Staphylococcus Aureus Superantigen-Like Protein SSL1: a Toxic Protease

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Article Staphylococcus aureus Superantigen-Like Protein SSL1: A Toxic Protease

Aihua Tang 1,*, Armando R. Caballero 1, Michael A. Bierdeman 1, Mary E. Marquart 1, Timothy J. Foster 2, Ian R. Monk 2,3 and Richard J. O’Callaghan 1

1 Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, MS 39216, USA; [email protected] (A.R.C.); [email protected] (M.A.B.); [email protected] (M.E.M.); [email protected] (R.J.O.) 2 Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, D02 PN40, Ireland; [email protected] (T.J.F.); [email protected] (I.R.M.) 3 Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, University of Melbourne, 3000 Melbourne, Australia * Correspondence: [email protected]; Tel.: +1-601-984-1700

Received: 7 November 2018; Accepted: 20 December 2018; Published: 1 January 2019

Abstract: Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. The present study characterized a previously unrecognized S. aureus toxin. This secreted toxin was purified from strain Newman ∆hla∆hlg, the N-terminal sequence determined, the gene cloned, and the purified recombinant protein was tested in the rabbit cornea. The virulence of a toxin deletion mutant was compared to its parent and the mutant after gene restoration (rescue strain). The toxin (23 kDa) had an N-terminal sequence matching the Newman superantigen-like protein SSL1. An SSL1 homodimer (46 kDa) had proteolytic activity as demonstrated by zymography and cleavage of a synthetic substrate, collagens, and cytokines (IL-17A, IFN-γ, and IL-8); the protease was susceptible to serine protease inhibitors. As compared to the parent and rescue strains, the ssl1 mutant had significantly reduced virulence, but not reduced bacterial growth, in vivo. The ocular isolates tested had the ssl1 gene, with allele type 2 being the predominant type. SSL1 is a protease with corneal virulence and activity on host defense and structural proteins.

Keywords: Staphylococcus aureus; protease; superantigen-like protein; dimerization; cytokine

1. Introduction Staphylococcus aureus causes infections in a wide variety of non-ocular sites and is a major ocular pathogen [1–6]. There are 27.6 cases of bacterial corneal infections per 100,000 people, and 130.4 cases per 100,000 contact lens users [7,8]. S. aureus is often described as the leading cause of corneal infections, including infections after LASIK surgery [4]. In a multi-center study, 42% of over 1,100 ocular S. aureus isolates were methicillin-resistant with a high concurrence of resistance to aminoglycosides, ﬂuoroquinolones, and macrolides [8]. S. aureus secretes over thirty biologically active substances, of which many are virulence factors favoring bacterial survival in host tissue [9–12]. Two hemolytic toxins encoded by ≥ 98% of isolates [13,14], alpha-toxin (encoded by hla) and gamma-toxin (encoded by hlg), have been shown to contribute signiﬁcantly to the pathology of experimental S. aureus keratitis [15,16]. Community- acquired MRSA strains may express another potential corneal virulence factor, Panton-Valentine leukocidin (PVL) [17]. In vitro, these secreted toxic proteins are produced late in the bacterial growth

Pathogens 2019, 8, 2; doi:10.3390/pathogens8010002 www.mdpi.com/journal/pathogens Pathogens 2019, 8, 2 2 of 17 cycle due to the genetic regulation by the accessory gene regulatory (agr) system [18,19]. A key problem in keratitis is that virulence factors causing tissue damage lead to permanent scarring that decreases vision. An antibiotic that kills the infecting bacteria cannot inhibit the corneal damage mediated by bacterial toxins or enzymes. No chemical or immunological reagent is available for clinical use to stop the damage mediated by these bacterial products. The success of S. aureus as a tissue infecting pathogen is mediated by its ability to avoid host defenses. Among the immune evasion mechanisms are multiple superantigen-like proteins that inhibit components of both the adaptive and innate immune responses [12]. Staphylococcal superantigen-like proteins (SSL) share structural similarities with superantigens, but unlike superantigens, the SSL proteins do not bind MHC receptors or T cell receptors to elicit a toxic cytokine response [12]. SSL proteins have been shown to bind specifically targeted host defense proteins such as IgA, IgG, and complement components [20–24]. Koymans et al. demonstrated in vitro that SSL1 and SSL5 limit neutrophil chemotaxis and migration by inhibiting the activity of matrix metalloproteases, which provides a rationale for further studies to examine virulence of these proteins in an in vivo model of infection [25]. Alpha-toxin and, to a lesser extent, gamma-toxin are the potent corneal virulence factors produced by S. aureus [2]. S. aureus strain Newman was reported to lack alpha-toxin production and, since this toxin is important for corneal virulence, strain Newman was expected to have significantly reduced virulence relative to other S. aureus strains. However, when injected into the rabbit cornea, strain Newman produced a severe infection [15]. When the alpha-toxin and gamma-toxin genes were mutated to prevent any production of these two toxins, strain Newman retained an unexpected amount of virulence. The present study was initiated to identify a novel virulence factor that contributes to S. aureus corneal infections. The findings demonstrate that the superantigen-like protein SSL1 exhibits protease activity and plays an important role in virulence during a corneal infection.

2. Results

2.1. Corneal Virulence of S. aureus Mutants A rabbit model of experimental keratitis was used to determine virulence. Intra-corneal injection of 100 colony-forming units (CFU) of S. aureus strain Newman resulted in severe ocular pathology as measured by a slit lamp examination (SLE) score of 13.44 ± 0.43 at 24 h post-infection (PI). The Newman mutant deﬁcient in both alpha- and gamma-toxins caused signiﬁcantly less pathology than the wild-type strain (SLE score: 8.63 ± 0.35; P ≤ 0.001), but did mediate substantial pathology (Figure1A) relative to normal eyes (SLE score: 0; data not shown). Both the wild-type and ∆hla∆hlg strains grew to similar numbers of viable bacteria in the cornea (7.36 ± 0.06 and 7.30 ± 0.09 log CFU per cornea, respectively; P = 0.717). PathogensPathogens 2018, 72018, x , 7, x 3 of 173 of 17 Pathogens 2019, 8, 2 3 of 17

1 1 2 2 16 16BacteriaBacteria CultureCulture 14 14 SupernatantsSupernatants 12 12

10 10 8 8 SLE SLE 50 kDa50 kDa 6 6 4 4 37 kDa37 kDa 2 2 0 0

B B C C A A FigureFigure 1. Ocular 1. Ocular pathology pathologyFigure of parental 1. ofOcular parental and pathology double and double mutant of parental mutant S. aureus and S. aureus doublestrain strain Newman mutant NewmanS. and aureus their andstrain culturetheir Newman culture and their culture supernatantssupernatants in rabbit in rabbit corneassupernatants corneas and theand in rabbitprotein the protein corneas contributing contributing and the to protein virulence. to virulence. contributing (A) The (Ato) SLEThe virulence. scoreSLE scoreat ( A24) Theat 24 SLE score at 24 h PI hourshours PI of PI S. ofaureus S. aureusof strainS. aureus strain Newmanstrain NewmanNewman double double doublemutant mutant mutant(Δhla Δ(Δ (hlg∆hlahla; Δ∆hlghlg; ; ) lacking )) lacking lacking alpha- alpha- alpha- and and and gamma-toxins, which gamma-toxins,gamma-toxins, which which producedproduced produced substantial substantial substantial pathology pathology pathology as ascompared comparedas compared to normal to normalto normal eyes, eyes, eyes,was was significantly was significantly significantly less than that of the less thanless thanthat ofthat the of wild-type thewild-type wild-type strain strain strain ( ( ()( ) (PP ≤ ≤) 0.001).0.001).(P ≤ 0.001). WhenWhen When theirtheir culture theirculture culture supernatants supernatants supernatants were were injected were directly into the injectedinjected directly directly into theintocorneal corneal the stroma,corneal stroma, thestroma, SLEthe scoreS theLE scoreS ofLE the scoreof double the of double mutantthe double mutant at 24 mutant h afterat 24 injection athours 24 hours after was substantialafter compared to injectioninjection was substantial was substantialnormal compared compared eyes, to but normal significantlyto normal eyes, eyes,but lower significantly but thansignificantly that lower of the lower than wild-type thatthan of that strainthe of wild-type the (P = wild-type 0.004). (B ) Non-denaturing strainstrain (P = 0.004).(P = 0.004). (B) Non-denaturing (no(B) SDS)Non-denaturing zymogram (no SDS) of(no the SDS)zymogram pooled zymogram fractions of the of ofpooled the concentrated pooled fractions fractions culture of concentrated supernatantof concentrated of the S. aureus double cultureculture supernatant supernatant of themutant of S. the aureus (NewmanS. aureus double double∆ mutanthla∆hlg mutant) (Newman containing (Newman Δ toxichlaΔ Δhlg activitieshla) Δcontaininghlg) tocontaining the toxi rabbitc toxiactivities cornea.c activities (toC) A to zymogram with SDS the rabbitthe rabbit cornea. cornea. (C) A (showing Czymogram) A zymogram standard with SDSwith proteins showingSDS (Laneshowing standard 1) and standard the proteins extracted proteins (Lane toxin (Lane 1) and (Lane 1) the and 2). extracted the extracted toxin toxin(Lane (Lane 2). 2). 2.2. Corneal Toxicity of Culture Supernatants 2.3. Identification2.3. Identification of the of Virulence theIntra-corneal Virulence Factor Factor injection of stationary phase culture supernatant of the wild-type or double mutant ± ± The concentratedThe concentrated( ∆supernatanthla∆ supernatanthlg) resulted of the of in double SLEthe double scores mutant ofmutant 10.31 was fractionatedwas0.40 fractionated or 8.75 on0.68, a ongelrespectively afiltration gel filtration column (P = column 0.004) (Figure 1A). Thus, and thenand thenfractions fractions weredespite were injected lackinginjected into alpha- rabbitinto rabbit andcornea gamma-toxins, corneas. Zymographys. Zymography the doubleof the of fractions mutantthe fractions and containing its containing supernatant toxic toxic both demonstrated activitiesactivities under under non-denaturing non-denaturingcorneal virulence, conditions conditions implying demonstrated demonstrated an additional a single a virulence single proteolytic pr factoroteolytic band was band (Figure involved. (Figure 1B). This1B). This proteaseprotease extracted extracted from from the gelthe and, gel and,on a onzymo a zymogramgram under under denaturing denaturing conditions, conditions, was wasfound found to to 2.3. Identification of the Virulence Factor migratemigrate at ~46 at kDa ~46 kDa(Figure (Figure 1C). This1C). Thisprotease protease on a ondenatured a denatured and reducedand reduced SDS-PAGE SDS-PAGE migrated migrated at at ~23 kDa~23 kDa(Figure (Figure 2A), 2A),suggestingThe suggesting concentrated that thatdimerization supernatant dimerization was of the wasimportant double important mutant for activity.for was activity. fractionated The TheN-terminal onN-terminal a gel filtration column and sequencesequence of 14 of amino 14 aminothen acids fractionsacids (i.e., (i.e.,A-E-V-K-Q-Q-S-E-S-E-L-K-H-Y) were A-E-V-K-Q-Q-S-E-S-E-L-K-H-Y) injected into rabbit corneas. yielded Zymography yielded in a BLASTin a of BLAST the search fractions search a 100% containing a 100% toxic activities matchmatch with withthe SSL1the underSSL1 protein protein non-denaturing of strain of strain Newm conditions Newman (froman demonstrated(from gene geneNWMN_0388; NWMN_0388; a single proteolytic GenBank GenBank bandAccession Accession (Figure 1B). This protease NumberNumber BAF66660.1). BAF66660.1).extracted Also, Also, analysis from analysis the of gel the of and, SSL1the on SSL1 aprotein zymogram protein by massby under mass spectrometry denaturing spectrometry conditions,revealed revealed only was only found to migrate at peptidespeptides matching matching the~46 sequ the kDa encesequ (Figure enceof the of1 NewmanC). the ThisNewman proteaseSSL1 SSL1 protein on protein a (data denatured (data not shown). not and shown). reduced The ssl1The SDS-PAGEgene ssl1 generesides resides migrated at ~23 kDa withinwithin the SaPIn2the SaPIn2 pathogenicity(Figure pathogenicity2A), suggestingisland island [12,26] that [12,26] dimerization(Figure (Figure 2B) and2B) was andencodes important encodes for for afor activity.25.6 a 25.6kDa The kDaprotein N-terminal protein sequence of 14 containingcontaining a signal a signal peptide.amino peptide. acidsCleavage (i.e.,Cleavage A-E-V-K-Q-Q-S-E-S-E-L-K-H-Y)of the of signalthe signal sequence, sequence, as determined as determined yielded by in aExPASy by BLAST ExPASy Signal search Signal P a 100% P match with the program,program, theoretically theoreticallySSL1 results protein results in a secreted ofin straina secreted protein Newman protein with (from witha molecular gene a molecular NWMN_0388; weight weight of 22.6 GenBank of kDa22.6 (FigurekDa Accession (Figure 2C), Number 2C), BAF66660.1). whichwhich agrees agrees with withtheAlso, SDS-PAGE the analysis SDS-PAGE ofsize the determinatsize SSL1 determinat proteinion byofion massthe of isolated the spectrometry isolated protein protein revealed (Figure (Figure only 2A). peptides 2A).The ssl1The matching ssl1 the sequence gene,gene, according according to a BLAST toof a the BLAST Newmansearch, search, is SSL1not is present not protein present in (data other in notother bacterial shown). bacterial species, The species,ssl1 butgene is but invariably resides is invariably within found found the in SaPIn2 in pathogenicity all S.all aureus S. aureus strains strains examined,island examined, [12 e.g.,,26] 88/88e.g., (Figure 88/88 sequenced2B) sequenced and encodes genomes genomes for [26]. a 25.6 [26]. kDa protein containing a signal peptide. Cleavage of the signal sequence, as determined by ExPASy Signal P program, theoretically results in a secreted protein with a molecular weight of 22.6 kDa (Figure2C), which agrees with the SDS-PAGE size determination of the isolated protein (Figure2A). The ssl1 gene, according to a BLAST search, is not present in other bacterial species, but is invariably found in all S. aureus strains examined, e.g., 88/88 sequenced genomes [26].

Pathogens 2019, 8, 2 4 of 17 Pathogens 2018, 7, x 4 of 17

Figure 2. The virulence factor, SSL1, of S. aureus strain Newman. (A) SDS-PAGE of molecular weight Figure 2. The virulence factor, SSL1, of S. aureus strain Newman. (A) SDS-PAGE of molecular weight standards and the isolated native protein under reducing and denaturing conditions showing a single standards and the isolated native protein under reducing and denaturing conditions showing a band at about 23 kDa. The N-terminal sequence of the single band is shown below the gel. (B) The single band at about 23 kDa. The N-terminal sequence of the single band is shown below the gel. (B) gene ssl1 for the protease is the first gene located in pathogenicity island 2 (SaPln2). (C) The amino The gene ssl1 for the protease is the first gene located in pathogenicity island 2 (SaPln2). (C) The acid sequence of SSL1. The theoretical signal sequence is underlined. The amino acids obtained from amino acid sequence of SSL1. The theoretical signal sequence is underlined. The amino acids N-terminal analysis are in red. obtained from N-terminal analysis are in red. 2.4. Recombinant SSL1: Immunogenicity and Toxicity 2.4. Recombinant SSL1: Immunogenicity and Toxicity The ssl1 gene was cloned and expressed in E. coli as a recombinant protein with a 6-His tag on theThe C-terminus. ssl1 gene was Recombinant cloned and SSL1 expressed was purified in E. coli from as a therecombinant culture supernatant protein with by a metal 6-His affinity tag on chromatographythe C-terminus. Recombinant followed by gelSSL1 filtration was purified (Figure 3frA).omThe the purifiedculture recombinantsupernatant monomerby metal fractionaffinity (~23chromatography kDa) was used followed as an by immunogen gel filtration to (Figure induce the3A). production The purified of recombinant polyclonal antibody monomer in fraction rabbits. Corneas(~23 kDa) injected was used with as active an immunogen recombinant to SSL1 induce (12 µ thg),e butproduction not the heat-inactivated of polyclonal antibody recombinant in rabbits. protein, Corneas injected with active recombinant SSL1 (12 µg), but not the heat-inactivated recombinant protein, produced substantial pathology (Figure 3B). These pathological effects caused by the active

Pathogens 2018, 7, x 5 of 17

recombinant SSL1 were also produced in eyes of rabbits immunized with the SSL1 monomer (Data not shown).

2.5. Recombinant SSL1: Polymerization Recombinant SSL1 protein was purified from culture supernatant by affinity and gel filtration chromatography. SDS-PAGE of the recombinant protein showed a major band at ~23 kDa (monomer); however, some preparations also contained a dimer of ~46 kDa (Figure 3A). Zymography of the recombinant protein under denaturing conditions exhibited gelatinase activity

Pathogensat2019 a ,molecular8, 2 weight of ~46 kDa and, in some cases, a protease band at a higher molecular5 of 17 size (~80 kDa; data not shown), but not at 23 kDa (Figure 3C, Lane 2). The protein band migrating at ~46 kDa was also recognized on a Western blot by the polyclonal antibody raised against the SSL1 monomer produced(Figure substantial 3C, Lane pathology 3). This (Figure 46-kDa3B). band These was pathological excised from effects the causedgel and by analyzed the active by recombinant mass spectrometry SSL1 wereconfirming also produced the identity in eyes as of SSL1. rabbits immunized with the SSL1 monomer (Data not shown).

Figure 3. Recombinant SSL1 proteolytic activity and polymerization. (A) SDS-PAGE showing the Figure 3. Recombinant SSL1 proteolytic activity and polymerization. (A) SDS-PAGE showing the standards (left lane), the dimer form of the recombinant protein at ~46 kDa, and the monomer at standards (left lane), the dimer form of the recombinant protein at ~46 kDa, and the monomer at ~23 ~23 kDa (right lane). (B) Photographs of rabbit corneas injected with heat-inactivated (left photo) or kDa (right lane). (B) Photographs of rabbit corneas injected with heat-inactivated (left photo) or active recombinant SSL1 (12 µg) at 24 h after injection (right photo). (C) Analysis of purified SSL1 active recombinant SSL1 (12 µg) at 24 hours after injection (right photo). (C) Analysis of purified including zymography (standards in Lane 1; recombinant SSL1 in Lane 2) and a Western blot of SSL1 including zymography (standards in Lane 1; recombinant SSL1 in Lane 2) and a Western blot of recombinant SSL1 in Lane 3. Please note that the protease activity is at the molecular size of a dimer recombinant SSL1 in Lane 3. Please note that the protease activity is at the molecular size of a dimer (46 kDa), but not at the monomer size (23 kDa) and that both the monomer and dimer reacted with (46 kDa), but not at the monomer size (23 kDa) and that both the monomer and dimer reacted with the antibody. the antibody. 2.5. Recombinant SSL1: Polymerization 2.6. Recombinant SSL1: Enzymatic Properties Recombinant SSL1 protein was purified from culture supernatant by affinity and gel filtration chromatography.The active SDS-PAGE recombinant of the recombinant SSL1 of strain protein Newman showed was a major tested band for at proteolytic ~23 kDa (monomer); activity in vitro however,against some several preparations host proteins also contained important a dimerto the i ofmmune ~46 kDa response (Figure and3A). was Zymography found to degrade of the IL-17A, recombinantIFN-γ protein, and IL-8 under (Figure denaturing 4A). The conditions 11-kDa breakdown exhibited gelatinaseproduct of activity IL-17A atwas a molecular N-terminally weight sequenced of ~46 kDa(Figure and, 4B) in someand, from cases, this a protease sequence, band we atconclude a higher that molecular SSL1 cleaves size (~80 human kDa; IL-17A data not onshown), the C-terminal but not at 23 kDa (Figure3C, Lane 2). The protein band migrating at ~46 kDa was also recognized on a Western blot by the polyclonal antibody raised against the SSL1 monomer (Figure3C, Lane 3). This 46-kDa band was excised from the gel and analyzed by mass spectrometry confirming the identity as SSL1.

2.6. Recombinant SSL1: Enzymatic Properties The active recombinant SSL1 of strain Newman was tested for proteolytic activity in vitro against several host proteins important to the immune response and was found to degrade IL-17A, IFN-γ, and IL-8 (Figure4A). The 11-kDa breakdown product of IL-17A was N-terminally sequenced (Figure4B) Pathogens 2019, 8, 2 6 of 17 and, from this sequence, we conclude that SSL1 cleaves human IL-17A on the C-terminal side of a lysine (K61). The P ,P , and P positions are threonine-58, asparagine-59, and proline-60, respectively. 4 3 2

Figure 4. Degradation of host proteins by recombinant SSL1. (A) SDS-PAGE of human recombinant IL-17A (16 kDa), IFN-γ (17 kDa), or IL-8 (8.3 kDa) after incubation in phosphate buffer (-) or in the buffer plus active recombinant SSL1 (+; dimer: 100 ng) for two hours at 37 ◦C. Black arrows indicate the position of the un-cleaved substrate protein. White arrows indicate the position of the SSL1 dimer. (B) Amino acid sequence of human IL-17A. The 11-kDa breakdown product was sequenced by N-terminal analysis and the ﬁrst ﬁve amino acids are RSSDY (shown in bold).

A library of 29 chromogenic substrates was tested for cleavage by SSL1 with a focus on substrates ending with either a lysine or arginine. The substrate N-(p-Tosyl)-Gly-Pro-Lys-4 nitroanilide, known as Chromozym PL (CPL), was found to be susceptible and useful for assaying protease activity. Kinetic analysis of the hydrolysis of CPL determined that the Km and Vmax were 771 µM and 1.2 µM/min, respectively (Figure5A). The effect of pH or temperature on SSL1 activity was also analyzed. The optimum pH for SSL1 activity was found to be pH 9, and the optimum temperature was 50 ◦C (Figure5B,C). Eleven protease inhibitors were tested for their ability to block the hydrolysis of CPL by SSL1 (Table1). The inhibitors demonstrating complete inhibition were AEBSF, TLCK, and chymostatin, all known to inhibit serine proteases. Partial inhibition (17 to 22%) was mediated by the metalloprotease inhibitor EDTA (10 mM) and the serine protease inhibitor aprotinin. Furthermore, the recombinant SSL1 protease was found to cleave collagen type I and type IV (Figure6A,B), which are structural components of the cornea [27,28].

2 Pathogens 2019, 8, 2 7 of 17 Pathogens 2018, 7, x 7 of 17

FigureFigure 5. Enzymatic5. Enzymatic properties properties ofof SSL1.SSL1. (A (A) )Kinetic Kinetic analysis analysis of ofthe thehydrolysis hydrolysis of the of substrate the substrate ChromozymChromozym PL. PL. Km K(771m (771µ M)µM) and and V Vmaxmax (1.2 µM/min)µM/min) were were determined determined by incubating by incubating the enzyme the enzyme withwith varying varying concentrations concentrations of of the the substratesubstrate ranging ranging from from 0 to 0 to2.4 2.4 mM. mM. (B) (TheB) Theoptimal optimal pH of pH SSL1 of SSL1 activityactivity was was determined determined using using the CPL CPL assay. assay. The The maximum maximum protease protease activity activity was found was at found pH 9. ( atC) pH 9. The optimal temperature for SSL1 activity was found to be 50 °C using the CPL assay. (C) The optimal temperature for SSL1 activity was found to be 50 ◦C using the CPL assay.

TableTable 1. 1.Inhibitors Inhibitors tested tested forfor SSL1 enz enzymaticymatic activity activity using using CPL CPL assay. assay. Inhibitor Target Protease Class Concentration Used % Activity of Control1 1 InhibitorNone Target Protease- Class Concentration - Used % Activity of 100 Control ChymostatinNone Cys/Ser - 0.1 - mM 100 0 ChymostatinAEBSF Cys/SerSerine 0.1 10 mM mM 0 0 TLCKAEBSF SerineSerine 10 1 mM mM 0 0 EDTATLCK SerineMetallo 1 10 mM mM 0 78 AprotininEDTA MetalloSerine 10 0.001 mM mM 78 83 AntipainAprotinin SerineCys/Ser 0.001 0.1 mMmM 83 100 PepstatinAntipain Cys/SerAspartic 0.1 0.001 mM mM 100 101 LeupeptinPepstatin AsparticCys/Ser 0.001 0.1 mMmM 101 110 LeupeptinBestatin Cys/SerMetallo 0.1 0.1 mM mM 110 119 BestatinE-64 MetalloCysteine 0.10.01 mM mM 119 130 PhosphoramidonE-64 CysteineMetallo 0.01 1 mMmM 130 137 Phosphoramidon Metallo 1 mM 137 1 These values indicate the changes of SSL1 activity in the presence of an inhibitor relative to the inhibitor-free control using the Chromozym PL assay. Pathogens 2018, 7, x 8 of 17

Pathogens1 2019These, 8, values 2 indicate the changes of SSL1 activity in the presence of an inhibitor relative to the 8 of 17 inhibitor-free control using the Chromozym PL assay.

B FigureFigure 6. 6.Cleavage Cleavage of of collagens collagens byby SSL1.SSL1. ( A)) Recombinant SSL1 SSL1 (20 (20 µg),µg), SSL1 SSL1 plus plus TLCK TLCK (1 (1mM; mM; negativenegative control), control), or or collagenase collagenase of ofClostridium Clostridium histolyticumhistolyticum (0.2 U/ml; U/ml; positive positive control) control) was was incubated incubated ◦ withwith fluorescent fluorescent collagen collagen Type Type I (40 I (40µg/ml) µg/ml) at at 37 37 C°C for for 6.5 6.5 h hours in a microtiter in a microtiter plate readerplate reader and the and change the in fluorescencechange in fluorescence was measured was everymeasured 30 minutes every 30 throughout minutes throughout the incubation. the incubation. Background Background fluorescence, determinedfluorescence, by no-enzymedeterminedreactions by no-enzyme (blanks) reactions was subtracted (blanks) fromwas subtracted each value. from Data each were value. expressed Data as thewere mean expressed± standard as deviation.the mean ± (B standard) SSL1 (20 deviation.µg), SSL1 ( plusB) SSL1 TLCK (20(1 µg), mM), SSL1 or collagenaseplus TLCK of(1 ClostridiummM), or histolyticumcollagenase(0.2 of U/ml)Clostridium was histolyticum incubated (0.2 with U/ml) fluorescent was incubated collagen with Type fluorescent IV (40 µg/ml), collagen as Type described IV (40 in Figureµg/ml),6A. as described in Figure 6A.

2.7.2.7. Corneal Corneal Virulence Virulence of of the the SSL1 SSL1 Deficient Deficient MutantMutant ToTo determine determine the the role role of of SSL1 SSL1 during duringS. S. aureusaureus cornealcorneal infection, an an isogenic isogenic mutant mutant lacking lacking the the functionalfunctionalssl1 ssl1gene gene was was constructed constructed usingusing thethe NewmanNewman Δ∆hlahlaΔ∆hlghlg asas the the parental parental strain. strain. This This new new mutantmutant strain, strain, Newman Newman∆ Δhlahla∆Δhlghlg∆Δssl1ssl1,, aa tripletriple mutant,mutant, was complemented complemented by by inserting inserting a functional a functional ssl1ssl1gene gene (i.e., (i.e., Newman Newman∆ Δhlahla∆Δhlghlg∆Δssl1/ssl1ssl1/ssl1)) atat thethe nativenative locus. The The correct correct phenotype phenotype of of these these strains strains waswas confirmed confirmed by by Western Western blotting blotting ofof thethe concentratedconcentrated culture supernatants supernatants using using the the antibody antibody againstagainst recombinant recombinant SSL1 SSL1 (Figure (Figure7 A).7A). Virulence Virulence analysisanalysis ofof the Newman wild-type, wild-type, Δhla∆hlaΔhlg∆hlg (the(the parentparent strain), strain), the thessl1 ssl1-deficient-deficient strain strain ((∆Δhlahla∆Δhlghlg∆Δssl1), and and the the rescue rescue strain strain demonstrated demonstrated that that the the ssl1-deficient strain had less virulence (SLE score: 4.56 ± 0.49) than that of the wild-type strain, the parent strain (Newman ∆hla∆hlg), and the rescue strain (SLE scores: 15.43 ± 1.10, 8.76 ± 0.59, and 10.82 ± 1.35, respectively; P ≤ 0.001) (Figure7B). The ssl1-deficient strain lacked the virulence, Pathogens 2018, 7, x 9 of 17 PathogensPathogensPathogens 2018 2018, 7 2018, x, 7 , ,x 7 , x 9 of9 17 of 9 17 of 17 ssl1-deficient strain had less virulence (SLE score: 4.56 ± 0.49) than that of the wild-type strain, the ssl1ssl1-deficientssl1-deficient-deficient strain strain strain had had less hadPathogens less virulence less virulence 2019virulence, 8(SLE, 2 (SLE (SLEscore: score: score: 4.56 4.56 ±4.56 0.49) ± 0.49) ± 0.49)than than thatthan that ofthat ofthe ofthe wild-type the wild-type wild-typeparent strain, strain, strain,strain the the (Newmanthe ΔhlaΔhlg9), of and 17 the rescue strain (SLE scores: 15.43 ± 1.10, 8.76 ± 0.59, and parentparentparent strain strain strain (Newman (Newman (Newman Δhla ΔhlaΔ ΔhlghlaΔhlg),Δ andhlg), and), theand the rescue the rescue rescue strain strain strain (SLE (SLE (SLEscores: scores: scores: 15.43 15.43 15.43± 1.10,± 1.10,± 1.10,8.76 8.76 10.82 ±8.76 0.59,± 0.59,±± 0.59,and1.35, and andrespectively; P ≤ 0.001) (Figure 7B). The ssl1-deficient strain lacked the virulence, ± ± ± 10.8210.82 10.82 1.35, 1.35, 1.35,respectively; respectively; respectively;especially P ≤P 0.001)≤P 0.001)≤ corneal0.001) (Figure (Figure (Figure infiltrates, 7B). 7B). The7B). The of ssl1The the ssl1-deficient otherssl1-deficient-deficient strains strain strain (Figure strain lacked lacked 7lackedB,C), the especiallythe yetvirulence, the virulence, grew virulence, incorneal the cornea infiltrates, as well of asthe the other strains (Figure 7B,C), yet grew in the cornea as well as the ± ± especiallyespeciallyespecially corneal corneal corneal infiltrates, infiltrates, infiltrates,other of theof strains ofthe other the other (6.95 otherstrains strains± strains0.07 (Figure log(F igure(F CFU igure7B,C), 7B,C), for 7B,C), yet the yet grewssl1 yet grew-deficient grewin thein inthe cornea strainthe cornea cornea as vsother aswell 6.93 aswell ± strainsaswell0.14 asthe asthe log(6.95 the CFU 0.07 for log the CFU wild-type, for the ssl1-deficient strain vs 6.93 0.14 log CFU for the wild-type, ± ± ± ± ± ± ± ± otherother otherstrains strains strains (6.95 (6.95 (6.95 0.07 0.07 log 0.076.77 log CFU log CFU± for0.11CFU for thelog for the ssl1 CFUthe ssl1-deficient ssl1-deficient for-deficient the strain Newman strain strain vs vs6.93∆ vs6.93hla 6.93∆ 0.14 hlg 0.14 parentlog 0.14 log CFU log CFU strain, forCFU for the andfor 6.77the wild-type, the 6.90wild-type, 0.11wild-type,± 0.21log CFU log CFU for the for Newman the rescue ΔhlaΔhlg parent strain, and 6.90 0.21 log CFU for the rescue ± ± ± ± ± ± 6.776.77 6.77 0.11 0.11 log0.11 log CFU log CFU forCFU for the for strain;the Newman the Newman NewmanP = 0.19).Δhla ΔhlaΔ ΔhlghlaΔhlg parentΔhlg parent parent strain, strain, strain, and and 6.90 and 6.90 6.90 0.21 0.21 log0.21 log CFU log CFU forCFUstrain; for the for the rescue Pthe rescue = 0.19).rescue strain;strain;strain; P = P 0.19). = P 0.19). = 0.19).

1 2 3 4 5 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5

25 kDa - 25 kDa25 kDa25 - kDa - - 20 kDa - * 20 kDa20 kDa20 - kDa - - * * *

A B A A A B B B Wild-Type ΔhlaΔhlg ΔhlaΔhlgΔssl1 ΔhlaΔhlgΔssl1/ssl1 Wild-Type Wild-Type Wild-Type Δ hla Δ hlaΔΔhlghlaΔhlg Δ hlg Δ hla Δ hlaΔΔhlghlaΔhlgΔΔssl1hlgΔssl1Δ ssl1 Δ hla Δ hla ΔΔhlghlaΔhlgΔΔssl1/ssl1hlgΔssl1/ssl1Δssl1/ssl1

C C C Figure 7. Phenotype confirmation and virulence of S. aureus strain Newman and the isogenic FigureFigureFigure 7. Phenotype7. Phenotype7. Phenotype confirmation confirmation confirmationFigure 7.and Phenotypeand virulenceand virulence virulence confirmation of ofS. ofaureusS. S.aureus andaureus strain virulencestrain strainNewman Newman ofNewmanS. aureusand and theandstrain the isogenic the Newmanisogenic mutants.isogenic and (A the) Western isogenic blot mutants. analysis of concentrated culture supernatants of wild-type strain Newman mutants.mutants.mutants. (A )( AWestern )( AWestern) Western blot blot analysis blot (analysisA) analysis Western of concentratedof concentratedof blot concentrated analysis cult culture of cult concentratedure supernatantsure supernatants supernatants culture of wild-typeof wild-typeof supernatants wild-type strain strain strainNewman of Newman wild-type Newmanand the strainmutants. Newman Lane 1: and molecular the weight standards; Lane 2: strain Newman; Lane 3: double mutant andand theand the mutants. the mutants. mutants. Lane Lane 1:Lane molecular 1: molecular 1:mutants. molecular weight weight Lane weight standards; 1:standards; molecularstandards; Lane Lane 2: weightLane strain 2: strain 2: strain standards;Newman; Newman; Newman; Lane Lane Lane 3:Lane2: double 3: strain double 3: double mutant Newman; mutant(Newman mutant Lane Δhla 3:Δ doublehlg); Lane mutant 4: triple mutant (Newman ΔhlaΔhlgΔssl1), and Lane 5: rescue strain (Newman(Newman(Newman Δhla ΔΔhla ΔhlgΔhla);hlg ΔLanehlg); Lane); Lane4:(Newman triple4: triple4: triplemutant ∆mutanthla mutant∆ (Newmanhlg (Newman); Lane(Newman Δ 4:hla Δ tripleΔhla ΔhlgΔhlaΔhlg mutantΔssl1hlgΔssl1),Δ ssl1and), (Newman and), Laneand Lane Lane5: ∆rescue5:hla rescue5:∆ hlgrescue strain∆ ssl1strain(Newman ),strain and Lane Δhla 5:Δhlg rescueΔssl1/ssl1 strain). The blot was developed using rabbit polyclonal antibody to (Newman(Newman(Newman Δ hlaΔ ΔhlaΔhlgΔhlaΔhlgΔssl1/ssl1hlgΔssl1/ssl1Δssl1/ssl1(Newman). The). ).The blotThe ∆hlablot was∆blot hlgwas ∆developedwasssl1/ssl1 developed developed). The using blotusing using wasrabbit rabbit developed rabbit polyclonal polyclonal polyclonal using antibody rabbit antibody antibody polyclonal recombinantto to to antibody SSL1. to recombinant(B) Slit lamp examination scoring (SLE) of rabbit eyes 24 hours after infection recombinantrecombinantrecombinant SSL1. SSL1. SSL1.(B ) (BSlit )( BSlit lamp) SSL1.Slit lamp lampexamination ( Bexamination) Slitexamination lamp scoring examination scoring scoring (SLE) (SLE) (SLE)of scoring rabbitof rabbitof (SLE)rabbit eyes eyes of 24eyes rabbit 24hours 24hours eyes hoursafter after 24 infectionafter h afterinfection infectionwith infection strain with Newman strain Newman( ), the double mutant ( ), the triple mutant ( ), or the rescue withwith strainwith strain strainNewman Newman Newman ( ( (), ( the), the ),),double thethe double doubledouble mutant mutant mutant mutant ( ( ( (), the),), the the ),triple the triple triple triplemutant mutant mutant mutant ( ( ( (),), or or), the orthe), rescueorthe rescue the rescuestrain strainrescue ( ( ).). TheThe SLESLE score score for for eyes infected with the triple mutant was significantly lower than strainstrain strain( ( (). The). The). SLE The SLE score SLE scoreeyes scorefor foreyes infected foreyes infectedeyes infected with infected thewith triplewith thwithe th mutanttriplee th triplee triplemutant was mutant significantlymutant was was significantly was significantly lower significantly than lower thatlower lowerthan ofthat eyesthan than of infected eyes infected with the with wild-type the wild-type Newman, the double mutant, or the rescue strain (*, P ≤ thatthat ofthat eyesof eyesof infectedeyes infected infected with with thewithNewman, the wild-type the wild-type wild-type the Newm double Newm Newman, mutant, an,thean, the double orthe double the double rescuemutant, mutant, mutant, strain or theor (*, orthe Prescue ≤the rescue0.001). rescue strain strain (C )strain(*, Photographs (*,P0.001). ≤(*,P ≤P ≤(C of) Photographs rabbit eyes 24 of h afterrabbit eyes 24 hours after intrastromal injection with 100 CFU of strain 0.001).0.001).0.001). (C )( CPhotographs )( CPhotographs) Photographs of rabbitofintrastromal rabbitof rabbit eyes eyes 24eyes injection24hours 24hours hoursafter withafter inaftertrastromal 100intrastromal in CFUtrastromal of injection strain injection injection Newman, with with 100with 100 theCFU 100 doubleCFU ofCFU strainof mutantstrainofNewman, strain (Newman the double∆hla mutant∆hlg), the (Newman ΔhlaΔhlg), the triple mutant (Newman ΔhlaΔhlgΔssl1), or the Newman,Newman,Newman, the the double the double double mutant mutant mutant triple(Newman (Newman mutant(Newman Δhla (NewmanΔΔhla ΔhlgΔhla),hlg Δthehlg), ∆the triple),hla the triple∆hlg triplemutant∆ mutantssl1 mutant), (Newman or (Newman the (Newman rescue Δhla Δ strainΔhla ΔhlgΔhlaΔhlg (NewmanΔssl1hlgΔssl1),Δ orssl1), theorrescue),∆ hlathe or ∆the hlgstrain ∆ssl1/ssl1 (Newman). These Δhla eyesΔhlgΔssl1/ssl1). These eyes contained similar log CFU per cornea (P = rescuerescuerescue strain strain strain(Newman (Newman (Newman Δhla ΔΔhlacontained ΔhlgΔhlaΔhlgΔssl1/ssl1hlgΔssl1/ssl1Δ similarssl1/ssl1). These). logThese). TheseCFUeyes eyes per containedeyes contained cornea contained (similarP =similar 0.19). similar log log CFU log CFU perCFU per cornea per cornea cornea (P0.19). =(P (=P = 0.19).0.19). 0.19). 2.8. ssl1 Alleles 2.8. ssl1 Alleles 2.8.2.8. ssl12.8. ssl1 Alleles ssl1 Alleles Alleles There are 12 known alleles of the ssl1 gene, with allele types 1, 2, and 3 being the most There are 12 known alleles of the ssl1 gene, with allele types 1, 2, and 3 being the most prevalent prevalent [26]. A set of clinical isolates including ones from ocular infections were analyzed to ThereThereThere are are 12 are 12known 12known known alleles alleles alleles of theof theof ssl1 the ssl1 gene, ssl1 gene, gene, with with allelewith allele alleletypes types types 1, 2,1, and 2,1, and2, 3 and being 3 being 3 being the the most the most[26]. mostprevalent prevalentA setprevalent of clinical isolates including ones from ocular infections were analyzed to determine the determine the presence and allele type of the ssl1 gene. As shown in Table2, the ssl1 gene was found [26].[26]. A[26]. setA set Aof set clinicalof ofclinical clinical isolates isolates isolates including including including ones ones fromones from fromocular ocular ocular infections infections infections were were wereanalyzed analyzed analyzed to determineto presencetodetermine determine the and the allelethe type of the ssl1 gene. As shown in Table 2, the ssl1 gene was found in all isolates in all isolates tested (27/27) and six different allele types were identified with type 2 being the most presencepresencepresence and and allele and allele alleletype type oftype theof theof ssl1 the ssl1 gene. ssl1 gene. gene. As As shown As shown shown in Tablein Tablein Table 2, the2, the2, ssl1 the ssl1 gene ssl1 gene genewas was found was found found intested allin allinisolates all(27/27)isolates isolates and six different allele types were identified with type 2 being the most prevalent testedtestedtested (27/27) (27/27) (27/27) and and sixand six different six differentprevalent different allele allele among alleletypes types thetypes were ocularwere wereidentified identified isolates identified with (13/20). with withtype type The type2 being2 amino being2 being the acid the most the sequencemost mostprevalent prevalent prevalent of SSL1 of MW2 (type 1 allele) shows 68.6% identity to that of Newman (type 3 allele) and the amino acid sequence of a type 2 ocular

Pathogens 2018, 7, x 10 of 17 Pathogens 2019, 8, 2 10 of 17 among the ocular isolates (13/20). The amino acid sequence of SSL1 of MW2 (type 1 allele) shows 68.6% identity to that of Newman (type 3 allele) and the amino acid sequence of a type 2 ocular strain, strain 295.236, was 83.2% identical to that of strain Newman. The SSL1 protein of strain MW2 shares 78% identity with that of strain 295.236. To determinedetermine ifif proteaseprotease activity activity is is limited limited to to only only allele allele type type 3, of3, of which which Newman Newman is a is member, a member, the theprotease protease activity activity of the of recombinantthe recombinant protein protein of the ofssl1 thegene ssl1 ofgene strain of strain MW2 (typeMW2 1),(type and 1), ocular and isolateocular isolate295.236 295.236 (type 2), (type was assayed.2), was assayed. Figure8A Figure and 8B 8A show and the 8B migration show the of migration the three differentof the three recombinant different recombinantproteins on SDS-PAGE proteins on and SDS-PAGE a Western blot,and a respectively. Western blot, Figure respectively.8C shows thatFigure the 8C recombinant shows that SSL1 the recombinantproteins of allele SSL1 types proteins 1, 2, of and allele 3 have types protease 1, 2, and activity. 3 have protease activity.

Table 2. The ssl1 allele types of Staphylococcus aureus clinical isolates. Table 2. The ssl1 allele types of Staphylococcus aureus clinical isolates.

OcularOcular Isolates Strain Strain AlleleAllele Type Type1 1 StrainStrain Allele AlleleType1 Type 1 05.4144† 2 82936§ 2 † § 05.414406.6451† 2 1 8352382936§ 2 2 † § 06.645111.14697† 1 5 8527883523§ 2 2 † § 11.1469713.9793† 5 3 8539785278§ 2 2 † § 13.9793190.164‡ 3 7 9173185397§ 2 2 190.164279.274‡ ‡ 7 3 SD2212591731§ § 3 2 279.274295.236‡ ‡ 3 2 CO19432SD22125§ § 1 3 295.236 ‡43506§ 2 2 CO61879CO19432§ § 2 1 43506 §67993§ 2 2 CO63355CO61879§ § 2 2 67993 §70468§ 2 2 CO63825CO63355§ § 2 2 70468 § 2Non-Ocular IsolatesCO63825 § 2 Strain Allele Type1 Strain Allele Type1 Non-Ocular Isolates MRSA 301|| 3 76.137‡ 2 StrainMRSA 306Allele|| Type3 1 103.93Strain‡ 3Allele Type 1 44.255‡ 1 116.60‡ 9 MRSA 301 || 3 76.137 ‡ 2 63.21‡ 2 MRSA 306 || 3 103.93 ‡ 3 1 Genomic DNA of S.44.255 aureus‡ clinical isolates1 was used 116.60as temp‡ late for PCR 9reactions to amplify the ssl1 gene. The PCR product63.21 ‡ was sequenced2 and compared to the previously published ssl1 alleles [26].1 Genomic † These DNA strains of S. were aureus providedclinical isolates by Eric was G. used Romanowski as template and for PCRRegis reactions P. Kowalski to amplify of the the Campbellssl1 gene. LaboratoryThe PCR product of the was University sequenced andof comparedPittsburgh. to the‡ previouslyThese strains published weressl1 isolatedalleles [26at]. the† These University strains were of Mississippiprovided by EricMedical G. Romanowski Center. and§ RegisThese P. Kowalskistrains ofwere the Campbellprovided Laboratory by Dr. of David the University Stroman, of Pittsburgh. Alcon ‡ These strains were isolated at the University of Mississippi Medical Center. § These strains were provided by Laboratories,Dr. David Stroman, Inc. Alcon|| These Laboratories, strains Inc.were || Theseisolated strains at Louisiana were isolated State at Louisiana University State Health University Sciences Health Center.Sciences Center.

A B C

Figure 8. Properties of SSL1 proteins of ssl1 alleles 1, 2, and 3. (A) SDS-PAGE of recombinant Figure 8. Properties of SSL1 proteins of ssl1 alleles 1, 2, and 3. (A) SDS-PAGE of recombinant SSL1 SSL1 proteins of alleles 1, 2, and 3 from strain MW2, 295.236, and Newman, respectively. Proteins proteins of alleles 1, 2, and 3 from strain MW2, 295.236, and Newman, respectively. Proteins were were puriﬁed from culture supernatants by metal afﬁnity and molecular sieve chromatography. purified from culture supernatants by metal affinity and molecular sieve chromatography. The The bands seen at approximately 23 kDa are the monomeric forms of the protein whereas the bands at bands seen at approximately 23 kDa are the monomeric forms of the protein whereas the bands at approximately 46 kDa are the dimeric forms. M, molecular weight standards. (B) Western blot analysis approximately 46 kDa are the dimeric forms. M, molecular weight standards. (B) Western blot of the proteins analyzed in Figure8A using antibody to the SSL1 monomer of strain Newman (allele

Pathogens 2019, 8, 2 11 of 17

type 3). The bands seen at approximately 46 kDa are the dimers and the bands at approximately 23 kDa are the monomers. (C) Zymography (gelatin) of the proteins analyzed in Figure8A. Despite having similar molecular weights, the proteolytic forms of recombinant proteins of alleles 1, 2, and 3 migrate differently, probably reflecting their differences in amino acid sequences. 3. Discussion The present study demonstrates that SSL1 mediates ocular virulence and can proteolytically cleave host proteins, including two types of collagens and the human recombinant cytokines IL-8, IL-17A, and IFN-γ. The protease activity of SSL1 is a most unexpected finding because SSL proteins have been characterized to act by binding to and inhibiting molecules needed for multiple host functions including neutrophil migration, complement activity, blood clotting, wound healing, and antibody functions [12,19–24]. There has been no prior suggestion that any SSL protein has enzymatic activity; however, Fraser and Proft reported that dimer formation could be common among SSL proteins and that no function has been assigned to SSL dimers [12]. The significance of SSL1 is demonstrated by its corneal toxicity and the reduced virulence of the mutant lacking SSL1 production (Figure7). SSL1 can cleave in vitro collagen types I and IV (Figure6), both of which are structural components of the cornea [ 27,28]. Although SSL1 can cleave several proinflammatory cytokines (Figure4) and is reported to inhibit the migration of neutrophils by inhibiting the matrix metalloproteases involved in neutrophil migration in vitro [25], overall the SSL1 protein causes an inflammatory reaction in the cornea. The inflammation could result from cleavage of protease activated receptors [29,30]. Corneal inflammation is known to contribute to the overall tissue damage and scarring of an infected cornea. The significance of SSL1 is also confirmed by the presence of the ssl1 gene in all sequenced S. aureus genomes previously analyzed (88/88) [26]. The importance of SSL1 to corneal virulence was conclusively established by the significant reduction in virulence of the mutant lacking SSL1 production relative to the isogenic parent and rescue strains (Figure7). SSL1 is a 226 amino acid protein sharing sequence homology with the other superantigen-like proteins. The structure, as derived from strain NCTC 8325, has recently been solved by Dutta et al. (available in the Protein Data Bank, ID#: 4O1N) [31]. The sequence of the ssl1 gene of NCTC 8325 is identical to that of Newman indicating that the Newman SSL1, like that of NCTC 8325, has the beta-grasp domain at the C-terminus [31]. The beta-grasp domain has been reported to mediate dimerization of SSL7 and SSL11 by the internalization of hydrophobic residues allowing hydrogen bond formation by neutral residues of the two SSL monomers [23,32]. Al-Shangiti et al. suggested that SSL polymerization occurs adjacent to the external bacterial surface [32], a mechanism for SSL1 that could prevent its proteolytic activity within the bacterium. Because dimer formation is essential for SSL1 protease activity, such dimer formation could aid proteolysis by positioning together sequences distant from each other in the monomer. SSL1 is a unique and novel protease sharing little sequence homology with any known protease. Searching the Pfam database, three hypothetical domains have been identified in SSL1 (Table3), including a staphylococcal superantigen-like OB-fold domain, an MHC class II analogous protein (MAP) domain, and a staphylococcal/streptococcal toxin beta-grasp domain. No protease domain was found. Our inhibitor and host protein cleavage studies indicated that SSL1 could be a serine protease, employing the Asp-His-Ser triad in the active site. In the SSL1 sequence, there are 5 histidines, 15 aspartic acids, and 17 serines, so identifying the catalytic triad via site-directed mutagenesis will be time-consuming. Another complication in identifying the triad is that, whereas the structure of the SSL1 monomer has been determined [31], the structure of the dimer is unknown. To form a dimer, Al-Shangiti et al. suggested that the beta-grasp domain must undergo alterations in the location of multiple amino acid residues [33]. Apparently, since only the dimer is proteolytic, the available structural work on the monomer has been of limited value in identifying the triad. There is an example of another serine protease produced by S. aureus that undergoes a conformational change to produce Pathogens 2019, 8, 2 12 of 17 a proteolytic triad. The exfoliative toxin is a serine protease whose active site is inactive until it undergoes a conformational change upon its interaction with a substrate protein [34].

Table 3. Bioinformatic analysis of SSL1 domains (motifs) using the Pfam database.

Amino Motif ID 1 Deﬁnition E-Value Acids pf: SSL_OB 41–123 Staphylococcal superantigen-like OB-fold domain 4.9 × 10 −32 pf: MAP 142–209 MAP domain 0.014 pf: Stap_Strp_tox_C 147–206 Staphylococcal/Streptococcal toxin, beta-grasp domain 0.0002 1 Motif search was performed using the Pfam database (available in the public domain at https://www.genome.jp/ tools/motif/).

The ssl1 gene lies ﬁrst in a series of ten contiguous superantigen-like genes in pathogenicity island two (SaPIn2) [12,26,35]. There are 12 known alleles of the ssl1 gene with alleles 1-3 being the most prevalent [26]. In the present study, the ssl1 gene was found in all of the 20 ocular isolates and 7 non-ocular isolates examined (Table2); these ssl1 genes were of six allelic types. Zymography revealed the proteolytic activity of the recombinant proteins of allele types 1, 2, and 3, despite the rather large differences (68.6% to 83.2% identity) in their sequences. These three proteins have similar but not identical molecular weights (22.622 kDa, 22.730 kDa, and 22.687 kDa, respectively) and they migrate at somewhat different rates on SDS-PAGE (Figure8A). Their proteolytic forms also migrate differently on zymograms (Figure8C), which is possibly due to the differences in their amino acid compositions. Additional allele types other than types 1-3, could also be proteolytic, but these have not yet been tested. Numerous secreted proteins of S. aureus are regulated by the accessory gene regulator (agr) such that their production is limited to late log phase and stationary phase of growth [18]. The expression of the ssl1 gene is up-regulated by two other regulatory systems, rot and sae, and down-regulated by agr, therefore SSL proteins are produced in the log phase, but not later in the growth cycle [10,36,37]. Benson et al. concluded that SSL proteins are active early in an infection [36]; thus, the ability of SSL1 to degrade pro-inﬂammatory cytokines could limit the initial host response to a developing infection. Naturally occurring agr-defective human isolates were found to over-produce SSL proteins that contribute to the virulence of the agr-defective strains [36]. Research to date shows that, when the cornea is infected with S. aureus, corneal pathology is mediated by at least three proven virulence factors; namely, alpha-toxin, gamma-toxin, and SSL1. For CA-MRSA strains, the corneal infection could involve a fourth virulence factor, Panton-Valentine leukocidin (PVL) [17]. Inhibition of the action of such aggressive molecules during keratitis would be an ideal means of limiting tissue damage while an antibiotic kills the infecting bacteria. Because SSL1 requires dimerization to achieve proteolytic activity, the application of an inhibitor of dimerization or of the protease activity could reduce the toxic effects of Staphylococcus keratitis.

4. Materials and Methods

4.1. Bacteria and Growth Conditions S. aureus strains Newman, Newman ∆hla∆hlg, Newman ∆hla∆hlg∆ssl1, Newman ◦ ∆hla∆hlg∆ssl1/ssl1 were grown to equivalent optical density (OD600) at 37 C for 24 h in tryptic soy broth or M9 minimal medium supplemented with 20 amino acids, casamino acids, and vitamins [15,16]. Antibiotics (10 µg/mL tetracycline and 10 µg/mL erythromycin) were added to the medium for strains with hla and hlg mutations. E. coli M15 [pREP4] with the expression vector pQE-60 was grown in LB medium with ampicillin 100 µg/mL and kanamycin 25 µg/mL. Pathogens 2019, 8, 2 13 of 17

4.2. PAGE and Gelatin Zymography Gelatin (0.1%) zymograms were performed using 10% acrylamide gels under non-denaturing conditions (no SDS) or denaturing conditions (with SDS) and stained with Coomassie blue [38]. Brieﬂy, the gel containing gelatin was electrophoresed at 4 ◦C and 20 mA, then incubated in a reaction buffer ◦ consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM CaCl2, and 1 µM ZnCl2 at 37 C for 24 h. After staining with Coomassie blue, the area where proteolysis of gelatin occurred reveals clear (white) bands against a blue background.

4.3. Identiﬁcation of S. aureus Virulence Factor Culture supernatants were prepared, concentrated, and then fractionated by molecular sieve chromatography using Sephacryl S-100 [39]. Fractions (10 µL) were injected intrastromally into rabbit corneas and toxic fractions were analyzed by SDS-PAGE and zymography. Protein was extracted from the proteolytic band on zymogram and analyzed by SDS-PAGE. The band on SDS-PAGE was excised for N-terminal sequencing and mass spectrometry (University of Texas Medical Branch Biomolecular Resource Facility, Galveston, TX) [39,40].

4.4. Recombinant Protein Production The gene was translated and the signal peptide cleavage site was identified using ExPASy’s software. For cloning into plasmid pQE-60, the NcoI and BglII restriction sites were incorporated into the PCR product. Genomic DNA of strain Newman ∆hla∆hlg, ocular isolate 295.236, or strain MW2 was used as template in PCR reactions. The forward primer was 50-AACCATGGGTAAATTTAAAGCGATAGCA-30 and the reverse primer was 50-TTAGATCTTTTCATTTCTACTAGAAT-30. The recombinant protein was secreted by E. coli. All DNA constructs were verified by sequencing (MCLAB, South San Francisco, CA). The recombinant protein was purified from culture supernatants by metal affinity (TALON Resin; Clontech Laboratories, Mountain View, CA) and Sephacryl S-100 chromatography.

4.5. Production of Polyclonal Antibody Antibody to the recombinant Newman SSL1 protein monomer was produced in rabbits (n = 3) by methods described previously [16]. Brieﬂy, rabbits were bled before immunization to acquire control sera (pre-bleed) and then they were injected subcutaneously with 100 µg SSL1 recombinant protein mixed 1:1 (vol/vol) with TiterMax Gold adjuvant (Sigma-Aldrich, St. Louis, MO). The immunization was repeated every 21 days for a total of three immunizations. For a negative control, rabbits were injected with adjuvant without the immunogen (i.e., mock immunized).

4.6. In Vitro Degradation of Host Proteins Recombinant Newman SSL1 (dimer, 0.1 µg) was incubated for 2 h at 37 ◦C with 1 µg of recombinant IL-17A, IFN-γ, or IL-8 (Applied Biological Materials Inc., Richmond, BC, Canada) and analyzed by SDS-PAGE. Recombinant SSL1 was also incubated with fluorescent collagen type I or type IV (Molecular Probes; Thermo Fisher Scientific, Waltham, MA) at 37 ◦C and the fluorescence (excitation at 485 nm and emission at 520 nm) was measured every 30 min for 6.5 h.

4.7. Enzyme Activity Assay, Kinetics, and Optimal Conditions Chromozym PL (CPL; Sigma-Aldrich) was used to determine SSL1 susceptibility to protease inhibitors. The substrate CPL mixed with enzyme samples was assayed in triplicate in a 96-well plate at 37 ◦C. Optical density (OD) at 405 nm was measured. Samples without enzyme or with heat-inactivated enzyme served as negative controls. The enzyme kinetics determination was performed by incubating ◦ SSL1 (20 µg) with varying concentrations of the substrate CPL at 37 C and measuring OD405 in a microtiter plate reader every 10 minutes for 2 h. The Km and Vmax were determined by plotting Pathogens 2019, 8, 2 14 of 17 the substrate concentration versus velocity using the GraphPad Prism 6 software. The optimum pH and temperature for activity were determined as described previously [39]. The optimal pH of SSL1 activity was determined by the CPL assay using potassium chloride - hydrochloric acid buffer (pH 1–2), citric acid - Na2HPO4 buffer (pH 3–7), Tris buffer (pH 8–9), sodium bicarbonate buffer (pH 10–11), or potassium chloride - sodium hydroxide buffer (pH 12–13). Brieﬂy, the substrate was added to a reaction mixture of SSL1 (10 µg) and a buffer of a certain pH and OD405 was measured every 30 min for a 2.5-h period at room temperature. Controls consisted of the substrate at the pH tested without SSL1. To determine the optimal temperature for SSL1 activity, puriﬁed recombinant SSL1 (5 µg) was incubated with the substrate CPL at various temperatures ranging from 5 ◦C to 65 ◦C for 5 h. After incubation, OD405 was measured. Controls consisted of the substrate at the temperature tested without SSL1.

4.8. ssl1 Allele Typing of S. aureus Clinical Isolates The ssl1 gene of the S. aureus isolates was ampliﬁed by PCR and sequenced in both directions (MCLAB, South San Francisco, CA). A pair of universal ﬂanking primers was used: 50-ATAGATTGGGGCTAAAAATT-30 and 50-ATAGCGCTTTGTTGTTAGAAAGTA-30. PCR reactions were performed using 0.1 µg S. aureus genomic DNA, 50 picomoles of each primer, and the GoTaq green master mix from Promega (Madison, WI). The cycling conditions were as follows: 94 ◦C for 1 min, then 30 cycles of 94 ◦C for 20 s, 54 ◦C for 20 s, and 68 ◦C for 45 s. The resulting ssl1 gene sequences were compared to the allele type sequences as described by McCarthy and Lindsay [26].

4.9. Construction of Isogenic Mutants The Newman ∆hla∆hlg double mutant (EryR/TetR) was created by phage transduction of the antibiotic markers from S. aureus strain 8325-4 ∆hla∆hlg with phage 85. To create the triple mutant (Newman ∆hla∆hlg∆ssl1), the ssl1 gene was deleted from the chromosome of the double mutant by allelic exchange without an antibiotic marker. A construct for the unmarked deletion of ssl1 was made by SOE-PCR [41]. An upstream AB fragment (A primer 50-TATATCTAGAATATTCATCAATTCTGGTAAATATGG-30 XbaI; B primer 50-CATAATTTTTAGCCCCAATCTATTTAAATTTG-30) and downstream CD fragment (C primer 50-AGATTGGGGCTAAAAATTATGTAATACTTTCTAACAACAAAGCGCTATG-30; D primer 50-ATATCTGCAGTACGGTCTTCTTGTAACTTTTTATCC-30 Pstl) were amplified and joined by PCR using Phusion polymerase (New England Biolabs, Ipswich, MA). The product was digested with XbaI/PstI and ligated into pIMC5 digested with SpeI/PstI and transformed into E. coli DH10B. The 2-kb insert was verified by sequencing. The construct pIMC5-ssl1 was transformed into S. aureus RN4220 and then transduced into Newman ∆hla∆hlg with phage 85. Allelic exchange was conducted as described previously [41] with potential Newman ∆hla∆hlg∆ssl1 mutants screened with OUT primers (50-TAATTAATACGACCATCGCCAGC-30 / 50-TCGTCATCAAGTATATGTGTTATGC-30). To restore the ssl1 gene in the Newman ∆hla∆hlg∆ssl1 background, the ssl1 gene and flanking sequence were amplified from Newman genomic DNA with the above A and D primers. The product was cloned into pIMC5 producing pIMC5ssl1+ and allelic exchange performed in the Newman ∆hla∆hlg∆ssl1 background yielding Newman ∆hla∆hlg∆ssl1/ssl1.

4.10. Corneal Toxicity Model The use of animals, New Zealand white rabbits (2.0–3.0 kg) of either sex, adhered to the protocol (1060D) approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Rabbits were anesthetized by subcutaneous injection of a mixture of xylazine (100 mg/mL; Butler Co., Columbus, OH) and ketamine (100 mg/mL; Fort Dodge Animal Health, Fort Dodge, IA). Proparacaine (Bausch and Lomb, Tampa, FL) was topically Pathogens 2019, 8, 2 15 of 17 applied to the eyes prior to intrastromal corneal injections (10 µL) of recombinant toxin, concentrated supernatant (50-fold), or 100 CFU of S. aureus in 10 µL tryptic soy broth (n ≥ 10 eyes/group) [16].

4.11. Slit Lamp Examination Scoring and Quantification of Viable Bacteria Rabbit eyes underwent slit lamp examination scoring (SLE) as described by Arana et al. [42]. Briefly, SLE scoring involved two masked observers grading on a scale of 0 to 4 for each of seven ocular parameters (conjunctival injection, conjunctival chemosis, corneal edema, corneal infiltration, iritis, fibrin accumulation in the anterior chamber, and hypopyon formation). The parameter scores were added to yield a SLE score that ranged from 0 for a normal eye to a theoretical maximal score of 28 for a highly inflamed and damaged eye. SLE scores were not allowed to go to or above 20. Log CFU per cornea was determined as previously described [16]. Briefly, rabbit corneas were harvested at 24 h PI and homogenized in 3.0 mL sterile phosphate-buffered saline (PBS). A 0.1-ml aliquot of the homogenate was serially diluted 1:10 in PBS and plated on tryptic soy agar (TSA) in triplicate. Plates were incubated at 37 ◦C for 24 h. Colonies were counted and expressed as base 10 logarithms.

4.12. Statistical Analysis Mean and standard error of the mean (SEM) were calculated using Microsoft Excel. Statistical analyses of SLE scores and CFU determinations were performed using SAS software [15]. For SLE scores, statistical analyses of inter-group differences were performed using non-parametric one-way analysis of variance (Kruskal-Wallis test). For CFU determinations, one-way analysis of variance (ANOVA) and Student’s t tests between least-squared means from each group were performed. P ≤ 0.05 was considered signiﬁcant.

Author Contributions: Conceptualization, A.T., A.R.C., and R.J.O.; methodology, A.T., A.R.C., M.A.B., and I.R.M.; data curation, A.T., A.R.C., M.E.M., and T.J.F.; writing—original draft preparation, A.T., A.R.C., and R.J.O.; writing—review and editing, A.T. and R.J.O.; funding acquisition, T.J.F. and R.J.O. Funding: This research was funded in part by the National Eye Institute of the National Institutes of Health (EY010974) and the Science Foundation Ireland (08/IN.1/B1845). Acknowledgments: The authors wish to thank Joseph Dajcs for his contributions to this study. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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