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ANT IM ICROB IALS

1Krebs Institute, Department of Molecular Biology and Biotechnology, Universityof Sheffield, Sheffield S10 2TN, UK. 2AvidBiotics Corp., South San Francisco, CA94080, USA. 3Institute of Infection, Immunity and Inflammation, College of Med-ical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK.*Corresponding author. Email: ggovons@gmail.com (G.R.G.); r.fagan@sheffield.ac.uk (R.P.F.)

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

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New class of precision antimicrobials redefines role ofClostridium difficile S-layer in virulence and viabilityJoseph A. Kirk,1 Dana Gebhart,2 Anthony M. Buckley,3 Stephen Lok,2 Dean Scholl,2

Gillian R. Douce,3 Gregory R. Govoni,2* Robert P. Fagan1*

There is amedical need for antibacterial agents that donot damage the resident gutmicrobiota or promote the spreadof antibiotic resistance. We recently described a prototypic precision bactericidal agent, Av-CD291.2, which selectivelykills specific Clostridium difficile strains and prevents them from colonizing mice. We have since selected two Av-CD291.2–resistant mutants that have a surface (S)-layer–null phenotype due to distinct point mutations in the slpAgene. Using newly identified bacteriophage receptor binding proteins for targeting, we constructed a panel of Avidocin-CDs that kills diverse C. difficile isolates in an S-layer sequence-dependentmanner. In addition to bacteriophage receptorrecognition, characterization of the mutants also uncovered important roles for S-layer protein A (SlpA) in sporulation,resistance to innate immunity effectors, and toxin production. Surprisingly, S-layer–nullmutants were found to persist inthe hamster gut despite a complete attenuation of virulence. These findings suggest antimicrobials targeting virulencefactors dispensable for fitness in the host force pathogens to trade virulence for viability and would have clear clinicaladvantages should resistance emerge. Given their exquisite specificity for the pathogen, Avidocin-CDs have substantialtherapeutic potential for the treatment and prevention of C. difficile infections.

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INTRODUCTION

New antibacterial agents are needed to counteract the impending loss ofeffective treatment options for multidrug-resistant bacteria. Furtheringthis need is the realization that dysbiosis caused by broad-spectrum an-tibiotic use contributes to the prevalence of diseases/disorders such asinflammatory bowel disease, obesity, and gastrointestinal infections (1).Strategies to overcome these threats include use of narrow-spectrum orprecision agents and the design of drugs that target virulence instead ofin vitro viability (2, 3). One pathogen for which alternative treatment ap-proaches are needed isClostridium difficile. This spore-forming, obligateanaerobe is the leading cause of nosocomial infectionsworldwide. About450,000 cases and 29,000 deaths each year are attributed to this pathogenin theUnited States alone (4). As a result, theCenters forDiseaseControland Prevention has identified C. difficile as an urgent threat to humanhealth (5). This opportunistic pathogen exploits a reduction in gut mi-crobiota diversity that often follows broad-spectrum antibiotic use toproliferate, release toxins, and cause life-threatening colitis (6). Althoughthe toxins have been studied in great detail, other aspects of C. difficilevirulence, including colonization of the gut, are not well understood (6).TheC. difficile cell surface is covered by a paracrystalline surface (S)–layer largely composed of S-layer protein A (SlpA) and sparsely in-terspersed by 28 related cell wall proteins (7). The S-layer precursorSlpA is proteolytically processed on the cell surface to generate the low–molecular weight (LMW) and high–molecular weight (HMW) S-layerproteins (SLPs). The SLPs interact with high affinity to form a hetero-dimer, the basic unit of the mature S-layer (8). The slpA gene is locatedwithin a highly variable S-layer cassette consisting of five genes; 13 dis-tinct S-layer cassette types (SLCTs) have been described to date (9). Thevariation that defines individual cassette types is largely confined to theLMWSLP-encoding region of slpA (4). TheHMWSLP region is highlyconserved and includes the cell wall binding motifs that anchor the S-

layer to the cell wall (10). The S-layer and several associated cell wall pro-teins have been implicated in colonization of host tissues (7) and in stim-ulation of the host immune response via Toll-like receptor 4 (TLR4)signaling (11).

AlthoughC. difficile is not significantly resistant to the frontline anti-biotics used to treat C. difficile infections (CDIs) (vancomycin, metro-nidazole, and fidaxomicin), the use of these antibiotics causes furtherdisruption of the resident microbiota leading to frequent CDI relapse(6). To be safe and effective, new agents to treat and prevent CDIs mustnot harm the diverse gut microbiota and the colonization resistance itprovides. Avidocin-CDs represent one such potential agent (12). Thesebactericidal proteins are genetically modified versions of natural R-typebacteriocins (also known as diffocins) produced by C. difficile to killcompetingC. difficile strains (13). Diffocins resembleMyoviridae phagetails and consist of a contractile sheath, nanotube core, baseplate, andtail fiber structures. However, instead of deliveringDNAacross the bac-terial membrane as does a bacteriophage, R-type bacteriocins functionas killingmachines by injecting a nanotube core through the bacterial cellenvelope and creating a small pore that dissipates the cell’s membranepotential (14). Killing specificity is determined by the receptor-bindingproteins (RBPs) located at the tail fiber tips that trigger sheath contractionuponbindingwith a cognate receptor on thebacterial cell surface.Geneticreplacement or fusion of the RBP gene with homologs from other strainsor RBP sources (such as C. difficile bacteriophages and prophage in-sertions)makes it possible to retarget killing (12, 15–17). Thesemodifiedbacteriocins are known as Avidocin-CDs. An Avidocin-CD prototype,Av-CD291.2, constructed with a bacteriophage RBP identified within aprophage insertion, was found to bemore stable than the natural parentdiffocin. Av-CD291.2 had a modified killing spectrum that includedall hypervirulent ribotype 027 (RT027)C. difficile strains tested, blockedC. difficile colonization in amousemodel of spore transmission, and didnot disrupt the resident gutmicrobiota (12). These properties encouragethe further development of Avidocin-CDs as oral human therapeutics.

Here, we further characterize the Av-CD291.2 mechanism of actionand describe an expanded panel of Avidocin-CDs that cover all clini-cally relevant C. difficile strain types using newly identified bacterio-phage RBPs. Analysis of rare Av-CD291.2–resistant mutants enabled

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the identification of SlpA as the cell surface receptor for all testedAvidocin-CDs and, by extension, the corresponding bacteriophageRBP sources. Unsuspected roles for SlpA inmultipleC. difficile processeswere also identified. Despite defects that include the complete attenua-tion of virulence, S-layer mutants remarkably were observed to colonizeand persist in the hamster gut for the duration of a 14-day study.

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RESULTSAv-CD291.2–resistant mutants lack an S-layerResistance to any antimicrobial agent can occur and be exploited tounderstand itsmode of action.We isolated two spontaneousmutants ofC. difficile strain R20291 (RT027), FM2.5 and FM2.6, that were resistantto killing by Av-CD291.2. These mutants appeared at a frequency of<1 × 10−9 and were found to encode independent point mutations inthe slpA gene (Fig. 1A). Bothmutations were predicted to truncate SlpAat a site N-terminal to the posttranslational cleavage site and therebyprevent formation of an S-layer. Both FM2.5 and FM2.6 lacked detect-able cell surface SLP subunits as predicted but still expressed minor cellwall proteins including Cwp2 and Cwp6 (Fig. 1B). These mutations didnot affect the growth rate of the bacteria in vitro (fig. S1); however,FM2.5 displayed a slight, but statistically significant earlier entry intothe stationary phase [maximum OD600nm (optical density at 600 nm):FM2.5 = 2.2; R20291 = 3.2;P= 0.000012].We attempted to complementthe FM2.5 and FM2.6mutations with a plasmid-bornewild-type R20291slpA; however, unexpected homologous recombination restored thewild-type slpA gene to the chromosome (fig. S2). Accordingly, we createdgenetically identifiable recombinants “watermarked”with synonymoussubstitutions in the R20291 slpA allele (Fig. 1A). The resulting strains,FM2.5RW and FM2.6RW, were found to have LMW and HMW SLPson their surface (Fig. 1B) and regained sensitivity toAv-CD291.2 (Fig. 1,C and D). These results confirmed that an intact S-layer is required forAv-CD291.2 killing but did not determine whether SlpA itself or a pro-tein dependent on the S-layer for surface localization was the receptorfor Av-CD291.2. To address this question, slpA alleles from the mostcommon non-R20291 SLCTs were individually cloned into an inducibleexpression plasmid and transferred into the S-layer–deficient FM2.5(Fig. 1E). Each resulting strain expressed the expected LMW and HMWSLPs on the cell surface, indicative of S-layer formation (9), but did notregain sensitivity to Av-CD291.2, thus ruling out the possibility that thesimple formation of an S-layer was responsible for sensitivity to Av-CD291.2. To address whether Av-CD291.2 directly interacts withSLCT-4 SlpA, plasmids encoding SlpA from eight different SLCTswere introduced into the non-isogenic laboratory strain 630 (SLCT-7;RT012), which is insensitive to Av-CD291.2. In this fully S-layer–competent SLCT-7 wild-type strain, only induction of SLCT-4 SlpAwas sufficient to confer sensitivity to Av-CD291.2 (fig. S3). Moreover,the degree of sensitivity was dependent on the level of induction, withonly 10 ng/ml of inducer required (fig. S3A). Together, these observa-tions clearly demonstrate that the SLCT-4 variant of SlpA is the cell sur-face target of Av-CD291.2.

Sensitivity to Avidocin-CDs is slpA allele–specificThese observations suggested that each variant of SlpA may serve as aspecific receptor for additional C. difficile bacteriophage RBPs. In anattempt to expand Avidocin-CD coverage beyond SLCT-4, we con-structed 10 new Avidocin-CDs using predicted bacteriophage RBPsmined from the genome sequences ofC. difficile clinical isolates or new-ly isolated bacteriophages (table S1). Source strains for RBP sequences

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

were chosen on the basis of their SLCTs because strain typing informa-tion for the RBP source often correlates with sensitivity to the corre-sponding Avidocin (for example, RT027 and sensitivity to Av-CD291.2and SLCT-1 of the phi-147 propagating strain and sensitivity to Av-CD147.1) (12, 15, 16). Preparations of Diffocin-4 (scaffold for allAvidocin-CDs), Av-CD291.2, and each of the new Avidocin-CDswere tested for killing activity on a panel of 62 C. difficile isolatescontaining all 12 known SLCTs, hybrid 2/6 SLCT, and a newly iden-tified 14th SLCT (fig. S4). Each new Avidocin-CD had a broaderkilling spectrum than the host range of the parental bacteriophagefrom which the RBP was derived (figs. S4 and S5), perhaps due tocircumvention of bacteriophage resistance systems. Only two strains,representing SLCT-3 and SLCT-5, were not killed by a single Avidocin-CD (fig. S4); otherwise, every isolate from all 12 other SLCTs was killedby at least one Avidocin-CD. A near-perfect correlation was observedbetween a strain’s SLCT and its sensitivity to each of the Avidocin-CDs. The only exceptions were SLCT-2 isolates with Av-CD027.2and Av-CD685.1. A strong correlation was also observed between ri-botype and sensitivity to a particular Avidocin-CDbecause all ribotypesin the panel, except RT012, RT014, and RT015, were found to associateexclusively with a single SLCT. A significant degree of sequence varia-bility was tolerated within an SLCT by some Avidocin-CDs. For exam-ple, both Av-CD684.1 and Av-CD685.1 kill every SLCT-7 strain testeddespite sequence identities between SLCT-7 SlpAs as low as 81% (figs.S4 and S6). Similar correlations with strain sensitivity and SLCT or ri-botype were not observed for killing with Diffocin-4, suggesting that thisnatural R-type bacteriocin binds to C. difficile via another receptor.

Having made these observations, we wanted to determine whethersensitivity to specific Avidocin-CDs was directly dependent on the var-iant of SlpApresent. The panel of isogenic FM2.5 strains complementedwith slpA alleles from the 11 most common SLCTs was tested for sen-sitivity to a panel of the most potent Avidocin-CDs (Fig. 2). The corre-sponding parental strains from which the slpA alleles were obtainedwere used as controls. Each complemented strain became sensitive tothe same Avidocin-CDs as the parental strain. To confirm that this en-gineered sensitivity did not result from altered cell surface architecture,we performed an analogous experiment in a second panel of isogenicstrains created when plasmids encoding SlpAs from eight SLCTs wereintroduced into the S-layer–competent strain 630 (fig. S3, B and C). Asbefore, the spectrumof killingwas identical to that of the parental strainsfrom which the slpA alleles were obtained. These data demonstrate thatthe polymorphic SlpA acts as the binding receptor for each of theAvidocin-CDs tested and therefore the corresponding bacteriophageRBPs.

S-layer–null mutants are abnormally sensitive to innateimmune effectors and display severe sporulation defectsBacterial S-layers serve many critical cellular functions (7). Given itslocation on the cell surface, the S-layer has been proposed to act as amolecular sieve to selectively limit exposure of the underlying cellenvelope to large biomolecules such as the innate immune effectorlysozyme (18). Until now, analysis of C. difficile S-layer function hasbeen hampered by an inability to isolate slpA-deficient mutants (19).Whereas C. difficile is highly resistant to killing by lysozyme, resistancehas been attributed to extensive peptidoglycan deacetylation (20). Todetermine whether the S-layer also plays a role in lysozyme resistance,we treated exponentially growing bacteria with a high concentration oflysozyme (500 mg/ml) and monitored the effects on growth (Fig. 3A).Upon addition of lysozyme, an immediate decrease in optical density

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Fig. 1. Mutations in slpA confer Av-CD291.2 resistance. (A) Alignment of the slpA sequence (nucleotides 268 to 294) from R20291, FM2.5, FM2.6, FM2.5RW, and FM2.6RW. Anucleotide insertion at position 283 of FM2.5 slpA results in a frameshift and premature stop codon (blue). A nucleotide substitution at position 280 of FM2.6 slpA results in anonsense mutation (red). To allow differentiation from the wild-type sequence, we introduced two synonymous mutations into slpA in FM2.5RW and FM2.6RW (green). (B) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S-layer extracts from R20291, FM2.5, FM2.6, FM2.5RW, and FM2.6RW. The positions of the LMW and HMW SLPs andminor cell wall proteins Cwp2 and Cwp6 are indicated. (C and D) The impact of Av-CD291.2 on exponentially growing R20291, FM2.5, FM2.5RW, and FM2.6RW wasmonitored by measuring the OD600nm. Av-D291.2 addition is indicated with an arrow. Experiments were carried out in triplicate on biological duplicates. Means andSDs are shown. (E and F) SDS-PAGE analysis and Av-CD291.2 sensitivity of FM2.5 complemented with slpA alleles from multiple SLCTs after induction with anhydrote-tracycline (20 ng/ml). R20291 and FM2.5RW are included as controls. A zone of clearance in the agar lawn indicates killing.

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consistent with cell lysis, followed by slower growth, was observed forthe S-layermutant FM2.5. In contrast, R20291 andFM2.5RWdisplayedonly a transient decrease in growth rate, consistent with natural resistance.

Having confirmed a role for the S-layer in lysozyme resistance, wethen tested for resistance to the human cathelicidin antimicrobial pep-tide LL-37 to determine whether S-layer–mediated resistance extendedto other innate immune effectors (Fig. 3B). LL-37 is found at mucosalsurfaces at concentrations of up to 5 mg/ml under normal conditions,with further expression induced in response to infection (21). Treat-ment with LL-37 (5 mg/ml) completely killed exponentially growingcultures of the S-layer mutant FM2.5; the same treatment caused onlya slight reduction in growth rates for R20291 and FM2.5RW. Together,these data demonstrate a role for the S-layer in resistance to both lyso-zyme and LL-37.

In addition to sensitivity to lysozyme and LL-37, it was noted thatthe S-layer mutant strains survived poorly in the standard charcoalmedium used to transport C. difficile strains. The ability of C. difficileto survive in the environment and be transmitted to new hosts is relianton the bacterium’s ability to produce a heat- and chemical-resistantspore (22). We analyzed sporulation efficiency by measuring thenumbers of heat-resistant spores as a percentage of total viable colony-forming units (CFUs) over 5 days (fig. S7A). Spore production by wild-type R20291 and FM2.5RW cultures was reproducible and equivalentand represented 73 and 85% of total viable counts on day 5, respec-

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

tively (Fig. 3C). In contrast, spore production by FM2.5 was significantlylower (P≤ 0.00001). Spores only represented 4.3% of total viable countson day 5, which is a 17- to 20-fold reduction compared to R20291 andFM2.5RW. These observed differences in FM2.5 spore formation werenot due to an inability to germinate efficiently (Fig. 3D). When the bilesalt germinant taurocholate was added to purified spore preparations,the speed and efficiency of germination initiation for FM2.5 spores wereindistinguishable from that of R20291 and FM2.5RW. Analysis of bac-terial cultures by phase-contrast microscopy also pointed to a reductionin sporulation efficiency (fig. S7, B and C), as 5.2-fold fewer phase-brightspores were observed in cultures of FM2.5 (FM2.5, 8.7% versus R20291,44.9%; FM2.5RW, 45.4%). We noted a discrepancy between the mag-nitudes of the sporulation defect determined microscopically (5.2-foldless than R20291) compared with direct counting of viable spore CFUs(20-fold less than R20291). This suggests that many of the microscop-ically counted spores were non-viable after the 65°C heat treatmentrequired to differentiate spores from vegetative cell CFUs. To test theFM2.5 spores for possible stress resistance defects, we exposed thecultures to a harsher heat treatment (75°C for 30 min). The 75°C heattreatment further reduced FM2.5 spore viability by 33-fold comparedwith the standard 65°C (Fig. 3C). The same treatment only reducedspore viability by 1.5-fold for R20291 and 2-fold for FM2.5RW. Collect-ively, these data indicate that the production and quality of the infectiousspores are severely impaired by the loss of the S-layer.

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Fig. 2. Avidocin-CD sensitivity correlates with SLCT. (A) SDS-PAGE analysis of SLPs extracted from a panel of strains representing the 11 most commonly isolated SLCTs.(B) Spot bioassays with eight Avidocin-CDs on the C. difficile strains used in (A), as well as FM2.5 alone (−) and FM2.5 complemented with slpA alleles from 10 SLCTs afterinduction with anhydrotetracycline (20 ng/ml). The zone of clearance caused by each Avidocin-CD is shown along with SLCT. H, H2/6.

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Transmission electron microscopy was used to identify potentialmorphological changes associated with these defects (fig. S8). In FM2.5cultures, we observed spores with disorganized material loosely attachedto the electron-dense core that lacked discernible, well-organized proteincoat layers (fig. S8A) (23). To determine whether these unusual sporemorphologies were responsible for the observed thermal sensitivity, werepeated these analyseswith spores purified on aHistodenz gradient. Afterpurification, spores of FM2.5 were morphologically indistinguishablefrom those of R20291 and FM2.5RW (fig. S8C). Surprisingly, purifiedFM2.5 spores still displayed increased thermal sensitivity. A 75°C heattreatment reduced FM2.5 spore viability by 37.1-fold compared to 10-and 18.1-fold for R20291 and FM2.5RW, respectively (fig. S8B). Severaldistinct biochemical and structural features of the spore, including theconcentric cortex peptidoglycan and protein layers and core dehydra-tion, have been independently linked to heat resistance (24–26). Minordefects in any of these features could explain the observed thermal sen-sitivity of FM2.5 spores.

S-layer–null mutants are completely avirulent despitepersistent gut colonizationWe tested the ability of the S-layermutant FM2.5 to cause disease in theGolden Syrian hamster model of acute CDI. As expected, all animalsinoculated with FM2.5RW behaved similarly to those inoculated withthe wild-type R20291 strain and succumbed to CDI within 102 hoursof infection, with mean times to cull for R20291 and FM2.5RW of

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

67 hours 36min and 57 hours 19min, respectively (P = 0.52; Fig. 4A).Both groups of hamsters showed typical signs of disease includingwet tail and drop in body temperature at experimental end point.In contrast, all animals inoculated with FM2.5 displayed no signsof disease and survived for the duration of the 14-day study (P =0.0018; FM2.5 versus R20291; Fig. 4A). Surprisingly, the lack of vir-ulence was not due to a colonization defect. FM2.5 was capable ofpersistent colonization; CFUs in the cecum and colon at the end ofstudy (14 days) were not statistically different from those observedfor R20291 and FM2.5RW in the same tissues taken at experimentalend point some 10 days earlier (Fig. 4B). Toxinmeasurements from gutcontents 14 days after inoculation with FM2.5 showed marked reduc-tions in both toxin A and B activity compared to samples taken fromhamsters that succumbed to infection with either R20291 or FM2.5RW(Fig. 4, C and D).

S-layer–null mutants produce less toxin in vitro andAvidocin-CD killing does not result in toxin releaseGiven the avirulence and low toxin activity observed in animals, weassayed the FM2.5 and control strains for toxin production in vitro(Fig. 5A). Toxin Bwas used as an indicator for both toxins because theyare coordinately expressed and released (6). As expected, both R20291and FM2.5RWproduced toxin upon entry into the stationary phase. Asmall amount of toxin B was detected intracellularly at 24 hours; there-after, toxin B was exclusively detected in the culture supernatant.

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Fig. 3. Phenotypic characterization of FM2.5. (A and B) Cultures of R20291, FM2.5, and FM2.5RW were challenged with lysozyme (500 mg/ml) (A) or LL-37 (5 mg/ml) (B) inexponential phase after 2.5 hours (indicated with arrows). Untreated control cultures were grown in parallel. Experiments were carried out in triplicate on biological duplicates.Means and SDs are shown. (C) Sporulation of R20291, FM2.5, and FM2.5RW after 5 days. Spore CFUswere determined after a standard 65°C heat treatment for 30min or a harsher75°C heat treatment for 30 min. Heat-resistant spore CFUs are expressed as a percentage of total viable CFUs (spores and vegetative cells). Experiments were carried out intriplicate on biological duplicates. Means and SDs are shown. *P < 0.01, determined using two-tailed t tests with Welch’s correction. (D) Germination of R20291, FM2.5, andFM2.5RW spores. Synchronous germination of purified spores was induced with the bile salt taurocholate. Germination initiation was monitored by measuring the resultingdecrease in OD600nm.

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Cultures of FM2.5 produced less toxin B at all time points, consistentwith in vivo observations.

For toxin-producing pathogens, antimicrobials that lyse bacteria orincrease toxin production can exacerbate disease severity and lessen theeffectiveness of the treatment by inadvertently releasing toxins (27). Weexamined culture supernatants of strain R20291 after treatment withAvidocin-CDs for the presence of extracellular toxin B to determinewhether killing by an Avidocin-CD could result in unwanted toxin re-lease (Fig. 5B). Despite effective killing, intracellular toxin B remainedconstant with no detectable release observed in the culture supernatants(Fig. 5C) at any concentration, including samples that exhibited totalkilling of all vegetative bacterial cells due to a large excess of Avidocin-CD. These results confirm that Avidocin-CDs are bactericidal but nei-ther lyse the target cell nor release harmful intracellular stores of toxin B.

DISCUSSIONThe bactericidal properties of Avidocin-CDs bode well for the clinicalapplication of this new class of antimicrobial agents for combating CDI.Previous studies demonstrated that killing by the prototypic Avidocin-CD, Av-CD291.2, was highly specific for BI/NAP1/RT027-type strainsand did not detectably alter the resident gutmicrobiota inmice (12). Forproduction purposes, fermentation of genetically modified Bacillus sub-

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

tilis expressing these agents is similar tomany established industrial pro-cesses and scalable to thousands of liters. We selected, isolated, andcharacterized rare C. difficilemutants resistant to Av-CD291.2 to betterunderstand its mechanism of action. In the process, we discovered theAvidocin-CD–binding receptor, the C. difficile S-layer. Our 10 newAvidocin-CDs were all found to target variants of SlpA associatedwith different SLCTs. Given that the great majority of the sequencevariation in SlpA is found within the surface-exposed LMW subunit(9), this relationship between SLCT and Avidocin-CD sensitivitystrongly suggests that the LMW SLP is the binding site for all thestudied Avidocin-CDs. Further, it indirectly identifies this portion ofthe SlpA as the binding receptor for many C. difficile myophages be-cause each Avidocin-CD is constructed with a different bacteriophage-derivedRBP.These findings help explain the limitedhost ranges observedfor many C. difficilemyophages (28, 29). It also suggests that antigenicvariation observed between SLCTsmay not be due to immune escapeas previously proposed (9) but rather due to a molecular arm race be-tween bacteria and bacteriophages.C. difficile is under selective pressureto change the bacteriophage receptor, which, in turn, puts selective pres-sure on the bacteriophages to evolve new RBPs.

Patterns of strain sensitivity to eachAvidocin-CD indicate that killingwas much broader than the parental bacteriophage’s host range. Apossible explanation for this observation is that a typical bacteriophage

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Fig. 4. In vivo analysis of slpAmutant in the Syrian Golden hamster. (A) Times to experimental end point of animals infectedwith R20291 (black line), FM2.5 (dark blue line),and FM2.5RW (light blue line), respectively. Each line represents six animals. (B) Total CFUs and spore CFUs (after heat treatment at 56°C for 20 min) were determined for lumen-associated (LA) and tissue-associated (TA) bacteria recovered from cecum (CE) and colon (COL) of infected animals and quantified at experimental end point (R20291 andFM2.5RW) or at 14 days after infection (FM2.5). Means and SEs are shown. The horizontal dashed line indicates the limit of detection. None of the observed differences arestatistically significant. (C and D) Relative toxin activity of filtered gut samples on HT-29 (toxin A) and Vero cells (toxin B), respectively. Values represent the reciprocal of the firstdilution in which cell morphology was indistinguishable from untreated wells. Samples were taken at experimental end point (R20291 and FM2.5RW) or at 14 days after infection(FM2.5). *P < 0.05 and **P < 0.01, determined using a two-tailed nonparametric Mann-Whitney test; NS, not significant.

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infection cycle is a seven-step process: attachment, genome injection,replication, transcription, translation, assembly, and lysis. Bacteria canbecome resistant to a bacteriophage by blocking any stage of the infec-tion cycle, for example, CRISPR-Cas or restriction-modification systemsthat prevent phage DNA from replicating (30). In contrast, Avidocin-CD killing is a two-step process: attachment to the target bacteriumfollowed by killing via creation of a small pore that dissipates the targetbacterium’s membrane potential. As observed with other Avidocins andR-type bacteriocins (16, 31), selected Avidocin-CD–resistant mu-tants survived because of mutations that caused the loss or modifica-tion of the SlpA-binding receptor and not because of mutations thatdirectly disrupted the killingmechanism, such as improper pore forma-tion, or caused proteolytic cleavage of the agent. The bias toward recep-tor mutations suggests that the Avidocin-CD killing mechanism issimple and robust.

Modification of the binding receptor to avoid Avidocin-CD killingthrough eithermissensemutations or S-layer switching, as evidenced bythe random association between clades and SLCTs (9), could also the-oretically lead to resistance. Although sequence variations between slpAalleles within each SLCT indicate that missense mutations do occur(9), our findings suggest that resistance due to this type of modifica-tion is unlikely because the bacteriophage RBPs used to construct theAvidocin-CDs have already evolved to counter this mode of potentialescape. A high degree of sequence variation is observed in SlpA withinSLCTs; for example, the SlpAs of SLCT-7 strains 630 andM68 display

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

only 81% amino acid identity. Despite this variation, both strains areefficiently killed by both Av-CD684.1 and Av-CD685.1. As for theemergence of resistance via horizontal transfer of the S-layer cassette,the administration of a cocktail of Avidocin-CDs that kill all thecommon SLCTs would make successful resistance via S-layer switchingextremely unlikely. It appears that the only likely means of resistanceto all the Avidocin-CDs is through complete loss of SlpA, as observedfor resistance to Av-CD291.2. As a consequence, the observed phe-notypes for FM2.5 are germane to all likely Avidocin-CD–resistantmutants.

For a precision medicine agent to be successful, knowing the molec-ular target (in this case, the binding receptor) is vital in designing accu-rate diagnostics to guide treatment decisions. If Avidocin-CDs were tobe administered individually, a diagnostic determining SLCT of infect-ing C. difficile would be highly accurate for informing Avidocin-CDtreatment decisions. However, an increase in the prevalence of hybridcassettes, as found in RT078 strains (9), would decrease the accuracyfor this typing method. Strain ribotyping could also be used to avoid de-velopment of new diagnostics, but predicting sensitivity to a particularAvidocin-CDmay be challenging because ribotypes are not consistentlylinked to a single SLCT (that is, RT012, RT014, and RT015, as notedabove). SlpA typing would provide the most accurate diagnosticand prove more illuminating than ribotyping because SlpA is directlyrelated to the physiology of C. difficile, whereas ribotyping detects phys-iologically inconsequential ribosomal RNA gene polymorphisms. Itmay

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Fig. 5. Toxin production and release in vitro. (A) In vitro cell lysate and culture supernatant samples from R20291, FM2.5, and FM2.5RW cultures were normalized to anequivalent optical density and separatedon6%SDSpolyacrylamidegels. Toxin BwasdetectedbyWestern immunoblot using an anti–toxin Bmonoclonal antibody. Samplesweretaken at the indicated time points. (B) To determine whether Avidocin-CD killing released intracellular toxin, R20291 was incubated for an hour either with Av-CD291.2 at theindicated ratio of agent to cells or Av-CD684.1, which does not kill strain R20291, at a 500:1 ratio (IS), or left untreated (UT). After treatment, viable bacteria were enumerated (bargraph), and the percentage killed relative to the untreated control was determined (numbers above each bar). The number of spores present in the untreated sample wasdetermined after heat treatment at 65°C for 30 min to kill vegetative cells (HT). (C) After Avidocin-CD treatment, released toxin B in culture supernatants (ExC) was detected byWestern immunoblot using an anti–toxin B monoclonal antibody. As a positive control for toxin release, R20291 was treated with the phiCD27L bacteriophage endolysin (41). Theamount of remaining intracellular toxin B (IntC) was determined by lysing cells with CD27L endolysin and detection byWestern immunoblot as before. A fresh sample of untreatedR20291 was lysed with CD27L endolysin to show normal intracellular toxin quantities (EL). The original uncropped images for each Western immunoblot can be found in fig. S9.

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also be possible to administer a cocktail of five to six Avidocin-CDs thattarget 12 of the 14C. difficile SLCTs. If such a cocktail of Avidocin-CDswere to be administered, a point-of-care diagnostic would only need todetect the presence of C. difficile to guide treatment decisions.

The strong selective pressure afforded by Av-CD291.2 allowed iso-lation of spontaneous C. difficile S-layer–null mutants. In addition toenabling identification of the Avidocin-CD cell surface receptor, thesemutants also provide an unprecedented opportunity to study S-layerfunction (fig. S10). Given the ubiquity of S-layers in both Bacteria andArchaea, includingmany pathogenic species, surprisingly little is knownabout their function. It has been suggested that the S-layer could act asa molecular sieve to exclude certain large biomolecules from the cellenvelope (18); however, this has not been confirmed in live cells. Wehave demonstrated that an intact S-layer is required for resistance totwo components of the innate immune system: lysozyme and the anti-microbial peptide LL-37. Assembled S-layers are highly symmetrical withregular repeating pores. The size of the pores in theC. difficile S-layeris not yet known, but in other species, pore sizes of between 2 and 6 nmhave been reported (7). A pore size of 2 nm could conceivably excludethe16-kDa globular protein lysozyme, but a small peptide such as LL-37would experience no such steric hindrance. It is possible that chargedsurfaces on the assembled S-layer serve to sequester the cationic peptideaway from the cell envelope in amanner analogous to capsular polysac-charides (32). Our data have identified other pleiotropic functions forthe C. difficile S-layer (fig. S10). It is clear that the S-layer is the cell sur-face receptor for all of theAvidocin-CDs described here and, by extension,the receptor for the bacteriophages from which the RBP-encoding geneswere cloned. Although a Bacillus bacteriophage has been found to bindSLP Sap (33), this is the first time a receptor for aC. difficile bacteriophagehas been identified. Furthermore, our data imply that S-layer recognitionis a common feature of bacteriophages that infect this species.

Surprisingly, the S-layer mutant also displayed severe sporulationdefects, with fewer andmorphologically defective spores produced. De-spite a number of well-studied spore-forming organisms producing S-layers, including Bacillus anthracis (33), S-layer biogenesis has not beenreported previously to affect sporulation. There is currently no evidenceto suggest that the SLPs are a structural part of themature spore, and themechanisms bywhich the S-layer can influence sporulation are currentlyunknown. However, this observation has serious ramifications for theability of an Avidocin-CD–resistant, S-layer–defectivemutant to surviveand be transmitted to other hosts. The spore is an absolute requirementforC. difficile survival in the aerobic environment and is critical for trans-mission (22). In addition to sporulation defects, the S-layer mutant wasalso found to produce less toxin in vitro. There have been previous sug-gestions of feedback between sporulation and the complex regulatorynetwork controlling toxin production (22). Although the mechanism bywhich these processes are affected in SlpA-nullmutants is far from clear, itis possible that the S-layer feeds into a point of cross-talk between regula-tion of virulence and transmission. Given the poor toxin production andother diverse phenotypes identified for the S-layer mutant, it is probablynot surprising that themutantwas entirely avirulent in the hamstermodelof acute infection.What did come as a surprise, however, was the ability ofthe S-layermutant to stably colonize and persist in the hamster gut for the14-day duration of the experiment. Previous reports have pointed to a rolefor the S-layer in epithelial cell adhesion; however, these earlier studieswere performed without access to an slpA-defective strain (34). The S-layer–defectivemutants and isogenic controls expressing the SLCT-specificslpA alleles provide the ideal controls to test these conflicting findingsand better define the effect SlpA type has on these functions.

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

There are several study limitations that should be considered whenevaluating the data presented. The activity of each new Avidocin-CDwas observed in vitro and needs to be confirmed in an animal treatmentmodel, as performed forAv-CD291.2. Similarly, the exquisite specificityof each agent for distinctC. difficile SLCTs also needs to be tested in vivoto confirm that treatment with these agents will not alter the diversity ofthe gut microbiota. The avirulent phenotype and long-term persistenceof theAvidocin-CD–resistant slpAmutants in the hamstermodel shouldalso be confirmed in other animal species. Finally, the implications of themutants’ observed sporulation defects on transmission and the spread ofresistance remain to be tested in vivo.

After accounting for these limitations, analysis of the data clearlydemonstrates that acquisition of Avidocin-CD resistance results in lossof toxin production and complete loss of virulence in the hamster. Veryfew described mutations located outside of the PaLoc locus have re-sulted in complete avirulence in this model (35). The ability of the S-layer mutant to stably colonize the gut in the absence of clinical diseasereveals that the in vivo lifestyle of the organism is independent of toxinproduction and virulence. The prevalence of nontoxigenic, avirulentC. difficile strains in the general population (36) supports this hypothesis.Virulence factors make attractive targets for new antimicrobials becausethey tend to be species-specific. If their loss does not affect pathogenfitness, as appears to be the case for the C. difficile S-layer, emergenceof resistance is likely to reduce virulence (3). As a result, we predictthat there will be no competing selective pressure to restore virulencein the context of Avidocin-CD resistance.

In summary, we have developed and characterized multiple newAvidocin-CDs, providing crucial insights into their potential advan-tages in the clinic. The precise killing activity of Avidocin-CDs makesthem attractive agents for both treatment and prevention because theycan be administered to patients without altering the diversity of the com-plex gutmicrobiota. In addition, when resistance does emerge, Avidocin-CDs force the pathogen to sacrifice virulence for viability, making thepotential clinical impact of resistance inconsequential.

MATERIALS AND METHODSStudy designThe objective of this studywas to characterize a panel of Avidocin-CDs,antibacterial protein complexes constructed to specifically target andkill C. difficile. During these experiments, the isolation of Avidocin-CD–resistant slpAmutants allowed detailed analysis of S-layer func-tion for the first time. Starting from the prototypic Av-CD291.2 (12),we constructed a panel of new Avidocin-CDs using bacteriophageRBPswe identified. Each newAvidocin-CDdisplayed a unique spectrumof killing activity with a strong correlation to SLCT. Sensitization of twoinsensitive C. difficile strains by heterologous expression of a cognateSLCT SlpA alone allowed identification of the S-layer as the receptorfor all described Avidocin-CDs. Analysis of the slpA mutant identifiedpreviously unsuspected in vitro roles for the S-layer in resistance to theimmune effectors lysozyme and LL-37 and in the production of matureheat-resistant spores. Finally, use of the Golden Syrian hamstermodel ofacute infection demonstrated that the slpAmutant was entirely avirulentdespite persistent infection. Greatly reduced toxin activity was detectedin intestinal contents from animals colonized with the slpAmutant, andthis observation was supported by identification of a toxin productiondefect in vitro. The design and execution of these animal experimentsare described in detail in Supplementary Materials and Methods.Primary data are reported in table S4.

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Strains, bacteriophage, and culture conditionsBacterial strains used in this study are described in fig. S2. DNA oligo-nucleotides are described in table S2. Escherichia coli strains wereroutinely grown in Luria broth (LB) and on LB agar (VWR). C. difficilestrains were routinely grown under anaerobic conditions on BrainHeart Infusion (BHI) or BHI-S agar and in tryptone yeast (TY) broth(37), except where otherwise stated. Cultures were supplemented withchloramphenicol (15 mg/ml), thiamphenicol (15 mg/ml), or anhydrote-tracycline (20 ng/ml) as required. C. difficile SLCTs were determinedby analyzing the nucleotide sequence of the slpA gene. When neces-sary, the slpA gene was sequenced using oligonucleotide primers previ-ously described (38, 39) or primers 023-F and 023_010-R or 014+++-Fand 014_002+-R (nucleotide sequences; table S2). The variable region ofstrain 19142 (RT046) slpA gene did not display high sequence identitywith other slpA alleles andhas been designated SLCT-13.Apartial strain19142 slpA sequence was deposited in GenBank (accession numberKX610658). Details of plasmid and strain construction are given in Sup-plementary Materials and Methods (fig. S11).

Bioassays to determine Avidocin-CD killing activityAvidocin-CD bactericidal activity was assayed by a semiquantitativespot method as previously described (12, 13). For broth-based killingassays, C. difficile strains were grown overnight in TY broth and thensubcultured to anOD600nm of 0.05 in 1-ml fresh TY broth supplementedwith 1 mM CaCl2. Av-CD291.2 (50 ml) was added to each culture after2.5 hours. Growth was monitored by measuring the OD600nm hourly.

Extraction of S-layer and associated proteinsSLPs were extracted using low-pH glycine as previously described (8)and analyzed by SDS-PAGE using standard methods.

Quantitative analysis of sporulation and germinationQuantitative analysis of sporulation was carried out as previously de-scribed (19) andmonitored by phase-contrast and transmission electronmicroscopy as described in Supplementary Materials and Methods.

Analysis of resistance to lysozyme and LL-37Broth-based killing assays were carried out as described above but withlysozyme (500 mg/ml) or LL-37 (5 mg/ml) added after 2.5 hours ofgrowth. Cell density was monitored by measuring the OD600nm hourly.Assays were carried out in triplicate on biological duplicates.

Animal experimentsThe Golden Syrian hamster model was performed as previously de-scribed (40). All procedures were performed in strict accordance withthe Animals (Scientific Procedures) Act 1986 with specific approvalgranted by the Home Office, UK (PPL60/4218). Further detail is givenin Supplementary Materials and Methods.

Quantification of toxin expressionQuantification of toxin activity was performed using Vero cells as de-scribed previously (40) and byWestern immunoblot using anti–toxin Bantibody (MA1-7413, Thermo Fisher). Further details are given in Sup-plementary Materials and Methods.

Statistical analysesDatawere analyzed usingGraphPadPrism software (GraphPad SoftwareInc.). Toxin production was compared using a two-tailed nonparametricMann-Whitney test, and animal survival curves were analyzed using a

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

log-rank (Mantel-Cox) test. All other statistical analyses were performedusing two-tailed t tests with Welch’s correction.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/406/eaah6813/DC1Materials and MethodsFig. S1. Characterization of growth.Fig. S2. Restoration of wild-type slpA to the chromosome of FM2.5 and FM2.6.Fig. S3. Avidocin-CD sensitivity correlates with SLCT.Fig. S4. C. difficile strain sensitivity patterns to Avidocin-CDs and Diffocin-4.Fig. S5. Comparison of C. difficile bacteriophage host range versus Avidocin-CD sensitivity.Fig. S6. Clustal Omega alignment of SlpA sequences from strains 630 and M68.Fig. S7. Characterization of sporulation.Fig. S8. Spore morphology and thermal sensitivity.Fig. S9. Expression of toxin B in vitro.Fig. S10. Schematic diagram summarizing the roles of S-layer in C. difficile biology andpathophysiology.Fig. S11. Schematic describing Avidocin-CD construction.Table S1. Newly identified C. difficile phages.Table S2. Primers used in this study.Table S3. GenBank accession identifiers.Table S4. Primary data.Reference (42)

REFERENCES AND NOTES1. J. R. Marchesi, D. H. Adams, F. Fava, G. D. A. Hermes, G. M. Hirschfield, G. Hold,

M. N. Quraishi, J. Kinross, H. Smidt, K. M. Tuohy, L. V. Thomas, E. G. Zoetendal, A. Hart, Thegut microbiota and host health: A new clinical frontier. Gut 65, 330–339 (2016).

2. D. A. Rasko, V. Sperandio, Anti-virulence strategies to combat bacteria-mediated disease.Nat. Rev. Drug Discov. 9, 117–128 (2010).

3. R. C. Allen, R. Popat, S. P. Diggle, S. P. Brown, Targeting virulence: Can we make evolution-proof drugs? Nat. Rev. Microbiol. 12, 300–308 (2014).

4. F. C. Lessa, Y. Mu, W. M. Bamberg, Z. G. Beldavs, G. K. Dumyati, J. R. Dunn, M. M. Farley,S. M. Holzbauer, J. I. Meek, E. C. Phipps, L. E. Wilson, L. G. Winston, J. A. Cohen,B. M. Limbago, S. K. Fridkin, D. N. Gerding, L. C. McDonald, Burden of Clostridium difficileinfection in the United States. N. Engl. J. Med. 372, 825–834 (2015).

5. “Antibiotic resistance threats in the United States, 2013,” (Threat Report 2013, Centers forDisease Control and Prevention, 2013).

6. W. K. Smits, D. Lyras, D. B. Lacy, M. H. Wilcox, E. J. Kuijper, Clostridium difficile infection.Nat. Rev. Dis. Primers 2, 16020 (2016).

7. R. P. Fagan, N. F. Fairweather, Biogenesis and functions of bacterial S-layers. Nat. Rev.Microbiol. 12, 211–222 (2014).

8. R. P. Fagan, D. Albesa-Jové, O. Qazi, D. I. Svergun, K. A. Brown, N. F. Fairweather,Structural insights into the molecular organization of the S-layer from Clostridium difficile.Mol. Microbiol. 71, 1308–1322 (2009).

9. K. E. Dingle, X. Didelot, M. A. Ansari, D. W. Eyre, A. Vaughan, D. Griffiths, C. L. C. Ip,E. M. Batty, T. Golubchik, R. Bowden, K. A. Jolley, D. W. Hood, W. N. Fawley, A. S. Walker,T. E. Peto, M. H. Wilcox, D. W. Crook, Recombinational switching of the Clostridium difficileS-layer and a novel glycosylation gene cluster revealed by large-scale whole-genomesequencing. J. Infect. Dis. 207, 675–686 (2013).

10. S. E. Willing, T. Candela, H. A. Shaw, Z. Seager, S. Mesnage, R. P. Fagan, N. F. Fairweather,Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifsthat recognise the anionic polymer PSII. Mol. Microbiol. 96, 596–608 (2015).

11. A. Ryan, M. Lynch, S. M. Smith, S. Amu, H. J. Nel, C. E. McCoy, J. K. Dowling, E. Draper,V. O’Reilly, C. McCarthy, J. O’Brien, D. Ní Eidhin, M. J. O’Connell, B. Keogh, C. O. Morton,T. R. Rogers, P. G. Fallon, L. A. O’Neill, D. Kelleher, C. E. Loscher, A role for TLR4 inClostridium difficile infection and the recognition of surface layer proteins. PLOS Pathog.7, e1002076 (2011).

12. D. Gebhart, S. Lok, S. Clare, M. Tomas, M. Stares, D. Scholl, C. J. Donskey, T. D. Lawley,G. R. Govoni, A modified R-type bacteriocin specifically targeting Clostridium difficileprevents colonization of mice without affecting gut microbiota diversity.mBio 6, e02368-14 (2015).

13. D. Gebhart, S. R. Williams, K. A. Bishop-Lilly, G. R. Govoni, K. M. Willner, A. Butani,S. Sozhamannan, D. Martin, L.-C. Fortier, D. Scholl, Novel high-molecular-weight, R-typebacteriocins of Clostridium difficile. J. Bacteriol. 194, 6240–6247 (2012).

14. P. Ge, D. Scholl, P. G. Leiman, X. Yu, J. F. Miller, Z. H. Zhou, Atomic structures of abactericidal contractile nanotube in its pre- and postcontraction states. Nat. Struct. Mol.Biol. 22, 377–382 (2015).

9 of 10

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

by guest on February 29, 2020

http://stm.sciencem

ag.org/D

ownloaded from

15. D. Scholl, M. Cooley, S. R. Williams, D. Gebhart, D. Martin, A. Bates, R. Mandrell, Anengineered R-type pyocin is a highly specific and sensitive bactericidal agent for thefood-borne pathogen Escherichia coli O157:H7. Antimicrob. Agents Chemother. 53,3074–3080 (2009).

16. D. Scholl, D. Gebhart, S. R. Williams, A. Bates, R. Mandrell, Genome sequence of E. coliO104:H4 leads to rapid development of a targeted antimicrobial agent against thisemerging pathogen. PLOS ONE 7, e33637 (2012).

17. S. R. Williams, D. Gebhart, D. W. Martin, D. Scholl, Retargeting R-type pyocins togenerate novel bactericidal protein complexes. Appl. Environ. Microbiol. 74,3868–3876 (2008).

18. M. Sára, U. B. Sleytr, Molecular sieving through S layers of Bacillus stearothermophilusstrains. J. Bacteriol. 169, 4092–4098 (1987).

19. M. Dembek, L. Barquist, C. J. Boinett, A. K. Cain, M. Mayho, T. D. Lawley, N. F. Fairweather,R. P. Fagan, High-throughput analysis of gene essentiality and sporulation in Clostridiumdifficile. mBio 6, e02383-14 (2015).

20. T. D. Ho, K. B. Williams, Y. Chen, R. F. Helm, D. L. Popham, C. D. Ellermeier, Clostridiumdifficile extracytoplasmic function s factor sV regulates lysozyme resistance and isnecessary for pathogenesis in the hamster model of infection. Infect. Immun. 82,2345–2355 (2014).

21. D. M. Bowdish, D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, R. E. Hancock, Impact of LL-37on anti-infective immunity. J. Leukoc. Biol. 77, 451–459 (2005).

22. L. J. Deakin, S. Clare, R. P. Fagan, L. F. Dawson, D. J. Pickard, M. R. West, B. W. Wren,N. F. Fairweather, G. Dougan, T. D. Lawley, The Clostridium difficile spo0A gene is apersistence and transmission factor. Infect. Immun. 80, 2704–2711 (2012).

23. D. Paredes-Sabja, A. Shen, J. A. Sorg, Clostridium difficile spore biology: Sporulation,germination, and spore structural proteins. Trends Microbiol. 22, 406–416 (2014).

24. J. Barra-Carrasco, V. Olguin-Araneda, Á. Plaza-Garrido, C. Miranda-Cárdenas,G. Cofré-Araneda, M. Pizarro-Guajardo, M. R. Sarker, D. Paredes-Sabja, The Clostridiumdifficile exosporium cysteine (CdeC)-rich protein is required for exosporiummorphogenesis and coat assembly. J. Bacteriol. 195, 3863–3875 (2013).

25. P. Setlow, Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat andchemicals. J. Appl. Microbiol. 101, 514–525 (2006).

26. P. C. R. Strong, K. M. Fulton, A. Aubry, S. Foote, S. M. Twine, S. M. Logan, Identification andcharacterization of glycoproteins on the spore surface of Clostridium difficile. J. Bacteriol.196, 2627–2637 (2014).

27. C. S. Wong, S. Jelacic, R. L. Habeeb, S. L. Watkins, P. I. Tarr, The risk of the hemolytic–uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N. Engl.J. Med. 342, 1930–1936 (2000).

28. J. Y. Nale, J. Spencer, K. R. Hargreaves, A. M. Buckley, P. Trzepiński, G. R. Douce, M. R. J. Clokie,Bacteriophage combinations significantly reduce Clostridium difficile growth in vitro andproliferation in vivo. Antimicrob. Agents Chemother. 60, 968–981 (2016).

29. O. Sekulovic, J. R. Garneau, A. Néron, L.-C. Fortier, Characterization of temperate phagesinfecting Clostridium difficile isolates of human and animal origins. Appl. Environ.Microbiol. 80, 2555–2563 (2014).

30. J. E. Samson, A. H. Magadán, M. Sabri, S. Moineau, Revenge of the phages: Defeatingbacterial defences. Nat. Rev. Microbiol. 11, 675–687 (2013).

31. J. M. Ritchie, J. L. Greenwich, B. M. Davis, R. T. Bronson, D. Gebhart, S. R. Williams, D. Martin,D. Scholl, M. K. Waldor, An Escherichia coli O157-specific engineered pyocin preventsand ameliorates infection by E. coli O157:H7 in an animal model of diarrheal disease.Antimicrob. Agents Chemother. 55, 5469–5474 (2011).

32. E. Llobet, J. M. Tomás, J. A. Bengoechea, Capsule polysaccharide is a bacterial decoy forantimicrobial peptides. Microbiology 154, 3877–3886 (2008).

33. R. D. Plaut, J. W. Beaber, J. Zemansky, A. P. Kaur, M. George, B. Biswas, M. Henry,K. A. Bishop-Lilly, V. Mokashi, R. M. Hannah, R. K. Pope, T. D. Read, S. Stibitz, R. Calendar,S. Sozhamannan, Genetic evidence for the involvement of the S-layer protein genesap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection ofBacillus anthracis. J. Bacteriol. 196, 1143–1154 (2014).

34. M. M. Merrigan, A. Venugopal, J. L. Roxas, F. Anwar, M. J. Mallozzi, B. A. P. Roxas,D. N. Gerding, V. K. Viswanathan, G. Vedantam, Surface-layer protein A (SlpA)

Kirk et al., Sci. Transl. Med. 9, eaah6813 (2017) 6 September 2017

is a major contributor to host-cell adherence of Clostridium difficile. PLOS ONE 8,e78404 (2013).

35. E. M. Ransom, K. B. Williams, D. S. Weiss, C. D. Ellermeier, Identification andcharacterization of a gene cluster required for proper rod shape, cell division, andpathogenesis in Clostridium difficile. J. Bacteriol. 196, 2290–2300 (2014).

36. J. K. Shim, S. Johnson, M. H. Samore, D. Z. Bliss, D. N. Gerding, Primary symptomlesscolonisation by Clostridium difficile and decreased risk of subsequent diarrhoea.Lancet 351, 633–636 (1998).

37. B. Dupuy, A. L. Sonenshein, Regulated transcription of Clostridium difficile toxin genes.Mol. Microbiol. 27, 107–120 (1998).

38. H. Kato, T. Yokoyama, Y. Arakawa, Typing by sequencing the slpA gene of Clostridiumdifficile strains causing multiple outbreaks in Japan. J. Med. Microbiol. 54, 167–171(2005).

39. T. Karjalainen, N. Saumier, M.-C. Barc, M. Delmée, A. Collignon, Clostridium difficilegenotyping based on slpA variable region in S-layer gene sequence: An alternative toserotyping. J. Clin. Microbiol. 40, 2452–2458 (2002).

40. A. M. Buckley, J. Spencer, D. Candlish, J. J. Irvine, G. R. Douce, Infection of hamsters withthe UK Clostridium difficile ribotype 027 outbreak strain R20291. J. Med. Microbiol. 60,1174–1180 (2011).

41. J. Peltier, H. A. Shaw, E. C. Couchman, L. F. Dawson, L. Yu, J. S. Choudhary, V. Kaever,B. W. Wren, N. F. Fairweather, Cyclic diGMP regulates production of sortase substrates ofClostridium difficile and their surface exposure through ZmpI protease-mediatedcleavage. J. Biol. Chem. 290, 24453–24469 (2015).

42. R. P. Fagan, N. F. Fairweather, Clostridium difficile has two parallel and essential Secsecretion systems. J. Biol. Chem. 286, 27483–27493 (2011).

Acknowledgments: We thank F. Tenover, T. Riley, T. Lawley, V. Young, and K. Dinglefor providing C. difficile isolates, N. Fairweather for a plasmid containing SLCT-11 slpA,H. Browne and T. Lawley for providing strain CD305 genome sequence, and C. Hill and theUniversity of Sheffield Electron Microscopy Unit for transmission electron microscopyanalysis. Funding: This work was supported by the National Institute of Allergy andInfectious Diseases of the NIH under award number R21AI121692. The content is solelythe responsibility of the authors and does not necessarily represent the official views ofthe NIH. Additional support was obtained from the Medical Research Council (grantnumber MR/N000900/1 to R.P.F.), AvidBiotics Corp. and the University of Sheffield viathe Higher Education Innovation Fund 2011–2015 (to R.P.F.), and the Wellcome Trust(grant number 086418 to G.R.D.). Author contributions: D.S., G.R.D., G.R.G., and R.P.F.designed and coordinated the study. J.A.K., D.G., A.M.B., S.L., and G.R.G. conducted theexperiments. G.R.D., G.R.G., and R.P.F. wrote the manuscript with contributions from allcoauthors. Competing interests: D.G., S.L., D.S., and G.R.G. are current or past employeesof and own stock in AvidBiotics Corp. R.P.F. received a research grant from AvidBioticsCorp. AvidBiotics Corp. hold the following patents: US8206971 (Modified bacteriocinsand methods for their use), US8673291 (Diffocins and methods of use thereof), US9115354(Diffocins and methods of use thereof), and EP2576604 (Diffocins and methods of usethereof). Data and materials availability: Nucleotide sequences have been depositedin GenBank with accession identifiers KX610658, KX557294, KX592438, KX592434, KX592441,KX592442, KX592443, KX592444, KX592439, KX592435, KX592437, KX592436, andKX592440 (table S3). Avidocin-CDs are available from AvidBiotics Corp. subject to amaterial transfer agreement.

Submitted 31 July 2016Resubmitted 6 February 2017Accepted 13 April 2017Published 6 September 201710.1126/scitranslmed.aah6813

Citation: J. A. Kirk, D. Gebhart, A. M. Buckley, S. Lok, D. Scholl, G. R. Douce, G. R. Govoni,R. P. Fagan, New class of precision antimicrobials redefines role of Clostridium difficile S-layer invirulence and viability. Sci. Transl. Med. 9, eaah6813 (2017).

10 of 10

virulence and viability S-layer inClostridium difficileNew class of precision antimicrobials redefines role of

and Robert P. FaganJoseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni

DOI: 10.1126/scitranslmed.aah6813, eaah6813.9Sci Transl Med

force bacteria into forgoing virulence in favor of survival., these findings showcase how making targeted antimicrobials canC. difficileaddition to revealing biology about

mutants had defensive defects and attenuated virulence but were still able to colonize the gut of hamsters. In isolated strains resistant to the treatment, which were found to have mutations in the surface layer. Theal.

etspecific antimicrobial derived from a genetically modified bacteriocin, Kirk −C. difficiletreat. While investigating a infects hundreds of thousands of people a year and is becoming increasingly difficult toClostridium difficile

icult decisiondiffForcing microbes to make a

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