Tables 12−14 in the Supporting Information also shown as graphs in Supplementary Figure 2S in the Supporting Information were used to generate the heatmaps with the Multi Experiment Viewer (MeV).56

proteins including 32 lipoproteins. The exoproteome consisted of 483 proteins and contained 27 lipoproteins (Table 3; Supplementary Tables 10 and 11 in the Supporting Information). The semiquantitative analysis of the lipoproteome showed that the lipoprotein distribution pattern of the double mutant shared similarities with the one observed for the Δlgt mutant. A considerable number of lipoproteins (SPD_0540, LivJ, SlrA, PiuA, GlnH or SPD_1609) were detected in reduced amounts in the surface proteome and accumulated in the culture supernatant (Figure 5; Supplementary Figure 2SC and Supplementary Table 14 in the Supporting Information). In addition, some lipoproteins such as SPD_0179, AmiA, and AdcA accumulated in the culture supernatant but were, nevertheless, identified in only slightly reduced amounts in the surface proteome.

Supplementary Figure 2SA and Supplementary Table 12 in the Supporting Information). Nevertheless, a considerable number of lipoproteins were detected in only slightly reduced amounts (MetQ, DacB, SPD_0792, and PpmA) or slightly increased amounts (TmpC, AdcAII, and PhtA) in the surface proteome of the Δlgt mutant. Lipoproteome of the Mutant Deficient in Lsp

Analysis of the surface proteome and exoproteome of the lipoprotein-specific signal peptidase-deficient mutant D39ΔcpsΔlsp resulted in the identification of 440 and 525 proteins, respectively (Table 3; Supplementary Tables 8 and 9 in the Supporting Information). 32 lipoproteins were identified in the surface proteome and 22 in the culture supernatant. The effect on the distribution of the lipoproteome was less clear than that in the Δlgt mutant (Figure 5; Supplementary Figure 2SB and Supplementary Table 13 in the Supporting Information). Similar to the Δlgt mutant, some lipoproteins such as SPD_0179, LivJ, and PiuA were detected in significantly reduced amounts in the surface proteome and in increased amounts in the culture supernatant. In contrast, the surface-associated and released amounts of a number of lipoproteins such as GshT and PpmA remained almost unaffected in the Δlsp mutant. Furthermore, several lipoproteins such as MetQ and PsaA were present in reduced amounts in both subproteomic fractions. PsaA was actually reduced to an undetectable level in the culture supernatant of the Δlsp mutant, indicating proteolytic degradation due to improper processing. An exception was the phosphate-binding protein PstS, which was present in increased amounts in both subproteomes of the mutant, suggesting an elevated expression level (Figure 5; Supplementary Figure 2SB in the Supporting Information).

Insights into Lipoprotein Processing in Pneumococci

To monitor whether lipoprotein processing in the mutants differs from processing in the parental strain D39Δcps and whether the protein species retained on the surface differs from the one released into the supernatant, immunoblots of whole cell lysates and corresponding supernatant fractions were performed using specific antibodies generated against representative lipoproteins. The effects of the lipoprotein maturation mutations on processing and localization of the two ABC transporter components MetQ and PsaA and the two peptidylprolyl cis/trans isomerases PpmA and SlrA were monitored (Figure 6; Supplementary Figure 3S in the Supporting Information). All four of these lipoproteins showed only one surfaceassociated protein species in strain D39Δcps, suggesting the presence of the mature lipid-anchored protein. Considerably lower protein amounts of MetQ, PpmA, and PsaA with the same apparent size were detectable in the corresponding supernatant fraction, most likely representing the same lipidated form (Figure 6). In the case of SlrA, the lipid moiety serves as a very efficient anchor because overexposure was needed to detect the SlrA protein in the culture supernatant of

Lipoprotein Distribution in the ΔlgtΔlsp Double Mutant

Analysis of the surface proteome of the double-knockout mutant deficient in both the diacylglyceryl transferase Lgt and the lipoprotein-specific signal peptidase Lsp using the biotinylation approach resulted in the identification of 470 660



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Figure 6. Effects of the lipoprotein maturation mutations on processing and localization of pneumococcal lipoproteins. Immunoblots of bacterial lysates and supernatants of the parental strain D39Δcps and the isogenic mutants D39ΔcpsΔlgt, D39ΔcpsΔlsp, and D39ΔcpsΔlgtΔlsp are shown. Specific polyclonal antibodies raised against the pneumococcal lipoproteins MetQ, PpmA, SlrA, and PsaA were used to study molecular weight and localization of the protein species observed in the parental strain and its mutants. Bacteria were harvested at an OD600 of 0.5, and samples were treated as described in the Experimental Procedures section.

The lipoprotein species detected in the lysate and the culture supernatant of the ΔlgtΔlsp double mutant was very similar to the higher molecular weight protein form detected in the Δlgt mutant, suggesting the presence of unprocessed lipoprotein precursor, which is nonlipidated and still contains the signal peptide (Figure 6). The protein species of lower molecular weight detected in the Δlgt mutant was missing in the double mutant, supporting the idea that nonlipidated lipoprotein precursors are processed by Lsp in the Δlgt mutant. Hence, in the case of MetQ, PpmA, and PsaA, the processing observed in the Δlgt mutant seems to be exclusively caused by the action of Lsp. In the bacterial lysate of the double mutant, two protein species of SlrA were detected after overexposure, suggesting alternative signal peptide processing by another signal peptidase or protease, as observed in the Δlsp mutant. In addition, a number of SlrA protein species were detected in the exoproteome after overexposure of the blot (Supplementary Figure 3S in the Supporting Information). In the supernatant fractions of all mutants and the parental strain, a degradation product or some alternatively processed PpmA lipoprotein was detected (Figure 6).

strain D39Δcps (Supplementary Figure 3S in the Supporting Information). In general, a slightly larger protein species than that in the parental strain was found to be surface-associated in the Δlgt mutant, suggesting the presence of unprocessed lipoprotein precursor, which is nonlipidated and contains the signal peptide (Figure 6). The immunoblots of PsaA, PpmA, and after overexposure SlrA also showed traces of a lower molecular weight protein species in the bacterial lysate most likely representing a protein form with cleaved signal peptide and suggesting processing by Lsp, another signal peptidase, or a specific membrane protease70 (Figure 6; Supplementary Figure 3S in the Supporting Information). In the supernatant fraction of the Δlgt mutant, the same two protein forms were detected, but contrary to the bacterial lysates the ratio was in favor of the smaller protein species, indicating that the protein form with cleaved signal peptide is released more efficiently. The change in the ratio between both protein species differed from lipoprotein to lipoprotein. While similar amounts of the protein species were detected for MetQ and PpmA, much more of the smaller processed protein form was detected for PsaA. SlrA released into the exoproteome exclusively consists of the smaller processed protein species without signal peptide. In addition, the loss of the lipid anchor led to an almost complete release of SlrA (Figure 6). In the bacterial lysate of the Δlsp mutant, a third protein species with the highest molecular weight was detected, suggesting the presence of signal peptide-containing and lipidated lipoprotein precursor (Figure 6). Slightly higher amounts of protein were detected in the lysate in comparison with the parental strain, suggesting more efficient retention of lipoproteins in the Δlsp mutant. Only for SlrA was a lower molecular weight protein species detected, indicating that in the absence of Lsp some alternative signal peptide processing takes place, which was not observed for the other lipoproteins. Potentially signal peptide-containing and lipidated lipoprotein precursors were also released into the supernatant fraction of the Δlsp mutant. In general, the lipoprotein amount detected in the exoproteome of the Δlsp mutant was comparable to the parental strain (Figure 6).

DISCUSSION Pneumococci are naturally competent bacteria and autolysis as well as fratricide are important features of these bacteria, leading to release of DNA and also of intracellular proteins.71 To avoid unspecific biotin labeling or trypsin treatment of nonsurface-associated proteins, autolysis had to be prevented during cultivation and sample preparation. As confirmed in this study by immunoblot analysis with antibodies against marker proteins, pneumococci showed only limited autolysis in THY media when cultured to early- and mid-exponential growth phase. In a previous surface proteome study, chemically defined medium (CDM) supplemented with either choline or ethanolamine was used,30 disregarding the fact that the replacement of choline by ethanolamine in CDM has been shown to cause unwanted changes in several cellular properties of the pneumococcus.72 In previous studies, pneumococci were incubated in PBS buffer with sucrose during mutanolysin treatment or shaving with soluble trypsin.26,30 However, in the present study, sucrose and other sugars had no stabilizing effect on pneumococci, whereas the addition of choline substantially reduced autolysis and stabilized the bacteria. Choline competes with the phosphorylcholine (PCho) of the cell wall for binding of CBPs. As a result, the presence of choline leads to the release of CBPs including autolysins from the pneumococcal surface.68 Although the loss of CBPs can be regarded as a disadvantage for surface proteome analysis, prevention of autolysis outweighs this. A further publication described difficulties to lyse pneumococci despite autolysis,73 a problem that was not observed in the present study. To obtain a first global overview of the composition of the pneumococcal surface proteome under defined culture conditions, we applied a biotinylation and a shaving approach to study the surface proteome of S. pneumoniae. Both methods benefitted considerably from the achieved reduction of autolysis. These two approaches differ substantially in their focus. The biotinylation approach was designed for enrichment and maximum coverage of surface proteins to monitor changes in the cell surface proteome on a global scale, while shaving with immobilized trypsin aimed at identifying exposed domains of surface proteins.16,17 The biotinylation reagent is membraneimpermeable but can easily penetrate the peptidoglycan and

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therefore label surface proteins exposed to the exterior of the bacteria and also proteins that are buried within the cell wall. In contrast, only surface proteins with domains directly exposed to the exterior are accessible to the immobilized trypsin in the shaving approach. Therefore, shaving with immobilized trypsin seems to be a more valuable tool to identify proteinaceous interaction partners of extracellular macromolecules and promising vaccine candidates. However, shaving with soluble trypsin, as recently also performed with pneumococci,30 seems to be more comparable to the biotinylation approach. The relatively small soluble protease may also penetrate the cell wall and release peptides of proteins that are actually buried and not exposed to the extracellular milieu, where they could interact with biomacromolecules of the host.16,17 The results show that the two methods established for pneumococci in this study complement each other. While 60 proteins were identified by both strategies, 41 were exclusively identified by shaving with trypsin beads and 262 were identified by biotinylation. When only proteins with a predicted noncytoplasmic localization were considered, 12 out of the 16 proteins detected by shaving were also identified by the biotinylation approach. In addition, 100 predicted noncytoplasmic proteins were exclusively detected by biotinylation. Approximately 16 and 35% of the proteins identified by shaving and biotinylation, respectively, were predicted to be surface-associated. The 35% predicted noncytoplasmic proteins identified by biotinylation represent the highest ratio described for pneumococci so far26,27,30 and indicate the successful enrichment of pneumococcal surface proteins by labeling with sulfo-NHS-SS-biotin. Nevertheless, not all identified cytoplasmic proteins are expected to fulfill an alternative function on the pneumococcal cell surface. Some might indeed represent false positively identified proteins, a point of view also emerging from other studies.26,30 Therefore, further studies such as electron microscopy or flow cytometry will be required to verify surface localization and to distinguish between genuine nonclassical surface proteins and cytoplasmic contaminations.14 Autolysis and fratricide are characteristic features of pneumococci,71 which might be responsible for carrying nonclassical surface proteins to their destination, as has been shown for some cytoplasmic proteins of S. aureus.74 Importantly, several classical surface protein classes were covered to a high degree in the present experiments. Approximately 95% of the predicted lipoproteins and 75% of the sortase substrates were identified by biotinylation. A similarly high percentage was obtained up to now only for the LPxTG cell wall-anchored proteins.30 Remarkably, the number of membrane proteins with one or more transmembrane segments was with 54 higher than the 47 proteins identified in a membrane proteome analysis of S. pneumoniae.27 The high coverage of lipoproteins and membrane proteins in the present study is most likely due to the implementation of an ultracentrifugation step in the biotinylation approach, the efficient protein extraction obtained with a mixture of the detergents ASB-14 and CHAPS,75,76 and the reduction of sample complexity by affinity purification. Combining the biotinylation approach with an exoproteome analysis extended the high coverage of surface proteins to the secreted proteins in the culture supernatant. Approximately 45% of the proteins predicted to be secreted by S. pneumoniae D39 were accessible to mass spectrometric identification. Analysis of the exoproteome of S. pneumoniae D39Δcps showed 16.4% proteins with a predicted noncytoplasmic localization, which is comparable to the percentage previously reported.29

The comparison of the total numbers of truly secreted proteins reveals that 15 secreted proteins were identified in this study and only up to 5 in the other study29 because PspC and LytC are CBPs and StrH is a sortase substrate. Numerous surfaceassociated proteins were also identified in the culture medium in the present study, indicating shedding of these proteins from the surface during growth. The high number of cytoplasmic proteins present in the culture supernatant may be attributed to lysis of a subpopulation of pneumococci during cultivation, a phenomenon that has also been observed for S. aureus and Bacillus subtilis.77,78 Surface-associated and secreted proteins identified by proteome analyses represent interesting candidates for vaccine development. Therefore, some of the 60 proteins identified by shaving with trypsin beads and the biotinylation approach were selected and further analyzed. Mapping of peptides identified by trypsin shaving to tertiary structure models of the corresponding proteins confirmed the specific identification of surface-exposed protein domains by the shaving approach. Trypsin shaving identified a single peptide of GshT, the binding protein of a glutathione uptake system. GshT was also detected in three clinical isolates studied in another proteomic approach. A GshT peptide immediately C-terminal of the peptide identified in this study was detected in the isolate 57H.30 The homologous lipoprotein of strain TIGR4 was previously identified,41 and the crystal structure has been solved (PDB ID: 4EQ9). In the tertiary structure of GshT, both peptides were mapped to a protein domain located sufficiently distant from the lipid anchor to be surface-exposed. Recently, the importance of glutathione utilization for resistance to oxidative stress and heavy metals has been shown.79 In addition, deficiency in GshT led to an attenuated phenotype in a mouse model of infection indicating its implication in pneumococcal virulence.79 Peptides identified by shaving with immobilized trypsin were also mapped to the tertiary structures of the lipoprotein MalX and the membrane-anchored sortase SrtA. Their location in sufficient distance from the membraneanchoring domains suggests a surface exposure of the corresponding domains, verifying the suitability of the shaving approach for the identification of surface-exposed protein domains. Taking this into account, the shaving approach should also allow the identification of exposed domains in nonclassical surface proteins whose orientation on the cell surface is not predictable. Three peptides of the pyruvate oxidase SpxB were detected by trypsin shaving, and all three were derived from the thiamine pyrophosphate binding domain of the enzyme. SpxB peptides were also identified on the surface of all clinical isolates studied by Olaya-Abril and colleagues.30 SpxB plays an important role in pneumococcal metabolism and virulence.80 The enzyme decarboxylates pyruvate to acetyl phosphate and the byproducts H2O2 and CO2. SpxB is required for survival during exposure to high levels of H2O2, suggesting that it contributes to pneumococcal viability during oxidative stress.80,81 Pyruvate oxidase also confers a selective advantage during colonization.82 An alternative function of the pyruvate oxidase on the pneumococcal cell surface has not been reported to date. The optimized biotinylation method was used to investigate the global impact of an impaired lipoprotein maturation pathway on the surface proteome and exoproteome, focusing on the distribution of the lipoproteins between pneumococcal cell envelope and culture supernatant. Lipoproteins are anchored to the outer leaflet of the membrane by the 662



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mutant. Remarkably, TlpA/Etrx1 was not detected at all in the exoproteome, suggesting rapid degradation of released TlpA/ Etrx1. Recently the operons encoding TlpA/Etrx1 and Etrx2 were shown to be involved in invasive pneumococcal disease and the protection of S. pneumoniae against external oxidative stress.38,39 It is therefore conceivable that the loss of virulence of the Δlgt mutant is partially attributed to improperly anchored and therefore malfunctioning thioredoxin family proteins. The lack of lipid modification of lipoprotein precursors in the absence of functional Lgt may result in direct release of these proteins or release after processing by Lsp. A recent immunoblot analysis of four pneumococcal lipoproteins displayed on the surface of an Δlgt mutant seems to support the first option.43 However, only the situation on the bacterial surface was shown, whereas the protein species released into the culture supernatant was not analyzed. The immunoblots of whole cell extracts and corresponding supernatant fractions in the present study confirm that unprocessed lipoprotein precursors are partially or at least transiently retained in the membrane by the uncleaved signal peptide, as has also been discussed for S. aureus and B. subtilis.86,87 As shown here for MetQ, PsaA, PpmA, and also SlrA, nonlipidated lipoprotein precursor cleaved prior to release by Lsp is released more efficiently into the culture supernatant, similar to the processing shown for other Gram-positive bacteria.88,89 Contrary to the strict order previously described for lipoprotein processing in B. subtilis,90 transfer of the lipid moiety to the lipoprotein precursor seems not to be an absolute requirement for proteolytic signal peptide processing by Lsp in S. pneumoniae (Figure 7).

lipoprotein diacylglyceryl transferase Lgt, and the signal peptide is removed by the lipoprotein-specific signal peptidase Lsp.31 Accordingly, the deletion of the corresponding genes should have an impact on anchoring and localization of the lipoproteome of S. pneumoniae. Two previous studies addressed the effects of an lgt knockout mutation on pneumococcal growth, ABC transporter function, and virulence in S. pneumoniae. 43,46 Growth of the D39ΔcpsΔlgt mutant in THY media was comparable to the growth of the parental strain D39Δcps and confirmed previous findings, where either no difference46 or only a slightly longer lag phase43 was reported, indicating a similar viability of mutant and isogenic parental strain in complex media. Thus, in contrast with the drastic effect of the lgt mutation in Gram-negative bacteria, lipid modification of lipoprotein precursors by Lgt is not essential for in vitro growth of S. pneumoniae.46 However, the lgt mutation has been shown to have a striking effect on pneumococcal virulence, probably caused by the impaired anchoring of lipoproteins.43,46 Mice infected with mutants deficient in Lgt were less susceptible to infection than mice infected with the wild-type. In the sepsis and pneumonia mouse infection models, lgt knockouts were substantially attenuated in virulence. Nasopharyngeal colonization can still be established by the mutant to a certain degree, suggesting that especially the development of invasive disease is considerably impaired in the Δlgt mutant, which is most likely attributed to the pleiotropic effect caused by a globally impaired ABC transporter function due to the lack of functionally active Lgt.43 In the present study, none of the ABC transporter substrate-binding proteins was found to be more abundant on the surface of the Δlgt mutant compared with the parental strain D39Δcps. The vast majority was less abundant on the surface and more abundant in the exoproteome, indicating their impaired anchoring and hence their release from the pneumococcal surface into the growth medium. Remarkably, some substrate-binding proteins were identified in comparable amounts on the surface of both the Δlgt mutant and the parental strain D39Δcps, while they were more abundant in the exoproteome of the mutant, suggesting release and higher expression of these proteins. This clearly demonstrates that cation and nutrient acquisition and, consequently, also bacterial fitness and virulence are impaired in the Δlgt mutant due to the global loss of properly anchored substrate-binding proteins on the pneumococcal cell surface. In addition to ABC transporter components, other lipoproteins, such as the peptidyl-prolyl cis/trans-isomerase SlrA, were less abundant on the surface of the Δlgt mutant and more abundant in the exoproteome. Remarkably, the abundance of PpmA, the other pneumococcal lipoprotein with homology to peptidylprolyl isomerases, remained unaffected. SlrA and even more pronounced PpmA are homologues of the PrsA lipoprotein in B. subtilis, which has previously been shown to be essential due to its importance for extracytoplasmic folding of secreted proteins.83,84 Contrary to this, SlrA has a minor impact on pneumococcal colonization, and PpmA has no impact at all. Loss of SlrA and PpmA function did not result in reduced binding of pneumococci to extracellular matrix and serum proteins,40,85 suggesting that the deletion of lgt has no obvious pleiotropic effect on protein secretion owed to improperly anchored SlrA and PpmA. In the Δlgt mutant, anchoring of the two extracellular thioredoxin family proteins TlpA/Etrx1 and Etrx2 is severely impaired. This is evidenced by a considerably reduced abundance of TlpA/Etrx1 on the surface and an increased abundance of Etrx2 in the exoproteome of the Δlgt

Figure 7. Lipoprotein maturation in S. pneumoniae. In the wild-type, lipoprotein processing is in accordance with the general model described for Gram-positive bacteria.34,97 In a Δlgt mutant, Lsp seems to have the capability to process nonlipidated lipoprotein precursors, which are released into the exoproteome. In a Δlsp mutant, lipoprotein precursors are lipidated by Lgt and anchored by the lipid moiety and the signal peptide. Specific lipoproteins appear to be subjected to alternative signal peptide processing by an unknown enzyme. In the double mutant, processing of lipoprotein precursors is abolished, further supporting a role of Lsp in the cleavage of unmodified precursors.

In contrast with previous findings,41 growth of the Δlsp mutant was slightly delayed in THY compared with the parental strain D39Δcps, suggesting that Lsp is not essential but has some influence on the in vitro growth behavior of S. pneumoniae. Despite causing lethal disease in mice, the Δlsp mutant showed significant attenuation of virulence, which is most likely due to the impaired ABC transporter function 663



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caused by the loss of Lsp.41 Interestingly, this study demonstrates that the absence of Lsp barely affects surface association and release of some lipoproteins, whereas other lipoproteins were present in reduced amounts in both subproteomic fractions, indicating their proteolytic degradation. Enhanced degradation might be attributed to misfolding and hence malfunctioning of these lipoproteins in the mutant deficient in Lsp. In contrast, the phosphate-binding protein PstS showed an increased abundance in both subproteomes of the Δlsp mutant. PstS is involved in penicillin tolerance of S. pneumoniae. In penicillin-resistant mutants, the protein was overexpressed, and deletion of the pstS gene led to a higher susceptibility to penicillin.91 The lsp mutation was shown to have a similar effect on the penicillin sensitivity of pneumococci.41 Hence, the function of PstS is impaired in the Δlsp mutant. Improper processing seems to lead to increased release and higher expression of PstS, suggesting that the Δlsp mutant attempts to compensate the lack of functionally active and surface-displayed PstS protein. The deficiency in Lsp results in a lipoprotein species of higher molecular weight, which is in accordance with the presence of lipidated lipoprotein precursors on the pneumococcal surface.41 The immunoblot analyses shown in this study also suggest the presence of signal peptide-containing and lipidated lipoprotein represented by the protein species of the highest molecular weight detected. In addition to the lipid moiety, uncleaved signal peptide might support anchoring of the lipoprotein precursors to the membrane. However, the presence of a hydrophobic signal peptide might also cause changes in protein conformation and impede oligomerization, thereby leading to functionally inactive proteins prone to proteolytic degradation. No alternative signal peptide processing by signal peptidase I or another protease was observed for MetQ, PsaA, and PpmA, whereas a smaller SlrA protein species was detected, suggesting that some lipoproteins are processed alternatively in the absence of Lsp, as was first described in B. subtilis (Figure 7).87,92,93 Previous findings also indicate alternative processing of SlrA in the Δlsp mutant.41 In contrast with the data presented here, PpmA was also shown to be processed.41 In vitro growth of the generated ΔlgtΔlsp double-knockout mutant was severely impaired in complex THY media, indicating that the deficiency of Lsp and Lgt is not lethal but constitutes an enormous burden for S. pneumoniae. This is the first time that an isogenic double mutant lacking both Lgt and Lsp is shown to be viable in vitro for S. pneumoniae. Double mutants have been described for Streptococcus uberis94 and Streptococcus agalactiae.88 The ΔlgtΔlsp mutant of S. agalactiae was reported to grow like the wild-type in rich media,88 whereas nothing has been mentioned about the in vitro growth behavior of the S. uberis double mutant.94 In general, the changes in the lipoprotein abundance of the ΔlgtΔlsp mutant shown here seem to be less drastic in their extent but comparable in their tendency to the changes induced by the Δlgt mutant, which is indicated by a similar overall distribution pattern. In comparison with the parental strain D39Δcps, most of the lipoproteins were identified in similar abundance or were less abundant on the surface of the double mutant and more abundant in the exoproteome, indicating an increased release into the culture supernatant. The presented immunoblots confirm that the absence of Lsp in the double mutant results in less alternative processing of nonlipidated lipoprotein precursor compared with the Δlgt mutant, leading to the less drastic

increase in lipoprotein release observed. Again, signal peptide processing was observed only for SlrA, suggesting that also nonlipidated SlrA lipoprotein precursor is processed alternatively in the absence of both Lsp and Lgt (Figure 7).

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CONCLUSIONS This study presents a comprehensive analysis of the pneumococcal surface proteome. The analysis is based on results obtained by the application of two approaches, namely, the exclusive biotinylation of surface proteins and the shaving of surface-exposed protein domains with immobilized trypsin, which were both optimized for pneumococci in this work. Both methods complement each other, and each provides useful insights into the pneumococcal surface proteome. In addition, the biotinylation approach was applied to study the impact of mutations in the biogenesis of lipoproteins on the distribution of the lipoproteome between surface and exoproteome in S. pneumoniae. Finally, alternative lipoprotein processing in the lipoprotein maturation mutants was confirmed by immunoblot analysis of bacterial lysates and supernatant fractions, and a model for lipoprotein processing in S. pneumoniae is proposed.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1S. Growth curves of S. pneumoniae D39Δcps and its isogenic lipoprotein maturation mutants. Supplementary Figure 2S. Changes in the distribution of lipoproteins between bacterial cell surface and exoproteome of D39Δcps and its isogenic lipoprotein maturation mutants. Supplementary Figure 3S. Effects of the lipoprotein maturation mutations on processing and localization of pneumococcal lipoproteins. Supplementary Table 1. Proteins identified on the surface of D39Δcps using the biotinylation approach. Supplementary Table 2. Proteins identified on the surface of D39Δcps via the shaving approach. Supplementary Table 3. Peptides identified on the surface of D39Δcps via the shaving approach. Supplementary Table 4. Peptides identified for the proteins shown in Figure 4 on the surface of D39Δcps via the biotinylation approach. Supplementary Table 5. Proteins identified in the exoproteome of D39Δcps. Supplementary Table 6. Proteins identified on the surface of D39ΔcpsΔlgt using the biotinylation approach. Supplementary Table 7. Proteins identified in the exoproteome of D39ΔcpsΔlgt. Supplementary Table 8. Proteins identified on the surface of D39ΔcpsΔlsp using the biotinylation approach. Supplementary Table 9. Proteins identified in the exoproteome of D39ΔcpsΔlsp. Supplementary Table 10. Proteins identified on the surface of D39ΔcpsΔlgtΔlsp using the biotinylation approach. Supplementary Table 11. Proteins identified in the exoproteome of D39ΔcpsΔlgtΔlsp. Supplementary Table 12. Log2 ratio of mean NSAFs (D39ΔcpsΔlgt/D39Δcps). Supplementary Table 13. Log2 ratio of mean NSAFs (D39ΔcpsΔlsp/ D39Δcps). Supplementary Table 14. Log2 ratio of mean NSAFs (D39ΔcpsΔlgtΔlsp/D39Δcps). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Dörte Becher: E-mail [email protected]. Tel +49 3834 864230. Fax +49 3834 864202. *Sven Hammerschmidt: E-mail [email protected]. Tel +49 3834 864161. Fax +49 3834 864172. 664



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Author Contributions ∥

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M.M. A.D., and J.J.E.B. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Landesregierung Mecklenburg-Vorpommern, BMBF Medizinische Infektionsgenomik FKZ 0315828A, EU CAREPNEUMO FP7-HEALTH2007-B, Top Institute Pharma project T4-213, and a Rosalind Franklin fellowship of the University Medical Center Groningen. We thank Karsta Barnekow (Greifswald, Germany) for excellent technical assistance, Regine Landmann (Basel, Switzerland) for kindly providing the R6Δlgt mutant, and Tim Mitchell (Birmingham, U.K.) for the anti-Ply antiserum. We also thank Haike Antelmann (Greifswald, Germany) for stimulating discussions.

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REFERENCES

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