Cellular Microbiology (2014) 16(11), 1646–1665

doi:10.1111/cmi.12313 First published online 19 June 2014

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display Cindy Fevre,1 Jovanka Bestebroer,1 Mirjam M. Mebius,1 Carla J. C. de Haas,1 Jos A. G. van Strijp,1 J. Ross Fitzgerald2 and Pieter-Jan A. Haas1* 1 Department of Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. 2 The Roslin Institute and Edinburgh Infectious Diseases, University of Edinburgh, Easter Bush Campus, Midlothian, UK. Summary In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high-throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL-1 on neutrophils and thereby inhibits the interaction between PSGL-1 and P-selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti-cancer therapy. Our findings provide new insights into the pathogenesis

Received 7 March, 2014; revised 2 May, 2014; accepted 5 May, 2014. *For correspondence. E-mail [email protected]; Tel. (+31) 88 7555555; Fax (+31) 88 7555865.

of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.

Introduction To successfully colonize, invade and cause disease in their host, pathogenic bacteria produce a wide variety of small secreted or surface-associated proteins. Some of them interact with immune system components to inhibit host defences and reduce the inflammatory response. This process, referred to as immune evasion, is a key stage in bacterial pathogenesis and virulence (Ernst, 2000; Hartleib et al., 2000; Rooijakkers et al., 2005; Chung et al., 2006; Sibbald et al., 2006; Fraser and Proft, 2008). Identifying the immune evasion molecules and understanding their mechanisms of action may lead to the development of new anti-infective strategies and the discovery of new targets in treatment of inflammatory diseases and cancer (Waldner and Neurath, 2008). A bacterial secretome comprises secreted extracellular proteins, membrane proteins and cell wall-anchored proteins. These proteins are synthesized in the cytoplasm as protein precursors encoding various membrane-targeting motifs, such as signal sequences, that address proteins to different secretion systems, cell wall-anchoring motifs and/or transmembrane domains. The type and combination of these motifs determine the ultimate location of the protein. Identification and characterization of the bacterial secretome is challenging. Increasing numbers of algorithms and databases (Emanuelsson et al., 2007; PSORT programs for localization prediction; SPdb: A Signal Peptide Database) to predict the presence of specific motifs in a protein sequence were developed which allow the identification of many secretome proteins (Nielsen et al., 1997; Antelmann et al., 2001; Zhang and Henzel, 2004; Sibbald et al., 2006; Gupta et al., 2007; Leversen et al., 2009; Ravipaty and Reilly, 2010). However, in silico predictions of the Bacillus subtilis secretome identified only 50% of the proteins secreted in the supernatant of bacterial cultures (Antelmann et al., 2001). Sequence analysis of the Staphylococcus aureus secretome

© 2014 John Wiley & Sons Ltd

cellular microbiology

Bacterial protein-cell interaction by phage display identified in vitro, showed that 50% of the proteins did not have a predictable signal sequence and that 36% were annotated as hypothetical proteins (Sibbald et al., 2006). Subsequent to the identification of secretome proteins, characterization of their function is essential in understanding their potential role in immune evasion. Classical approaches to study the function of secreted proteins are labour intensive, heavily depend on growth conditions, level of protein expression and can only be applied to culturable bacteria (de Haas et al., 2004; Jongerius et al., 2007). Currently a high-throughput method for functional characterization of secreted proteins is not available. Phage display technology is the process of expressing proteins fused to phage coat proteins on the surface of a filamentous phage. A phagemid vector containing the gene encoding the fusion protein is encapsidated in the phage particle. A phage library contains a large amount of different phage clones each expressing a different protein resulting in a heterogeneous mixture of phages. The displayed protein often retains the behaviour of its free counterpart which allows for affinity selection of the phage displayed molecules. The selected phages can be amplified to perform further round of affinity selection under more stringent conditions. Successive rounds of selection allow for identification of phages displaying proteins with high affinity for a specific target and the displayed protein is then identified through sequencing of the phagemid vector. Secretome phage display, also called signal sequence phage display, is based on whole-genome phage display and constitutes a very promising alternative to in silico secretome analysis and classical functional characterization methods (Zhang et al., 1998; Rosander et al., 2002; Wall et al., 2003; Karlström et al., 2004). In this strategy, only secretome proteins of a bacterial genome are displayed, which is well suited to identify and characterize immune evasion proteins. In this study we describe the design and construction of an S. aureus secretome phage display library and its use to discover new immune evasion proteins interaction with cellular and humoral immune components. Staphylococcus aureus is a common human pathogen causing a variety of infectious diseases ranging from localized skin and soft tissue infections to life-threatening systemic diseases like septic shock, endocarditis and necrotizing pneumonia. To colonize and cause infection, S. aureus needs to overcome local and systemic host defences. S. aureus is known to produce over 200 secretome proteins many of which are involved in modulation of innate immune responses (Sibbald et al., 2006; Ravipaty and Reilly, 2010). In our newly designed phage display library, we confirmed that fusion protein display is secretome specific and for the first time we successfully performed functional © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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screening. Using our technique we identified two previously uncharacterized S. aureus secreted proteins, SElX and SSL6, that interact with neutrophil surface receptors. SSL6 was found to interact with CD47 and induced phagocytosis of erythrocytes while SElX bound PSGL-1 and inhibited the binding of neutrophils to P-selectin, an essential step in neutrophil recruitment to the site of infection.

Results Secretome phage display strategy By far, the most commonly used phage coat protein for phage display is the protein III (pIII). The wild-type (WT) pIII protein is made of two domains: the amino-terminal domain (N-pIII), required for phage infection and the carboxy-terminal domain (C-pIII), required for phage stability. As the fusion with C-pIII allows display of larger protein as compared to the full-length pIII (Jankovic et al., 2007), in the strategy described here, displayed proteins were fused to the C-pIII. In our phage display system an Escherichia coli is co-infected with a phage library and a helper phage. Each bacterium encodes a helper phage vector and one of the phagemid vectors from the phage library (Fig. 1). The helper phage DNA encodes all proteins required for the production of new phage particles, but is devoid of a packaging signal, which is a motif driving the incorporation of DNA into new phage particles. The phagemid vector carries the packaging signal, as well as randomly sheared chromosomal DNA fragments from the bacterial genome of interest, here S. aureus. This exogenous DNA is inserted upstream the carboxy-terminal part of the gene III (C-gIII) encoded on the phagemid vector (Fig. 1B). When both genes are in frame, it gives rise to a fusion protein. The specificity of secretome phage display, as compared to whole-genome phage display, is based on the absence of a membrane-targeting motif in the phagemid vector. Since addressing of a fusion protein to the E. coli membrane is required for the incorporation into new phage particles, only exogenous DNA encoding its own membrane-targeting motif, i.e. genes of the secretome, will lead to the production of phages displaying a fusion protein (type A and B phages Fig. 1A). Since the helper phage encodes for the WT-gIII, WT-pIII can also be incorporated into the new phages giving rise to phages displaying both the fusion protein and the WT-pIII (type B phages Fig. 1A) or only to WT-pIII (type C phages Fig. 1A). The latter will be referred to as background phages. The three types of phages can be used for affinity selection and consecutive phage amplification through E. coli co-infection with helper phages. Since the N-pIII is required for E. coli infection, the type A phages will not be

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Fig. 1. A. Secretome phage display strategy. For phage production, a helper phage DNA encoding all proteins necessary for the production of new phage particles is required. Phage proteins are addressed to the E. coli membrane, by their signal sequence, and assemble together to form a phage assembly machinery. As the helper phage is devoid a packaging signal, this DNA is not incorporated into the phage particles but the additional phagemid vector containing a packaging signal is incorporated into the phage particle instead. This phagemid vector also carries randomly sheared exogenous DNA from the bacterial genome of interest. This DNA has been inserted upstream the C-gIII encoded on the phagemid vector. This vector does not encode a membrane targeting motif. When the exogenous DNA is in frame with C-gIII AND encodes a membrane targeting motif, the resulting fusion protein is addressed to the membrane and is subsequently incorporated into the phage particle by the phage assembly machinery. Three types of phages (A, B and C), with different protein display can be produced. Type A phages display only the fusion protein. As they lack the N-pIII, required to infect a bacterium, these phages are not infectious, and cannot be amplified in further screening assay. Type B phages display both the fusion and the WT-pIII, therefore they display a secretome protein and can be amplify in further screening. Type C phages display only the WT-pIII, they can be amplified in further screening but do not display a secretome protein. These phages are therefore considered as background phages. In all other cases, where the exogenous DNA sequence is not in frame with the C-gIII, contains a stop codon or does not encode a membrane-targeting motif, the fusion protein is not expressed or not incorporated into the phage particle. In these cases the bacteria produce only background phages. SS: signal sequence; PS: packaging signal; MTM: membrane-targeting motif; WT-gIII: wild-type gene III; WT-pIII: wild-type protein III; C-gIII: carboxy-terminal part of the gene III; C-pIII: carboxy-terminal part of the protein III; N-gIII: amino-terminal part of the gene III; N-pIII: amino-terminal part of the protein III. B. Vector map of the pDJ01 phagemid vector: RBS: ribosome binding site; MCS: multiple cloning site; C-gIII: gene encoding the carboxy-terminal domain of pIII; CmR: Chloramphenicol resistance cassette. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

Bacterial protein-cell interaction by phage display amplified. The aspecific selection of the type C phages decreases over the selection rounds which are performed under more stringent washing conditions. After the last round the selected displayed proteins can be identified by sequencing the insert from the phagemid vector. Functional display of secretome proteins To evaluate if the S. aureus proteins displayed on phages are capable of binding their immune targets we first performed experiments using phages displaying the Chemotaxis Inhibitory Protein of S. aureus (CHIPS). CHIPS is a secreted protein that specifically binds and blocks both the C5a receptor (C5aR) and formylated peptide receptor (FPR) (Postma et al., 2004). Correct folding of CHIPS is essential for activity (Haas et al.,

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2005). Phages displaying CHIPS (Fig. 2B) were tested for their interaction with the C5aR and FPR that were stably transfected and expressed in U937 monocytic cell lines. Flow cytometry analysis using an α-M13-phage antibody showed specific binding of phages displaying CHIPS to C5aR and FPR expressing cells compared to control phages (Fig. 2A). The binding affinity of CHIPS to the C5aR and FPR are 1.1 nM and 35.4 nM, respectively, explaining the binding difference observed by Chipsphages to both receptors. These results confirmed that a phage displayed secretome protein retains its capability to interact with its target receptor. The need for a signal sequence in protein display was examined using Chips-phages devoid of a signal sequence (NoSSChips-phages, Fig. 2B). CHIPS display was detected by ELISA using two different α-CHIPS

Fig. 2. Phages displaying CHIPS. A. Interaction of phages displaying CHIPS with U937-C5aR ( area) and U937-FPR ( area) cells compared with control phages ( U937-C5aR and U937-FPR). The percentage of cells bound by the different type of phages is shown. The figure is representative of three independent experiments. B. Mapping of the four constructs used to produce the phages displaying the CHIPS protein onto the S. aureus genome. Numbers indicate distance in bp from ATG. The location of the chp gene is shown by the black arrow. PromChips: chp gene preceded by its native promoter and RBS; RbsChips: chp gene preceded by its own RBS; CHIPS: chp gene; NoSS-Chips: chp gene without its Signal Sequence. C and D. Phages displaying CHIPS, produced from the four types of constructs, were tested for interaction with the 2G8 (C) and 2H7 (D) α-CHIPS mAb by ELISA. The data represent means of three independent experiments. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

1650 C. Fevre et al. monoclonal antibodies, 2H7 and 2G8, which recognize a linear and conformational epitope respectively. The absence of binding of the NoSSChips-phages as compared to Chips-phages (Fig. 2C and D) confirmed that fusion proteins are not displayed in the absence of a signal sequence. The pDJ01 phagemid vector encodes a psp promoter and ribosome binding site (RBS) upstream the multiple cloning site (Fig. 1B). They were previously described to result in efficient fusion protein expression and display on the phage surface (Jankovic et al., 2007). In the secretome library, genomic fragments are inserted, some containing their own promoter and/or RBS. To test if the presence of a native promoter or RBS has an impact on fusion protein display, three constructs were made: (i) the chp gene preceded by its promoter and RBS (PromCHIPS), (ii) the chp gene preceded by the RBS but lacking the chp promoter (RbsCHIPS), and (iii) the chp gene without its promoter and RBS (Fig. 2B). The display of CHIPS was detected by ELISA using the two different α-CHIPS monoclonal antibody. Figure 2C and D show that the binding abilities of all phages were similar, suggesting that the presence of a native promotor or RBS does not impact protein display. Whole-genome bacterial library and secretome phage display library To generate an S. aureus secretome library, randomly sheared chromosomal S. aureus DNA fragments were ligated into the multiple cloning site of the pDJ01 phagemid vector between the RBS and the C-gIII (Fig. 1B). Constructs were transformed into E. coli yielding 7.2 × 107 individual clones considered as unique and constituting the bacterial library. Insert amplification from 562 randomly selected clones showed that 99.8% carry an insert and that 56% have an insert larger than 600 bp (Fig. 3A). The diversity and complexity of this bacterial library was assessed by sequencing 122 amplified inserts, which were shown to represent unique sequences and were evenly distributed over the S. aureus genome (Fig. 3B). The secretome phage display library was created by infecting the bacterial library with the VCSM13 helper phage encoding all proteins required for production of new phage particles, including the WT-pIII. The combination of pDJ01 and VCSM13 results in the production of phages displaying the fusion protein, the WT-pIII or both (Fig. 1A). After concentration the secretome phage library had a titre of 9.8 × 1012 infectious phages ml−1. To evaluate the complexity (i.e. the total amount of unique sequences) and diversity (i.e. the total amount of different genes), of the secretome phage display library, phages displaying a fusion protein were isolated from

background phages and their inserts were sequenced. As the phagemid vector encodes a Myc-tag in frame with the C-gIII (Jankovic et al., 2007) only phages displaying a secretome protein have a Myc-tag. They were isolated using an α-Myc-tag monoclonal antibody. To decrease the amount of background phages, 2 rounds of selection and amplification were performed. After each round, 76 and 78 inserts were sequenced respectively. All inserts were unique even though several were found to encode the same protein (Supplementary Tables S1 and S2). We identified 65 unique proteins out of 76 (85%) analysed after the first round, and 50 out of 78 (65%) after the second round indicating the high complexity and diversity of our phage library. Displayed proteins correspond to the secretome of S. aureus To confirm that the displayed proteins correspond to the S. aureus secretome, sequences obtained after both selection rounds with the α-Myc-Tag Ab were further analysed. After the first and second rounds, 12 (15.8%) and 2 (2.6%) inserts, respectively, encoded genes out of frame with the C-pIII, and/or encoded a stop codon preventing fusion with the C-pIII and/or lacked a membrane-targeting motif (Fig. 3C). These were considered background phages. All other inserts encoded at least a signal sequence and/or a transmembrane domain, showing that protein display is specific for the S. aureus secretome (Supplementary Tables S1 and S2). As expected, all signal sequences were full-length. They were all predicted to be classical (type 1) or lipoprotein sequences, showing that display is specific for proteins dependent on the Sec secretion pathway and/or having a transmembrane domain (Rosander et al., 2002; 2003; Wall et al., 2003; Karlström et al., 2004). According to the combination of signal sequences, cell wall anchoring motifs and transmembrane domains, displayed proteins were classified into four groups: cell wall-anchored proteins, membrane bound lipoproteins, extracellular proteins and transmembrane proteins (Supplementary Tables S1 and S2). Percentages of phages displaying these four subgroups of proteins after each selection round are shown in Fig. 3C. An enrichment of extracellular proteins was observed after the second selection round and the size of the displayed proteins was significantly lower (Student’s t-test: P = 0.019). This selection pressure towards small-sized fusion proteins can happen during interaction, infection or during amplification when there is an advantage for low protein size. In contrast to previous studies (Rosander et al., 2002), a biased selection towards inserts encoding a promoter/RBS was not detected (data not shown). © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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Fig. 3. Bacterial library characteristics. A. Insert size of 562 randomly selected clones from the bacterial library. The percentage of clones per size range is shown. B. Position of the genes encoded by 122 randomly selected clones from the bacterial library on the S. aureus Newman strain genome. The number of clones for each 100 000 bp interval of the genome is shown. Pearson χ2 analysis showed no statistical difference (P = 0.388). C. Phages displaying a fusion protein have been selected after two rounds of screening using an α-Myc-tag mAb. All displayed proteins belonged to the S. aureus secretome and were classified into four groups. The percentage of phages displaying each type of secretome proteins is shown.

© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

1652 C. Fevre et al. Identification of staphylococcal immune evasion proteins To show that the secretome phage display library is suitable for identification of immune evasion proteins using immobilized ligands, the library was screened for interaction with the 2H7 α-CHIPS mAb and three immune system components: the complement factor C3b, the von Willebrand factor (vWF), and the immunoglobulin G isotype 1 (IgG1). These immune components have previously been described to be modulated by S. aureus secretome proteins favouring its survival and colonization ability (Zhang et al., 1998; Bjerketorp et al., 2002; Jongerius et al., 2007). After three selection rounds using C3b, the efb gene encoding the extracellular fibrinogen-binding protein (Efb) was present in 50% of the inserts (Fig. 4A). Additionally, 23% of the inserts harboured the sbi gene encoding the S. aureus binder of Ig (Sbi) (Fig. 4A). Three and four unique overlapping sequences were identified for both proteins respectively (Fig. 4B). Both Efb and Sbi were previously found to interact with C3b (Lee et al., 2004). For screening using the vWF, the vwb gene encoding the von Willebrand factor binding protein (vWbp) was present in 83% of the inserts and seven unique overlapping sequences were identified (Fig. 4A and B). Using purified IgG, 89% of the inserts encoded the S. aureus protein A (Spa) and 21 unique overlapping sequences were identified (Fig. 4A and B). This high number of unique inserts could be explained by the presence of five IgG-binding domains in Spa, which led to the selection of many different truncated variants. Using the α-CHIPS mAb for selection, 87% of the inserts contained the chp gene and 4 unique overlapping sequences were identified (Fig. 4A and B). The high number of unique inserts selected after three rounds illustrates the high diversity of the library. As expected, all inserts encoded at least a signal sequence. The absence of full-length protein in the selected phages was not surprising since there is no selection pressure towards the carboxy-terminal part downstream the binding domain. For all genes the smallest inserts ended on the carboxy-terminal limit of the binding domain (Fig. 4B). Altogether these results demonstrate that the screening method described here can be used to identify interactions between an S. aureus secretome protein and a host immune system component.

SElX and SSL6 interact with isolated neutrophils Neutrophils are essential for an effective inflammatory response against invading S. aureus. Disorders of neutrophil function lead to an increased susceptibility to S. aureus infection. Previously, different S. aureus proteins that interact and block receptors on the neutrophil

surface were identified (Prat et al., 2006; 2009; Wines et al., 2006; Bestebroer et al., 2009). To identify secretome proteins interacting with cell surface proteins we screened our secretome phage library for binding to isolated human neutrophils. Four selection rounds were performed. Two distinct displayed proteins were selected, SElX encoded by the selx gene and SSL6 encoded by the set6nm gene (Fig. 4A). Five and three unique overlapping sequences were identified respectively (Fig. 4B). Both proteins have an unknown function and are classified into different protein families based upon genetic and structural homologies. SElX belongs to the group of staphylococcal enterotoxin-like proteins and was recently described to possess superantigenic activity and is associated with lethality in community-associated MRSA necrotizing pneumonia (Wilson et al., 2011) whereas SSL6 belongs to a family of staphylococcal superantigenlike proteins which includes proteins involved in host immune evasion proteins (Langley et al., 2005; Bestebroer et al., 2009). SElX and SSL6 bind glycosylated PSGL-1 and CD47 respectively To identify the receptor involved in the SElX and SSL6 interaction with neutrophils, we performed a multiantibody inhibition assay on isolated neutrophils and peripheral blood mononuclear cells (PBMC). We used a panel of 57 different antibodies that specifically recognize a variety of leucocyte surface receptors including chemokine and cytokine signalling receptors, and receptors involved in adhesion, chemotaxis or phagocytosis. SElX or SSL6 were added to examine competition with antibody binding. SElX and SSL6 were found to specifically interfere with binding of the α-PSGL-1 (α-CD162) (Fig. 5A) and α-CD47 (Fig. 5B) antibodies respectively. Next, the binding of SElX to PSGL-1-Fc was confirmed using ELISA (Fig. 5C) and the binding of SSL6 to CD47 was confirmed by flow-cytometry analysis by comparing the binding of fluorescently labelled SSL6 to CD47positive and CD47-deficient Jurkat cells (Fig. 5D). PSGL-1 and CD47 are both heavily glycosylated on neutrophils. However, PSGL-1 is hardly glycosylated on resting T-cells due to a low expression level of glycosyltransferases. The α-PSGL-1 antibody used (Serotec clone 3E2.25.5) detects both glycosylated and unglycosylated forms. As SElX does not inhibit antibody binding to PSGL-1 expressed on lymphocytes (Fig. 6A) this suggests that PSGL-1 glycosylation plays a crucial role in SElX binding (Fig. 6A). Indeed, upon removal of sialic acid moieties on neutrophils using Vibrio cholerae neuraminidase SElX did not block α-PSGL-1 binding (Fig. 6B), and SSL6-FITC no longer bound to the cells (Fig. 6C). Therefore, glycosylation of both PSGL-1 and © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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Fig. 4. Screening of the secretome phage display library. A. Percentage of phages displaying the different staphylococcal proteins identified after the third round of selection against C3b, IgG1, vWF, α-CHIPS mAb and fourth round in case of isolated human neutrophils. The immune targets are indicated on the x-axis. The total number of unique clones is indicated in parentheses. Phages encoding an out-of-frame insert, an in-frame stop codon or lacking a membrane targeting motif are considered background. B. The different inserts found in the selected phages are mapped on the corresponding S. aureus gene. The diagonally stripped boxes represent the signal sequence and the vertically stripped boxes represent the binding domain of the staphylococcal proteins (if known). Numbers indicate base positions relative to the start codon. For spa and vwb only the most relevant inserts are shown.

© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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CD47 is an essential determinant in the interaction with SElX and SSL6 respectively. SSL6 induces phagocytosis of erythrocytes Since CD47 deficiency was found to induce phagocytosis, we hypothesized that blocking CD47 by SSL6 may induce phagocytosis of normal human erythrocytes. Pre-incubating isolated erythrocytes with SSL6 indeed induced phagocytosis by granulocytes in a dose-dependent manner (Fig. 7B). The S-component of staphylococcal gamma-haemolysin (HlgA), which also

binds erythrocytes (Kaneko et al., 1997; Perret et al., 2012), was used as a control and showed no induction of phagocytosis. SElX inhibits PSGL-1-mediated cell adhesion SElX directly binds PSGL-1 and competes with the binding of an inhibitory α-PSGL-1 antibody. However, this does not necessarily mean that SElX inhibits PSGL-1 binding to its natural ligand P-selectin. In order to determine if SElX inhibits binding of PSGL-1 to P-selectin, we measured neutrophil adhesion to immobilized P-selectin under static conditions in the presence or absence of © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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SSL6−FITC concentration ( μg/mL) Fig. 6. Binding of SElX and SSL6 is glycosylation dependent. A. Inhibition of α-PSGL-1 mAb binding to neutrophils, monocytes and lymphocytes by SElX using flowcytometry. Graph shows α-PSGL-1 mAb binding in presence of SElX relative to buffer-treated cells. Figure represents three independent experiments. B. Competition of α-PSGL-1 mAb and SElX binding to neutrophils before and after neuraminidase treatment measured by flowcytometry. C. Binding of fluorescently labelled SSL6 to neuraminidase treated neutrophils. Fluorescence was measured using flowcytometry.

SElX. SSL5 a known PSGL-1 inhibitor was used as a control. SElX inhibited neutrophil binding to P-selectin in a concentration-dependent manner (Fig. 7A). Discussion In this study, we describe a strategy to construct a new S. aureus secretome phage display library with a high diversity and complexity. We confirmed that protein display is specific for transmembrane proteins and proteins secreted through the Sec secretion pathway, which is present in both Gram-positive and Gram-negative bacte© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

ria and used for secretion of most of the extracytoplasmic proteins (Pugsley, 1993). For the first time, we successfully screened a secretome phage display library to identify secretome proteins that interact with soluble host immune components, antibodies and isolated cells. For all tested immune components known to be modulated by S. aureus, at least one S. aureus protein has been identified. For the complement factor C3b, two S. aureus proteins were identified, highlighting the efficiency of the screening method. Using isolated cells, we confirmed that secretome phage display is a successful high-throughput method to identify putative S. aureus immune-modulators

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Fig. 7. SElX inhibits P-selectin-mediated cell adhesion and SSL6 induces phagocytosis. A. Adhesion of fluorescent human neutrophils to P-selectin coated plates was measured in presence of increasing concentrations of SElX. SSL5, a known inhibitor of PSGL-1, was used as control. Figure is representative of three independent experiments. B. Phagocytosis by neutrophils of fluorescently labelled human erythrocytes pre-incubated with increasing concentrations SSL6 or HlgA. Fluorescence was measured by flowcytometry. Data show mean and SEM of four independent experiments.

since in a single screening, SElX and SSL6 were identified to interact with neutrophils. Five secretome phage display studies have previously been published (Rosander et al., 2002; 2003; Wall et al., 2003; Karlström et al., 2004; Jankovic et al., 2007). These studies aimed at identifying the secretome of several bacterial species and none of these studies described any screening. Four of these studies used the pG3DSS phagemid vector that does not contain a ribosome binding site and encodes the strong constitutive promoter of SpA (Rosander et al., 2002; 2003; Wall et al., 2003; Karlström et al., 2004). Consequently, the presence of an RBS on the insert is required for protein display, which decreases the proportion of phages displaying a fusion protein. The strength and constitutive activity of the SpA promoter constitute a major drawback for proteins that are toxic when overproduced (Beekwilder et al., 1999; Rosander et al., 2002). Moreover, as E. coli is known to be sensitive to overproduction of pIII (Krebber et al., 1996), high expression of fusion protein likely impairs bacterial growth. An advantage of our strategy is the use of the pDJ01 phagemid vector encoding an RBS and the weaker psp promoter that is activated upon phage infection, i.e. after E. coli growth and upon infection with helper phage (Rosander et al., 2002; 2003; Wall et al., 2003; Karlström et al., 2004). The pDJ01 vector with the psp promoter had been shown to improve the display of toxic proteins (Beekwilder et al., 1999), and we found that it does not induce any biased selection of inserts encoding their own promoter and RBS. The pDJ01 phagemid vector has also been used in the fifth secretome phage display library previously described (Jankovic et al., 2007). However, in

this study the helper phage used was deprived of the WT-pIII. This strategy leads to the production of noninfectious phages displaying only the fusion protein. Without the pIII amino-terminus, amplification of phages, which constitutes the strength of our screening method, is not possible. Therefore, libraries created using a pIIIdeficient helper phage are suitable for secretome identification but not for high-throughput screening. For the first time, we demonstrate that a secretome phage display strategy combines both identification and functional screening of secretome proteins. Compared to in silico analysis and the classical functional methods, based on genetic homologies or function-guided purification (Haas et al., 2004), genomic data and predictive algorithms are not required. It can be applied to bacterial species that are not culturable and it can be extended by using metagenomic DNA as the starting material. Moreover, most secretome proteins can be studied at the same time, since the protein expression level is not limited. We have demonstrated that secretome phage display is a powerful technique to identify immune evasion proteins interacting with immune components including complex targets such as cells. A broader knowledge of those proteins is essential to understand immune modulating mechanisms and the pathogenesis of infections, in general. Secretome phage display is also a potent tool to identify immunogenic bacterial antigens for vaccine development, especially extracellular antigens implicated in pathological processes. As we showed with the α-CHIPS mAb screening, secretome phage library can be screened using antibody, and incubation with human immune serum can be used to identify new antigens (Beghetto et al., © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

Bacterial protein-cell interaction by phage display 2009). Secretome phage display technology can easily be extended to simultaneously characterize secretomes of multiple organisms or even metagenomes and can become a valuable tool in development of antiinflammatory molecules and vaccine strategies. We showed that secretome phage display can be used to identify bacterial immune evasion proteins that interact with cell surface receptors. Although multiple S. aureus proteins binding neutrophil cell surface receptors were described earlier we identified two previously uncharacterized proteins in our secretome phage selection experiments. Multiple factors determine the outcome of the phage selections. First, selection depends on the presence of the gene in the initial library and its expression as a fusion protein. In the system used here the presence of a signal sequence or membrane targeting motif within the inserted fragment is essential for fusion protein expression. Proteins containing a signal sequence processed by another system than the Sec secretion pathway were not identified in the phage library. Also, factors limiting protein expression like protein size, solubility, (mis)folding and toxicity interfere with fusion protein display. The carboxy-terminal end of the displayed protein is fused to the pIII protein. When protein binding is dependent on a free carboxy-terminus or when the active site is located close to the carboxy-terminal the displayed protein cannot bind its target and will not be selected. Other factors like binding affinity, amount of binding domains, conformational change, temperature and formation of protein complexes influence the outcome of a selection experiment. All these factors combined will lead to the preferential selection of a single phage clone after multiple selection rounds and repeating the experiment may well lead to a different outcome. In-depth analysis of phage clones during early screening using nextgeneration sequencing will give a better understanding on selection dynamics and may very well lead to identification of additional proteins. Also, to prevent selection of phages expressing SElX or SSL6 and identify other neutrophil binders, purified SElX and/or SSL6 can be added during selection to compete for receptor binding, like we did for Spa. The identification of the cell surface receptors for the identified proteins is based upon an antibody inhibition assay. This assay is a screening test to identify potential binding receptors on the neutrophil surface. No inhibition of antibody binding does not rule out a receptor as a binding target. Indeed, the protein binding site and the antibody binding site do not necessarily overlap. In our experiment we observed an inhibition of the α-PSGL-1 and α-CD47 Abs binding to neutrophils in the presence of SELX and SSL6 respectively. Further experiments © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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were performed to confirm these interactions. About the SELX-PSGL-1 interaction, we performed an ELISA coating SElX and detecting with Fc-tagged PSGL-1. We indeed confirmed the binding, however, high concentrations of SElX are necessary to observe this interaction. That may indicate that the SELX-PSGL-1 interaction is not the primary interaction for SELX binding to neutrophils. A additional receptor, not identified here, could be present on the cell surface. This will be further studied. SElX was previously described as a virulence factor in MRSA necrotizing pneumonia, but a molecular mechanism was not identified (Wilson et al., 2011). We show that SElX binds PSGL-1 and inhibits PSGL-1-mediated cell adherence to P-selectin, an essential step during extravasation of neutrophils to the site of infection. As the binding of SElX to PSGL-1 is glycosylation dependent, this interaction is very likely unrelated to the superantigen activity of SElX. Next to SSL5, this is the second staphylococcal protein found to interfere with this interaction. The exact affinity of SElX compared to SSL5 needs to be determined, but from our ELISA experiments, it appears to have a lower affinity to immobilized PSGL-1. The fact that multiple staphylococcal proteins apparently compete for the same function is not unique (Zhang et al., 1998; Lee et al., 2004; Rooijakkers et al., 2005; Burman et al., 2008). It appears that for efficient immune evasion, a certain amount of redundancy is necessary. The second neutrophil binder identified in this study, SSL6, belongs to the family of staphylococcal superantigen-like (SSL) proteins. Currently 14 different SSL proteins have been identified in S. aureus. They are secreted proteins with a high sequence similarity and structural homology to staphylococcal and streptococcal superantigens but do not display superantigen activity. Members of this family have recently been described to play a role in staphylococcal immune evasion. SSL7 was found to bind complement component 5 and IgA (Wines et al., 2006). SSL5 binds to PSGL-1 and thereby interferes with initial neutrophil rolling along the endothelium, a crucial step in neutrophil extravasation and recruitment to the site of infection (Bestebroer et al., 2009, p. 5). SSL3 was recently described to inhibit Tolllike receptor 2 activation (Bardoel et al., 2012; Yokoyama et al., 2012). SSL4 and SSL11 were found to bind glycosylated proteins via interaction with sialyl Lewis X (Chung et al., 2007, 11; Hermans et al., 2012). SSL10 binds human IgG, prothrombin and factor Xa to inhibit complement activation and blood coagulation (Itoh et al., 2010, 10; 2013, p. 10). CD47 is a cell surface protein that is expressed on nearly all cells in the body. CD47 has been described to play a role in a wide variety of processes. It was initially

1658 C. Fevre et al. identified as a regulator of integrin function, but was later found to have effect on phagocytosis, cell–cell fusion, cell migration, T-cell activation and regulated cell death (Oldenborg, 2013). CD47 was found to bind the signal regulatory protein alpha (SIRPα) that is highly expressed in macrophage and monocytic cell lineages, but is also found on other myeloid cells like granulocytes and dendritic cells (Seiffert et al., 1999). SIRPα is involved in negative regulation of cell functions. By interacting with SIRPα, CD47 functions as a ‘marker of self’. Oldenborg et al. showed that erythrocytes harvested from CD47deficient mice when transfused into wild-type mice had a markedly reduced circulation time (Oldenborg, 2013). CD47-deficient erythrocytes were cleared from the circulation within 24 h, whereas the average lifespan of normal mice erythrocytes is 45 to 60 days. This clearance was complement- and antibody-independent but was mediated by splenic macrophages and splenectomy in the transfused mice effectively abrogated the clearance of CD47-deficient erythrocytes (Oldenborg et al., 2000). We show that SSL6 interferes with the ‘marker of self’ signal mediated by CD47 and induces phagocytosis of host cells. During an inflammatory response, macrophages become more phagocytically active in order to clear invading pathogens. Jaiswal et al. found that CD47 is upregulated on haematopoietic stem and progenitor cells upon an inflammatory stimulus. This increased CD47 expression protects migrating haematopoietic cells from early phagocytosis by activated macrophages (Jaiswal et al., 2009). Lindberg et al. show that CD47-deficient mice have a decrease resistance against bacterial infection due to an impaired neutrophil influx into the inflamed tissue (Lindberg et al., 1996). SSL6 may play a role in inhibiting these key processes. Besides the infectious process, massive and/or chronic inflammatory response plays a major role in the pathogenesis of inflammatory diseases like sepsis, autoimmune diseases, degenerative diseases, cardiovascular diseases and cancer. An increasing number of anti-inflammatory molecules have been described as interesting therapeutic targets for the treatment of those diseases (Humbles et al., 2000; Barnum, 2002; Gerard and Gerard, 2002; Adcock et al., 2008; Waldner and Neurath, 2008). Recent studies found that CD47 shows an increased expression on a variety of human tumour cells (Willingham et al., 2012). Upregulation of CD47 expression on tumour cells leads to an overexpression of anti-phagocytic signals compared to pro-phagocytic signals, thereby preventing phagocytosis by immune cells. Of note, CD47 expression on tumour cells was found to inversely correlate with survival. Blocking CD47 using an α-CD47 antibody increased survival, inhibited tumour growth and effectively prevented tumour metastasis in a mouse tumour model (Willingham et al., 2012).

Further experiments will be needed to determine the activity of SSL6 in promoting tumour phagocytosis. Although further studies are required, the identification of the SSL6–CD47 interaction describes a completely new immune evasion strategy in S. aureus and can lead to the development of unique anti-infective, antiinflammatory and anti-cancer therapies.

Experimental procedures Ethics statement Isolation of human neutrophils: written informed consent was obtained from all subjects and was provided according to the Declaration of Helsinki. Approval was obtained from the medical ethics committee of the University Medical Center Utrecht (Utrecht, the Netherlands)

Helper-phage, phagemid vector, bacterial strains and growth conditions VCSM13 phage (Agilent technologies) was used as a helper phage. The phagemid vector PDJ01 was a generous gift from Dr Rakonjac (Massey University, Palmerston, New Zealand). E. coli TG1 (Lucigen) was used to produce phages. The bacteria were cultured in Luria–Bertani broth (LB) liquid medium at 37°C with slow (120 rpm) or fast (250 rpm) shaking or on 2% LB-agar (LBA) solid medium. Kanamycin (Sigma) at 25 µg ml−1 (Km25) was added upon helper phage infection and chloramphenicol (Sigma) at 10 µg ml−1 (Cm10) was added after infection of phages containing the phagemid vector.

Isolation of human neutrophils Leucocytes were isolated by means of the Ficoll-Histopaque gradient method. Venous blood was obtained from healthy volunteers using sodium heparin as anticoagulant (Greiner). Heparinized blood was diluted with an equal volume of PBS, and subsequently layered onto a gradient of Ficoll-Paque PLUS (Amersham Biosciences) and Histopaque-1119 (Sigma-Aldrich). After centrifugation for 20 min at 400 g, peripheral blood mononuclear cells (PBMC) were collected from the Ficoll layer and neutrophils from the Histopaque layer. After washing with RPMI 1640 containing 25 mM Hepes (Invitrogen), L-glutamine (BioWhittaker) and 0.05% human serum albumin (HSA; Sanquin) (RPMI/HSA), the neutrophils were subjected to a hypotonic shock with water for 30 s to lyse remaining erythrocytes, after which the neutrophils and PBMCs were washed with RPMI/HSA.

Construction of the phages displaying CHIPS Four portions of the S. aureus genome, containing the chp gene, were amplified by PCR using Pwo polymerase (Roche) following the manufacturer’s recommendations. Four different forward primers were used (Fig. 2B) (PromChipsF: 5′-ATTGCTGTG TCTAAGATAAATATAG-3′; RbsChipsF: 5′-AAGGAGAATTAACA TCATTATGA-3′; ChipsF: 5′-ATGAAAAAGAAATTAGCAACAA CAG-3′; NoSSChips: 5′-CTTTTACTTTTGAACCGTTTCCTAC© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

Bacterial protein-cell interaction by phage display 3′) in combination with one unique reverse primer (ChipsR: 5′-GTATGCATATTCATTAGTTTTTCCA-3′) which 5′ end excludes the chp gene stop codon. Amplified PCR products were processed with T4 polynucleotide kinase (New England Biolabs) and purified with a PCR purification kit (Qiagen). The pDJ01 vector was digested at the SmaI restriction site located upstream the C-gIII. The digested vector was dephosphorylated with both Calf intestinal alkaline phosphatase (Invitrogen) and Shrimp alkaline phosphatase (USB). After purification with PCR purification kit (Qiagen), PCR products and vector were ligated together using T4 DNA ligase (Invitrogen) and transformed into E. coli TG1. Transformed E. coli were grown for 1 h at 37°C, 250 rpm and spread on LBA-Cm10. After an overnight incubation at 37°C, the inserts of several clones were sequenced. For each construction, one clone having an insert with the chp gene in frame with the C-gIII (Fig. 2) and without mutation, was used for the phage production.

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residues 16 and 30 of the mature CHIPS protein (Haas et al., 2005). The 2G8 mAb binds a conformational epitope of CHIPS (P.-J.A. Haas, pers. comm.). ELISA plates (Maxisorb; Nunc) were coated overnight with 10 µg ml−1 of each α-CHIPS mAb in PBS. Further incubations were carried out in a volume of 50 µl for 1 h at 37°C, always followed by three washes with PBS-0.05% Tween-20. Dilution buffer for all reagents was TBS-0.02% Tween-20 containing 2% bovine serum albumin (BSA) (SigmaAldrich). First, plates were blocked with 4% BSA in PBS-0.05% Tween-20. Second, plates were incubated with different amounts of the phage library and incubated with the α-M13 mAb diluted at 1/200. Finally, peroxidase-conjugated goat α-mouse IgG1 (SouthernBiotech) was added at a 1/2000 dilution. Peroxydase activity was detected with 3,3′,5,5′-tetramethylbenzine (TMB) for 40 s and the reaction was terminated using H2SO4.

Construction of the secretome phage display library Phage production and titration TG1 E. coli were grown in LB-Cm10 at 37°C for 2h30 shaking at 120 rpm prior to helper phage infection at a multiplicity of infection of 20–50 for 30 min at room temperature. Kanamycin was added and bacteria producing phages were grown overnight at 37°C shaking at 250 rpm. Bacteria were removed by centrifugation at 3000 g for 40 min. Supernatant was incubated with gentamicin (30 µg ml−1) (Gibco) at 37°C for 1 h in presence of the cOmplete EDTA-free protease inhibitor cocktail (Roche). After another centrifugation at 12 000 g for 40 min the phages were precipitated overnight at 4°C by adding 0.15 volume of 16.7% PEG/3.3M NaCl. Phages were pelleted by centrifugation at 28 000 g for 2 h at 4°C and resuspended in tris buffered saline (TBS). Phage titre was determined by infecting TG1 E. coli previously grown for 2h30 in LB at 120 rpm, with serial dilution of the phage preparation. After 30 min of incubation at room temperature, infected E. coli were spread on LBA-Cm10 and incubated overnight at 37°C. The number of colony-forming units corresponded to the number of infectious phages.

Binding of the phages displaying CHIPS to U937-C5aR and U937-FPR cell lines U937 cells (human promonocytic cell line) stably expressing the C5aR or FPR were a generous gift from Dr Eric Prossnitz (University of New Mexico, Albuquerque). They were cultured as previously described (Postma et al., 2004). For the binding assay, incubations were carried out in a volume of 100 µl for 1 h at 4°C, always followed by three washes with RPMI-0.05% HSA that was also used for antibody dilutions. First 5 × 106 cells were incubated with 1 × 1012 phages displaying CHIPS in RPMI-0.05% HSA at 4°C for 1 h. Then cells were incubated with a 1/200 dilution of the mouse α-M13 mAb (GE Healthcare), specific for the phage pVIII proteins, and finally with a FITC-labelled goat α-mouse IgG (SouthernBiotech). Fluorescence was measured by flow cytometry (FACSCalibur; Becton Dickinson) and data were analysed using FlowJo software (Tree Star, Ashland, OR).

Phage ELISA Two different mouse α-CHIPS mAbs were used (Haas et al., 2004). The 2H7 mAb binds a linear epitope located between © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

Chromosomal DNA was isolated from S. aureus Newman strain using a NucleoBond AXG 100 kit (Macherey-Nagel) according to the manufacturer’s recommendations. Isolated chromosomal DNA was sheared by sonication. The majority of the DNA fragments had a size ranging from 0.3 to 3 kb. To remove fragments below 300 bp, Chroma spin TE200 (Clontech) size exclusion columns were used. Inserts were prepared for blunt end ligation using T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs). The pDJ01 vector was prepared as described in the section Construction of the phages displaying CHIPS. After purification with PCR purification kit (Qiagen), ligation of inserts (10 µg) and vector (2 µg) was achieved with the Ready to go T4 DNA ligase (Amersham Pharmacia). After purification using a PCR purification kit (Qiagen) followed by an ethanol precipitation, ligation products were transformed into TG1 E. coli. An aliquot of 200 µl was kept to determine the number of transformants by plating serial dilutions. The remaining bacteria were spread on LBA-Cm10. Solid medium was used to avoid any competition between clones. In order to end up with individual colonies, 30 large Petri dishes were used (500 cm2, VWR). After an overnight incubation at 37°C, 562 clones were selected, their inserts were amplified by PCR, their sizes were evaluated after migration on an agarose gel and 122 PCR products were sequenced (see Insert amplification, sequencing and analysis). Using Blastn (Altschul et al., 1990), the identified genes were mapped on the S. aureus Newman strain genome in order to determine their location. The remaining clones, which constitute the bacterial library, were pooled in LB-20% glycerol, aliquoted and stored at −70°C. Phage production was performed using 1/200 portion of this bacterial library as described in Phage production and titration.

Screening The phage library was screened for interaction with the 2G8 α-CHIPS mAb and three host immune system components: complement factor C3b isolated from serum as previously described (Lambris et al., 1980), human IgG1 (SouthernBiotech) and vWF isolated from serum as previously described (Hulstein et al., 2007). Five wells from an ELISA plate (Maxisorb; Nunc) were coated with a total amount of 5 µg of each immune protein or antibody in PBS overnight at 4°C. Thereafter, wells were blocked

1660 C. Fevre et al. for 1 h at 37°C with PBS containing 40 mg ml−1 BSA-0.05% Tween-20. Proteins purified from serum and Ab, except the IgG1, were also blocked with PBS-0.05% Tween-20 containing 50 µg ml−1 Spa (Sigma) in order to prevent selection of phages displaying Spa. After washings, 100 µl per well of the phage library was added and incubated at room temperature for 4 h with a slight shaking, followed by three washes and elution of bound phages by addition of 0.05 M Na-citrate/0.15 M NaCl, pH 2 and neutralized with 2 M Tris-HCl pH 8.4. The selected phages were amplified by infecting TG1 E. coli and infected bacteria were spread on LBA-Cm10. After an overnight incubation at 37°C all colonies were pooled and phage production was induced by adding helper phages (see Phage production and titration). A second and a third round of selection were performed in the same way, except that incubation time with host immune system components lasted for 2 h and plates were washed 10 times. After the last selection round 48 clones of TG1 E. coli infected with the selected phages were randomly chosen, the inserts were amplified and sequenced to identify the displayed proteins. Selection of phages displaying a fusion protein was achieved using a rabbit polyclonal α-Myc-tag Ab (Abcam) and two selection rounds selection rounds were performed. After infection of E. coli with the selected phages followed by an overnight culture on LBA-Cm10, 85 clones were picked after each round in order to amplify and sequence the insert encoding the selected proteins.

Screening with neutrophils Isolated human neutrophils were resuspended in 1 ml ice-cold RPMI at 2 × 107 neutrophils ml−1. 40 mg ml−1 BSA and 50 µg ml−1 Spa were added and incubated for 30 min on ice. Cells were washed with 6 ml ice-cold RPMI and centrifuged at 300 g and 4°C for 10 min. Cells were resuspended in RPMI containing 20 mg ml−1 BSA and 25 µg ml−1 Spa, 300 µl phages were added and incubated for 45 min on ice. Cells were washed twice with ice-cold RPMI and resuspended in 500 µl elution buffer (0.05 M Na-citrate/0.15 M NaCl, pH 2) and incubated for 4 min in total (1.5 min shaking, 1.5 min spin down at 2000 rpm, 1 min to stop the centrifuge). The eluted phages were transferred into tubes containing 62.5 µl neutralization buffer (2 M Tris-HCl pH 8.4) and used to infect 10 ml TG1 E. coli.

Insert amplification, sequencing and analysis Selected clones were resuspended in 50 µl water, boiled for 10 min at 96°C, and centrifuged for 10 min at 14 000 g. The supernatant was used as DNA template to amplify the inserts contained in the phagemid vectors of the selected phages. PCR amplification was performed with HotStar Taq Master mix (Qiagen) using primers InsertF (5′-GGAAGAGCTGCAGCA TGATGAAA-3′) and InsertR (5′-CACCGTAATCAGTAGCGAC AGAA-3′). The inserts were sequenced using the same primers, with BigDye version 3.1 Cycle Sequencing kit (Applied Biosystem), and were analysed on an ABI 3730 DNA analyser apparatus. Sequences were edited with SeqMan program of the LaserGene package (DNA Star, Madison, Wisconsin). Only sequences with the full-length insert, including both vector limits, were further analysed. The encoded genes were identified with Blastn (Altschul et al., 1990). In order to determine if those genes

were in frame with the C-pIII, and which part of the gene was encoded by the insert, the sequences were translated, and analysed with BlastP (Altschul et al., 1990). The presence of a classical SS was evaluated using SignalP 3.0 (Dyrløv Bendtsen et al., 2004).

Cellular location of the displayed proteins The cellular location of the displayed protein was determined based on the annotation of the highest BlastP hit with the S. aureus Newman genome. If not informative the highest blast hit with another S. aureus was considered, with a minimum of 99% similarity. Presence of signal sequences, transmembrane domains and cell wall anchoring motifs was assessed using several prediction software packages. SignalP 3.0 software was used to identify classical signal sequences (Sibbald et al., 2006). When more than two output scores of the SignalP neural network prediction were below the cut-off values, the signal sequence prediction was based on the identification of the n-, h-, and c-region probability from hidden Markov model. LipoP 1.0 software (Juncker et al., 2003) was used to predict lipoprotein signal sequences and TatP 1.0 (Bendtsen et al., 2005) was used to predict Tat signal sequences, which are specific of proteins secreted through the Tat secretion system (Sibbald et al., 2006). Presence of a signal sequence specific for Comm and ABC transporter secretion systems were not tested, as no specific prediction software is available. The prediction of transmembrane helices was determined using TMHMM 2.0 (Krogh et al., 2001) software. Those four prediction tools were chosen among the others based on several comparative studies which describe them to give the best predictions (Möller et al., 2001; Bendtsen et al., 2005; Rahman et al., 2008; Ravipaty and Reilly, 2010). When a transmembrane helix was predicted to be present in the signal sequence, it was considered as a false prediction of the TMHMM 2.0 software (Emanuelsson et al., 2007). This is due to the way both SignalP and TMHMM software were trained and in all cases annotations and/or literature confirmed false prediction of a transmembrane helix. The presence of cell wall anchoring motifs, i.e. the Gram-positive specific YSIRK and LPxTG motifs as well as the S. aureus-specific NPQTN motif, was assessed manually. The presence of a LysM domain, which are widely distributed among bacteria, was deduced based on the annotation of the highest BlastP hit. When annotations and literature were not informative, about the natural location of the displayed proteins, proteins with both a classical signal sequence and a cell wall anchoring motif were classified as cell wall anchored. Proteins with both a classical signal sequence and a lipoprotein signal sequence were classified as membrane-bound lipoprotein (Emanuelsson et al., 2007). Proteins with only a classical signal sequence were considered to be secreted and proteins with at least one TM helix were considered to be transmembrane. Proteins described to be both secreted and cell wall anchored were classified here as secreted proteins, except Spa.

Cloning, expression and purification of recombinant proteins SElX was expressed and purified as described earlier (Wilson et al., 2011). Expression of SSL5 and SSL6 were performed as described earlier (Bestebroer et al., 2007). Briefly, the SSL5 gene © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

Bacterial protein-cell interaction by phage display (ssl5) and the SSL6 gene (set6-nm) of S. aureus strain NCTC8325, except for the signal sequence, were cloned into the expression vector pRSETB (Invitrogen) directly downstream of the enterokinase cleavage site. After verification of the correct sequence the constructs were transformed into RosettaGami(DE3)pLysS E. coli according to the manufacturer’s protocol (Novagen). Expression of HIS-tagged protein was induced with 1 mM isopropyl-γ-D-thiogalactopyranoside (IPTG; Roche) for 3 h. HIS-tagged protein was isolated under denaturing conditions on a HiTrap chelating HP column (Amersham Biosciences). The protein was renatured on the column by gradually exchanging denaturing buffer (8 M urea, 500 mM NaCl, 20 mM Na2HPO4, pH 5.3) for native buffer (500 mM NaCl, 20 mM Na2HPO4, pH 5.3). Bound protein was eluted using 50 mM EDTA. After dialysis, the HIS-tag was removed from the protein by cleavage with enterokinase according to the manufacturer’s instructions (Invitrogen). Proteins were stored in PBS at −20°C. Protein purity was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Competition for receptor binding To determine a putative receptor for SElX and SSL6, a mixture of neutrophils (5 × 106 cells ml−1) and PBMC (1 × 107 cells ml−1) was incubated with 10 µg ml−1 SElX or SSL6 for 15 m on ice. Subsequently, 57 FITC-, PE- or APC-conjugated mAbs directed against a series of cell surface receptors were added for 30 min on ice. After washing, fluorescence was measured using flow cytometry. Neutrophils, monocytes and lymphocytes were identified according to their FSC/SSC pattern. For specific SElX binding experiments, neutrophils and/or PBMC (5 × 106 cells ml−1) were incubated with increasing concentrations of SElX for 30 min on ice. After washing, cells were incubated with a PE-conjugated α-PSGL-1 (BD Pharmingen) mAb for 30 min on ice, washed and fluorescence was measured using flow cytometry. Where indicated, cells (2 × 106 cells ml−1) were first treated with 0.2 U ml−1 neuraminidase (from V. cholerae, Sigma) at 37°C for 45 min at pH 6.0.

PSGL-1 ELISA ELISA plates (Maxisorb; Nunc) were coated with 3 µg ml−1 SElX or SSL5 overnight at 4°C. Plates were washed with PBS-0.05% Tween-20 and blocked with PBS-0.05% Tween-20 5% BSA for 1 h at 37°C. Plates were washed and incubated with different concentrations PSGL-1-Fc for 1 h at 37°C. Bound PSGL-1-Fc was detected using a peroxidase-conjugated goat-α-human-Fc monoclonal antibody (Jackson Immunoresearch). Peroxydase activity was detected with TMB for 40 s and the reaction was stopped using H2SO4.

Binding of SSL6 to cells To determine binding of SSL6 to cells, SSL6 was labelled with FITC, as described earlier for SSL5 (Bestebroer et al., 2007). Briefly, 1 mg ml−1 SSL6 was incubated with 100 µg ml−1 FITC in 0.1 M sodium carbonate buffer (pH 9.6) for 1 h at 37°C. Unbound FITC was removed using a HiTrap desalting column. Human neutrophils (5 × 106 cells ml−1 in RPMI) were incubated with increasing concentrations of SSL6-FITC for 30 min on ice and © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1646–1665

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washed. SSL6-FITC binding to neutrophils was analysed by flow cytometry. To test whether SSL6 binding is dependent on glycosylation, neutrophils were pre-treated with neuraminidase, as described in the section Competition for receptor binding. To confirm the SSL6–CD47 interaction, Jurkat cells, naturally expressing CD47 or CD47-deficient Jurkat cells [Jurkat CD47deficient, clone JinB8 (Reinhold et al., 1999)] were incubated with 1 µg ml−1 SSL6-FITC on ice for 30 min. The binding of SSL6FITC to Jurkat cells was analysed by flow cytometry. The Jurkat and Jurkat CD47-deficient cell line were kindly provided by Dr Timo van den Berg and cultured in RPMI containing 5% FCS and 50 µg ml−1 gentamicin.

Adhesion of neutrophils to P-selectin under static conditions To investigate adhesion of neutrophils to P-selectin, neutrophils were loaded with 4 µM calcein-AM (Molecular Probes, Leiden, the Netherlands) in Hank’s buffered salt solution (HBSS, BioWhittaker) with 0.05% HSA. A 96-well plate (Greiner bio-one) was coated with 3 µg ml−1 P-selectin (R&D Systems) for 1 h at 37°C. After washing with PBS, the plate was blocked with 4% BSA for 90 min at 37°C. The plate was then washed, and 3 × 105 calcein-labelled neutrophils were added to duplicate wells and allowed to adhere for 15 min at room temperature. After washing twice, adherent cells were quantified using a platereader fluorometer (FlexStation).

Phagocytosis of human erythrocytes Isolated human erythrocytes were labelled with the PKH2 green fluorescent cell linker kit (Sigma) following manufacturers protocol. Cells were washed and resuspended in RPMI-0.05% HSA. Labelled erythrocytes (107 cells per 100 µl) were incubated with increasing concentrations of SSL6 or HlgA on ice for 30 min. Erythrocytes were washed with RPMI-0.05% HSA and 106 human neutrophils were added and incubated with vigorous shaking at 37°C for 15 min. Cells were fixed with 1% paraformaldehyde and fluorescence was measured using flowcytometry.

Acknowledgements The phagemid vector PDJ01 was a generous gift from Dr Rakonjac (Massey University, Palmerston, New Zealand).

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Supplemental information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Tables S1 and S2. Cellular location of the displayed proteins. The cellular location of the displayed protein from phages selected after one (Table S1) and two rounds (Table S2) of

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selection against the α-Myc-tag Ab is shown. (a) Annotation from the S. aureus Newman genome. When annotated as hypothetical protein or when not informative about the cellular location, the highest BlastP hit result is indicated. Accession numbers and % homologies are given in parentheses. (b) Gene names correspond to the Newman strain annotation or when more commonly used, from the second annotation. (c) Portion of the protein that is displayed. The first position corresponds to first amino acid of the immature protein. When a protein was identified several times the different portions that are displayed are indicated. (d) Prediction of classical SS and (e) lipoprotein SS. (f) Prediction of the number of TM helices in the WT protein and, in parenthesis, the portion of the protein that is displayed. (g) When location of a protein had been previously described reference is indicated. CW: cell wall-anchored protein; EC: extracellular protein; MB: membrane-bound protein; TM: transmembrane protein.

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display.

In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and chara...
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