Neisseria gonorrhoeae Evades Calprotectin-Mediated NutritionalImmunity and Survives Neutrophil Extracellular Traps byProduction of TdfH

Sophonie Jean,a* Richard A. Juneau,b* Alison K. Criss,b Cynthia N. Cornelissenc

Integrative Life Sciences Program, Department of Biology, Virginia Commonwealth University, Richmond, Virginia, USAa; Department of Microbiology, Immunology, andCancer Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia, USAb; Department of Microbiology and Immunology, Virginia CommonwealthUniversity Medical Center, Richmond, Virginia, USAc

Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by pro-duction of TonB-dependent transporters (TdTs)— outer membrane proteins that facilitate nutrient transport in an energy-de-pendent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including trans-ferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). Theroles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstratingthat production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evalu-ate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcalTdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector proteinhighly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfHdirectly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterialsurvival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a keyfunctional arm of NET-mediated killing of gonococci. We demonstrate for the first time that N. gonorrhoeae exploits this hoststrategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as azinc source and thereby evades nutritional immunity.

Neisseria gonorrhoeae is an obligate human pathogen and theetiological agent of the sexually transmitted infection (STI)

gonorrhea, which was estimated to infect 820,000 people in theUnited States alone in 2011 (1). Generally, symptomatic infectionpresents as cervicitis in women and urethritis in men and can beresolved with antibacterial drug treatment. However, infection isoften asymptomatic in women, resulting in the lack of treatmentand ascension to the upper reproductive tract. This can result inserious downstream sequelae, including pelvic inflammatory dis-ease (PID) and disseminated gonococcal infection (DGI), and caneven result in ectopic pregnancy and infertility. In addition to thehigh morbidity associated with gonococcal infection, the total di-rect cost of treating N. gonorrhoeae infections in the United Statesin 2008 was estimated to be between $81.1 and $243.2 million (2),representing a significant economic burden.

The ability to treat and control gonococcal infections has be-come increasingly challenging due to the development of antibi-otic resistance. Resistance to sulfonamides, penicillin, and fluoro-quinolones is widespread among gonococcal isolates, renderingextended-spectrum cephalosporins (ESCs) the last class of antibi-otics approved for use (3). However, recent reports of treatmentfailures with cefixime and ceftriaxone (4) have led to revised ther-apy recommendations, which include combination therapy ofceftriaxone plus azithromycin (5). Characterization of “super-bug” gonorrhea, resistant to all approved therapies, has legiti-mized the threat of untreatable gonorrhea (6, 7). As such, thedevelopment of novel strategies to treat gonorrhea has become atop international priority.

In spite of significant efforts, there is no vaccine to protectagainst gonococcal infection (reviewed in reference 8). Further-

more, natural gonococcal infection confers no protective immu-nity; as such, previously infected individuals remain susceptible tofuture infections (8). The ability of the gonococcus to phase andantigenically vary its surface structures is thought to contribute toits evasion of the host adaptive immune response (8, 9). Recentstudies have also demonstrated that the gonococcus proactivelyskews the host response away from humoral immunity and to-ward innate immunity, which is to the pathogen’s advantage (9).New vaccine development strategies will likely include immuno-modulatory therapy along with presentation of conserved surfaceantigens in an effort to develop a protective response (10). Thus,the identification of potential vaccine targets remains imperative.Given their surface exposure, conservation among gonococcal

Received 13 April 2016 Returned for modification 15 May 2016Accepted 25 July 2016

Accepted manuscript posted online 1 August 2016

Citation Jean S, Juneau RA, Criss AK, Cornelissen CN. 2016. Neisseria gonorrhoeaeevades calprotectin-mediated nutritional immunity and survives neutrophilextracellular traps by production of TdfH. Infect Immun 84:2982–2994.doi:10.1128/IAI.00319-16.

Editor: S. M. Payne, University of Texas at Austin

Address correspondence to Cynthia N. Cornelissen,Cynthia.cornelissen@vcuhealth.org.

* Present address: Sophonie Jean, Department of Microbiology and Immunology,Virginia Commonwealth University Medical Center, Richmond, Virginia, USA;Richard A. Juneau, School of Science, Technology, Engineering, and Mathematics,Virginia Western Community College, Roanoke, Virginia, USA.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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isolates, and their limited sequence variability due to their role inthe acquisition of essential nutrients, TonB-dependent transport-ers (TdTs) have the potential to be ideal targets for novel thera-peutic and/or preventative strategies.

TonB-dependent transporters are large outer membrane�-barrel proteins with loop regions that extend into the extracel-lular space to interact with external molecules. A conserved plugoccludes the lumen of the barrel and also extends into theperiplasmic space (reviewed in reference 11). TonB, an innermembrane protein in complex with ExbB and ExbD, interactswith the “ton box” region of the plug to energize passage of nutri-ents across the bacterial outer membrane through the barrel ofTdTs (12). N. gonorrhoeae encodes eight putative TdTs, five ofwhich have been at least partially characterized (reviewed in ref-erence 13). The best characterized gonococcal TdTs facilitate theacquisition of iron or iron chelates from host-derived proteins,including transferrin, lactoferrin, and hemoglobin, as well as fromsiderophores made by other bacteria. Iron acquisition is particu-larly important to pathogenesis, as genetically engineered gono-coccal strains incapable of simultaneously using transferrin andlactoferrin cannot cause experimental infection in human malevolunteers (14).

In addition to the ability to hijack host proteins for iron acqui-sition, Neisseria gonorrhoeae can survive a robust neutrophil re-sponse, which it specifically elicits during infection (15, 16). As thefirst line of defense in the innate immune response, polymorpho-nuclear leukocytes (PMNs) or neutrophils normally locate andeliminate invading microbes through phagocytosis and subse-quent degranulation or the formation of neutrophil extracellulartraps (NETs) (17). A growing body of evidence now indicates thatN. gonorrhoeae can overcome the antimicrobial functions ofPMNs by delaying maturation of phagosomes (18), resisting theoxidative burst (19, 20), and escaping NETs through excretion ofa nuclease (21).

The host defense strategy of nutritional immunity (reviewed inreference 22) refers to the sequestration of essential nutrientswithin the host to restrict the growth of pathogenic bacteria and,therefore, their ability to cause infection. Originally described as astrategy of iron limitation, the limitation of other transition met-als such as Zn and Mn in response to microbial infection has alsobeen described (23). Zn, in particular, provides structural or cat-alytic support to 5 to 6% of bacterial proteins (80% of which areenzymes) (24) that function in bacterial processes such as generegulation, cellular metabolism, and virulence (23). Calprotectin(CP) is a hetero-oligomeric protein complex that constitutes 45%of the cytosolic protein content of neutrophils (25). This innateimmunity protein has been demonstrated to have antimicrobialactivity against a wide range of pathogens (26–33), which has beenattributed to its Zn- and Mn-chelating ability. The depletion of Znwithin microbial tissue abscesses (26) due to CP and the attenua-tion of virulence or colonization by microbes with inactivated Zntransport systems (23) indicate the importance of Zn at the host-pathogen interface and underscore the need for better under-standing of bacterial mechanisms that overcome nutritional im-munity strategies.

Herein, we describe the mutagenesis and phenotypic analysisof two gonococcal TdTs: TdfH and TdfJ. The Neisseria meningiti-dis homologues of these proteins, CbpA and ZnuD, respectively,have been shown to contribute to growth under Zn-limited con-ditions (34, 35). CbpA was demonstrated to bind to CP and fur-

thermore to enable growth under Zn limitation. ZnuD was re-cently shown to bind to Zn in vitro (36). In the present study, weshow that gonococcal TdfH and TdfJ are Zn and Zn uptake regu-lator (Zur) repressed and contribute to gonococcal growth underlimited-Zn conditions. We demonstrate that TdfH binds directlyto CP in whole-cell binding assays and facilitates gonococcal in-ternalization of Zn from CP. Furthermore, we show that bothgonococcal growth in the presence of CP and CP binding to wholecells require TdfH production. Our studies further extend ourunderstanding of the biological significance for production ofTdfH in gonococcal pathogenesis, as we demonstrate that CP isaccessible to gonococci in NETs and that TdfH enhances gono-coccal survival in the presence of NETs. Finally, we show that Znsequestration is a key component of NET-mediated killing. This isthe first report to identify a bacterial surface protein that facilitatesCP resistance within a biologically relevant niche for N. gonor-rhoeae. Therefore, we present a novel mechanism of gonococcalsurvival in the presence of neutrophils wherein TdfH mediates Znutilization to overcome nutritional immunity by its obligate hu-man host.

MATERIALS AND METHODSBacterial growth conditions. Escherichia coli was cultured in Luria-Bertani medium with antibiotic selection at 100 �g/ml for ampicillin and50 �g/ml for kanamycin. N. gonorrhoeae was routinely maintained on GCmedium base (Difco) agar with Kellogg’s supplement I (37) and 12 �MFe(NO3)3 at 37°C in 5% CO2. Zn-restricted conditions in liquid culturewere achieved in both rich medium and defined medium. For each con-dition, the Zn chelator concentration added was the minimum requiredto inhibit the growth of the wild-type strain prior to addition of excess Zn.For Zn-restricted growth in rich medium, individual colonies from GCbroth (GCB) agar plates were inoculated into GCB treated with supple-ment I, 12 �M Fe(NO3)3, and 12.5 �M chelator N,N,N=N=-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), and cultures were grown untilthey reached log phase. At log phase, cultures were back-diluted with GCBplus supplement I, Fe, and TPEN and further treated with 18.75 �MZnSO4 for Zn-replete conditions or not further treated for Zn-depletedconditions. For Zn-restricted growth in defined medium, individual col-onies from GCB agar plates were inoculated into chemically defined me-dium (CDM) treated with Chelex-100 (Bio-Rad) (38), and cultures weregrown to log phase with or without Fe(NO3)3 before back-dilution andthe addition of TPEN and/or ZnSO4 at the stated concentrations. Allliquid cultures were grown at 37°C with 5% CO2 and vigorous shaking.For regulation studies, gonococcal strains were grown in GC broth me-dium treated with supplement I until log-phase growth was achieved. Atlog phase, ZnSO4 or TPEN was added at a final concentration of 25 �M forthe Zn-replete or Zn-depleted conditions, respectively. Cultures weregrown for 4 h under the described conditions before whole-cell lysateswere harvested for SDS-PAGE analysis. For NET bacterial survival stud-ies, viable exponential-phase gonococci were obtained by sequentialdilution in rich medium as previously described (19), except at the finaldilution, where cultures were resuspended in phenol red-free RPMI with1 �M TPEN and grown to an optical density at 550 nm (OD550) of 0.4.Bacterial suspensions were then centrifuged at 10,000 � g for 3 min andwashed once with RPMI before exposure to neutrophils.

Construction of gonococcal mutants. The strains and plasmids usedin this study are listed in Table 1. All plasmids were propagated in E. coliTOP10 cells (Invitrogen). To construct the gonococcal zur mutant, weobtained zur::kan from Alastair G. McEwan, which contains the full-length zur gene in the SmaI site of pUC19 interrupted by a kanamycinresistance cassette. This plasmid was linearized with ScaI and used totransform gonococcal strains FA19 and FA1090. Transformants were se-lected on GCB agar plates supplemented with kanamycin at 50 �g/ml, andthe location of the insertion in the chromosome was confirmed via PCR.

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The resulting strains were named MCV963 (FA19 zur) and MCV964(FA1090 zur). Gonococcal strain MCV927 contains an � insertion withinthe coding region of the tdfH gene in an FA19 background and has beendescribed elsewhere (39).

To generate a complemented derivative of MCV927, the full-lengthtdfH gene, including its native ribosome binding site (RBS) was PCRamplified from FA1090 chromosomal DNA using the following primers:oVCU807 (5=-GGG CAG TAC TGA GGA AAA TAT GAG ATC T-3= [ScaIsite underlined]) and oVCU798 (5=-CAC AGT TTA AAC AAA CGC GGGCTG-3= [PmeI site underlined]). The amplicon was cloned into pCR2.1using the TOPO TA Cloning kit (Invitrogen) according to the manufac-turer’s instructions to generate pVCU944. The tdfH sequence was con-firmed by sequencing, and pVCU944 was digested with restriction endo-nucleases ScaI and PmeI to isolate the tdfH fragment, which was thenpurified and inserted at the PmeI site of pGCC4 (40). The proper orien-tation of tdfH in the resulting plasmid, pVCU945, was verified by restric-tion mapping with ScaI and KpnI. For gonococcal transformation,pVCU945 was digested with NotI, and the fragment containing tdfH, lctP,aspC and the erythromycin resistance cassette was purified and used totransform MCV927. Transformants were selected on GCB agar platessupplemented with erythromycin at 1 �g/ml. The resulting strain,MCV956, contains the chromosomal tdfH mutation and an ectopicallyinserted copy of the wild-type tdfH gene preceded by its native RBS underthe control of a lac promoter.

To generate a double mutant incapable of expressing both TdfH andTdfJ in the FA1090 background, the XbaI- and SacI-flanked tdfJ fragment

from pVCU703 was first subcloned into pVCU403, which contains 10-bpgonococcal uptake sequence between the HindIII and PstI sites of pUC18(41). The resulting plasmid, pVCU937, was subjected to random trans-poson mutagenesis using the Ez-Tn5�Kan-2� kit (Epicentre). Kana-mycin-resistant clones were screened via restriction mapping forTn5�Kan-2� insertion within the tdfJ gene fragment; the insertionallymutagenized plasmid employed subsequently was named pVCU938.Gonococcal strain MCV661 contains an � insertion within the codingregion of the tdfH gene in an FA1090 background and has been describedelsewhere (42). MCV661 was transformed with pVCU938, and transfor-mants were selected on GCB agar supplemented with 50 �g/ml kanamy-cin, resulting in strain MCV936.

For NET survival studies, a tdfH mutant in the opacity (Opa) protein-deficient (Opaless) background (43) was constructed. To generate thismutant, the XbaI- and SacI-flanked tdfH fragment from pVCU702 wassubcloned into pVCU403. The resulting plasmid, pVCU947, was also sub-jected to Ez-Tn5�Kan-2� random transposon mutagenesis and restric-tion mapped for Tn5�Kan-2� insertion within the tdfH gene fragment,and the insertionally mutagenized plasmid was named pVCU948. TheOpaless version of strain FA1090 was then transformed with pVCU948,and transformants were selected on GCB agar supplemented with 30�g/ml kanamycin, resulting in strain MCV955. The complemented Opal-ess tdfH mutant strain was constructed by transforming MCV955 withchromosomal DNA from strain MCV956, selecting for erythromycin re-sistance. The transformant was back-crossed twice, again into MCV955.The final, selected transformant (strain MCV956A) was confirmed to

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Genotype and/or relevant characteristic(s) Reference or source

StrainsFA19 Wild type 68FA1090 Wild type (�lbpBA, HpuAB off) 69Opaless FA1090 �opaA to -K 43�nuc mutant FA1090 Opaless �nuc (Kanr) 21FA1090 misR FA1090 misR (Kanr) William M. Shafer, unpublished dataFA19 misR FA19 misR (Kanr) William M. Shafer, unpublished dataMCV650 FA19 tonB::� (Strr Spcr) 42MCV661 FA1090 tdfH::� (Strr Spcr) 42MCV662 FA1090 tdfJ::� (Strr Spcr) 42MCV927 FA19 tdfH::� (Strr Spcr) 39MCV928 FA19 tdfJ::� (Strr Spcr) 39MCV936 FA19 MCV661 transformed with pVCU938 (Kanr Strr Spcr tdfH tdfJ) This studyMCV955 Opaless FA1090 transformed with pVCU948 (Kanr tdfH) This studyMCV956 FA19 MCV927 transformed with pVCU945 (Ermr Strr Spcr tdfH tdfHc) This studyMCV956A Opaless FA1090 transformed with chromosomal DNA from MCV956

(Ermr Strr Kanr tdfH tdfHc)This study

MCV963 FA19 transformed with pUC19zur::kan (Kanr) This studyMCV964 FA1090 transformed with pUC19zur::kan (Kanr) This study

PlasmidspCR2.1 TOPO Cloning plasmid (Kanr Ampr) InvitrogenpUC18 Cloning plasmid (Ampr) InvitrogenpGCC4 Complementation plasmid (Kanr Ermr) 40pVCU403 pUC18 with gonococcal uptake sequence (Ampr) 41pVCU702 pCR2.1 containing tdfH fragment 42pVCU703 pCR2.1 containing tdfJ fragment 42pVCU937 pVCU403 containing tdfJ fragment from pVCU703 This studypVCU938 pVCU937 with tdfJ disrupted by Ez-Tn5�Kan-2� This studypVCU944 pCR2.1 containing full-length tdfH native RBS This studypVCU945 pGCC4 containing tdfH allele from pVCU944 This studypVCU947 pVCU403 containing tdfH fragment from pVCU702 This studypVCU948 pVCU947 with tdfH disrupted by Ez-Tn5�Kan-2� This studypUC19zur::kan pUC19 containing full-length zur disrupted by Kan cassette Alastair G. McEwan, unpublished data

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contain the tdfH gene disrupted by Ez-Tn5�Kan-2�, in addition to awild-type copy of tdfH in the ectopic expression locus.

Immunoblotting and antibodies. Gonococcal whole-cell lysates weregenerated by harvesting gonococcal strains at a standardized density basedupon the optical density of the culture. One milliliter of culture at a den-sity of 100 Klett units (KU) was centrifuged, and the standardized gono-coccal cell pellets were then resuspended in Laemmli solubilizing buffer(44) and stored at 20°C until use. Immediately prior to SDS-PAGE,whole-cell lysates were treated with 5% �-mercaptoethanol and boiled for3 min. Proteins were separated on a 7.5% polyacrylamide gel and trans-ferred to nitrocellulose. Equivalent loading of whole-cell lysates from wellto well was verified by Ponceau S staining of the nitrocellulose mem-branes. For TdfH detection, membranes were blocked with 5% bovineserum albumin (BSA) in a high-salt Tris-buffered saline (TBS) (20 mMTris, 500 mM NaCl [pH 7.5], 0.05% Tween 20), probed with polyclonalantiserum against TdfH (45) and washed with high-salt TBS followed byincubation with secondary antibody conjugated to alkaline phosphatase(AP) (Bio-Rad). Blots were developed with the nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) system (Sigma). ForTdfJ detection, we generated polyclonal antipeptide antibodies (NewEngland Peptides) against regions predicted to correspond to loop 2 andloop 5 after alignment of TdfJ to a two-dimensional (2D) topology modelof TbpA. Membranes were blocked with 5% skim milk in a low-salt TBS(50 mM Tris, 150 mM NaCl [pH 7.5]), probed with loop 2-specific sera,and washed with low-salt TBS before being incubated with secondaryantibody conjugated to horseradish peroxidase (Southern Biotech). Blotswere developed with the Pierce-ECL-2 kit (Thermo Scientific). For TbpBdetection, rabbit anti-TbpB primary antibody (46) was used after block-ing with 5% skim milk. Blots were then washed with low-salt TBS beforeincubation with secondary antibody conjugated to AP and developed withNBT/BCIP.

Enumeration of bacterial growth under Zn-replete and -depletedconditions. Gonococci were grown to log phase in Zn-restricted richmedium (GCB plus supplement I, Fe, and TPEN). At log phase, cultureswere back-diluted and were not further treated to represent Zn-depletedconditions or were treated with ZnSO4 (18.75 �M) for Zn-replete condi-tions. After growth for 6 h, bacterial cells were harvested by centrifugationat 4,000 � g for 15 min at 4°C, washed twice with cold PBS–1 mM EDTA,and resuspended in PBS before being serially diluted and spot plated onGCB agar plates supplemented with V-C-N inhibitor (BD BBL). Plateswere incubated at 37°C in 5% CO2 for 16 h before CFU were enumerated.

Growth with calprotectin. Gonococci were grown as described abovein Zn-restricted chemically defined medium (CDM) without Fe(NO3)3.After the optical density of each culture doubled, independent cultureswere diluted to the same optical density (100 KU) and then transferred toa 96-well microtiter plate. Each well contained 7.5 �M 30% saturatedhuman transferrin (hTf) as the sole iron source. hTf (Sigma) was dissolvedin a mixture of 40 mM Tris, 150 mM NaCl, and 10 mM NaHCO3 at pH8.6, to which ferric chloride was added to result in 30% saturation by mass.After equilibration, partially saturated hTf was dialyzed to remove resid-ual unbound iron. In addition to the sole iron source, 2.5 �M apo-bovinetransferrin (bTf) was added to sequester residual iron, and 1 �M TPENwas incorporated for Zn chelation. A 2 mM concentration of IPTG (iso-propyl-�-D-thiogalactopyranoside) was also present in all wells to inducetdfH expression in MCV956. Some wells were further supplemented witheither 10 �M CP plus 5 �M ZnSO4 or 5 �M ZnSO4 alone. Levels ofcalcium in CDM (0.25 mM) are consistent with excess calcium require-ments (0.2 mM) described for optimal Zn binding to CP (47). The micro-titer plate was incubated at 37°C in 5% CO2 with vigorous shaking, andOD600 readings were collected every 2 h for 6 to 8 h. CP was a kind giftfrom Walter Chazin and was purified as previously described (31).

Whole-cell calprotectin binding assay. Gonococcal strains weregrown as described for Zn restriction in CDM with 24 �M Fe(NO3)3.After one mass doubling (approximately 1.5 h of incubation), 1 �MTPEN and 2 mM IPTG were added, and cultures were incubated under

the described conditions for 4 h before being applied to nitrocellulosemembranes using a dot blot apparatus (Bio-Rad). Dried membranes wereblocked for 1 h with 5% skim milk in low-salt TBS before being incubatedwith CP plus Zn (0.17 �M CP, 0.01 �M ZnSO4) in blocker. After beingwashed with low-salt TBS, blots were probed for CP using rabbit anti-s100A9 polyclonal antibody (Thermo-Scientific) and secondary antibodyconjugated to AP. Dot blots were then developed with NBT/BCIP. Fordensitometry, replicate blots were scanned, and intensities of individualdots were quantitated using NIH Image J (48). Quantitated intensitieswere then normalized to the wild type and expressed as a percentage of CPbinding.

Zn accumulation assays. Gonococcal strains were grown as describedfor Zn restriction in rich medium (GCB plus supplement I, Fe, and TPEN)until log-phase growth was achieved, at which point cultures were back-diluted and either treated with 18.75 �M ZnSO4 (Zn replete) or no furtheraddition (Zn depleted). Cultures were grown for 6 h before being har-vested by centrifugation at 4°C for 15 min at 4,000 � g and washed twicewith cold, Chelexed PBS supplemented with 1 mM EDTA. Bacterial cellswere then resuspended in undiluted trace-metal-grade nitric acid, heatedat 95°C for 2 h, and cooled at room temperature overnight. For analysis,samples were diluted 12.5- or 22-fold in Chelexed distilled water (dH2O).Metal composition was determined by inductively coupled plasma-opti-cal emission spectrometry (ICP-OES) using an MPX Vista spectrometer(Varian, Inc.). To determine the concentration of metals associated withthe cells, a standard curve was generated with a 10 �g/ml multielementstandard (CMS-5; Inorganic Ventures) diluted in Chelexed dH2O andthen serially diluted 2-fold in 1% HNO3 to generate dilutions rangingfrom 0.640 �g/ml to 0.020 �g/ml. Cell pellets from parallel cultures wereresuspended in PBS, treated with 5% SDS, and vortexed for 30 s for lysis.Protein concentrations were determined using the bicinchoninic acid(BCA) protein assay kit (Pierce) according to the manufacturer’s instruc-tions.

Immunofluorescence to detect calprotectin in NETs. Poly-L-lysine-treated wells of chambered cover glasses (Nunc) were seeded with 1 � 106

primary human neutrophils and treated with 20 nM phorbol myristateacetate (PMA) for 30 min to induce NET formation. Opaless parentalgonococci were added to wells for 1 h at a multiplicity of infection (MOI)of 1. Samples were fixed in 4% paraformaldehyde (Electron MicroscopySciences) and then blocked overnight in 1% BSA–PBS (Fisher-Gibco).Primary antibodies against gonococci (Biosource) and calprotectin (SinoBiological, Inc.) and secondary antibodies anti-rabbit Alexa 555 and anti-Mouse Alexa 645 (Life Technologies), respectively, were used for staining.Following washes, samples were stained with Sytox green (Invitrogen) tovisualize DNA. Samples were mounted in Fluoromount G (SouthernBiotech) with 2.5 mg/ml n-propyl gallate (Acros Organics). Images wereacquired on a Zeiss LSM 700 confocal laser scanning microscope (63�/1.40 oil immersion) in the University of Virginia Advanced MicroscopyCore. z-stack slices were exported as TIF files from Zen 2012 image pro-cessing software (Zeiss).

NET bacterial survival assay. Survival of gonococcal strains uponexposure to PMA-stimulated neutrophils treated with cytochalasin D orcytochalasin D and DNase I was determined as previously described (21).Neutrophils were purified from venous blood collected from healthy do-nors according to a protocol approved by the University of Virginia Insti-tutional Review Board for Health Science Research. Briefly, 24-well plateswere seeded with neutrophils and treated with 20 nM PMA for 30 min at37°C in 5% CO2. One set of wells was treated with 10 �g/ml cytochalasinD (Sigma), while another set was treated with cytochalasin D and 1 U ofDNase I, each for 15 min. Neutrophils were then exposed to Opalessparent, MCV955 (tdfH), MCV956A (tdfHC) (the complemented strain),or �nuc gonococci grown as described above at an MOI of 1 for 1 h. Afterinfection, well contents were scraped, serially diluted, and plated for CFUenumeration. Bacterial survival is expressed as a percentage of the initialinocula per well.

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NET bacterial survival in the presence of Zn sulfate. Neutrophilswere PMA stimulated and cytochalasin D treated to generate NETs, asdescribed above, and infected with Opaless parent or MCV955 (tdfH)gonococci � 0.5 �M ZnSO4 at an MOI of 1 for 1 h. Well contents werescraped, serially diluted, and plated to enumerate CFU and to calculate thepercentage of bacterial survival relative to the initial inoculum per strain.

RESULTSTdfH and TdfJ production is Zn repressed and Zur regulated.We first sought to characterize the production of TdfH and TdfJ inresponse to Zn levels. Gonococcal wild-type strain FA1090 wasgrown in chemically defined media (CDM) in the presence of thezinc chelator TPEN, ZnSO4, and/or Fe(NO3)3 at the indicatedconcentrations. We found that TdfH and TdfJ levels were down-regulated in the presence of Zn (Fig. 1). Conversely, production ofboth proteins was upregulated in the presence of increasing con-centrations of TPEN. Consistent with previous reports (45), TdfHproduction levels did not change in response to iron. However,TdfJ production was further elevated when supplemented withiron, even at TPEN concentrations shown to derepress TdfJ pro-duction (Fig. 1), suggesting that TdfJ is also iron-induced. Toverify that TPEN treatment only altered Zn levels, we assessedproduction of TbpB (transferrin binding protein B), which isknown to be iron regulated. TbpB levels did not change at various

TPEN concentrations. TbpB production (except under iron-re-plete conditions) also serves as a loading control for the TdfH andTdfJ blots shown in Fig. 1A.

To assess whether zinc repression of TdfH and TdfJ was Zurmediated, we generated zur isogenic mutants in gonococcalstrains FA19 and FA1090, resulting in strains MCV963 andMCV964, respectively. FA1090, FA19, and their respective zurmutant derivatives were grown in Zn-replete or Zn-depleted me-dium. We found that production of both TdfH and TdfJ was up-regulated under Zn-depleted conditions in the wild-type back-grounds. Production was increased in zur mutant derivatives inboth backgrounds. Additionally, TdfH and TdfJ were not differ-entially produced under Zn-replete versus Zn-depleted condi-tions in zur mutants (Fig. 1B). These data indicate that Zn repres-sion of TdfH and TdfJ is Zur mediated. We also observed thatTdfH production was higher under all conditions in strainFA1090 compared to that in FA19, while levels of TdfJ productionwere similar in both backgrounds (Fig. 1B). Interestingly, the in-creased TdfH production in FA1090 versus FA19 was also ob-served in the respective zur mutants. These data suggest that whileZn repression of TdfH is Zur mediated in strains FA1090 andFA19, Zur does not contribute to the increased production ofTdfH in FA1090 versus FA19.

Next, we sought to determine whether production of gonococ-cal TdfH was altered by the two-component regulatory systemMisRS. Microarray studies have identified meningococcal TdfH aspart of the MisR regulon (49). Additionally, MisRS has beenshown to regulate the inner core structure of gonococcal lipo-oligosaccharide (LOS) and production of the hemoglobin recep-tor HmbR in N. meningitidis (50, 51). When gonococci weregrown under Zn-replete and Zn-depleted conditions, we foundthat the misR mutant derivative in the FA1090 backgroundshowed no difference in TdfH production compared to its parent(Fig. 1C). In contrast, in FA19 the misR mutant derivative pro-duced less TdfH than its parent under both Zn-replete and Zn-depleted conditions (Fig. 1C). MisR was not found to alter theproduction of TdfJ in either FA1090 or FA19. These data suggestthat TdfH production can be controlled by MisR in addition to Znand Zur, and this regulatory circuit varies depending on the ge-netic background.

TdfH, TdfJ, and TonB contribute to growth under Zn-re-stricted conditions. Given the regulation of the TonB-dependentreceptors TdfH and TdfJ by Zn and Zur, we sought to determinewhether these TdTs contribute to gonococcal Zn acquisition. Wehypothesized that those gonococcal strains lacking Zn receptorswould be deficient for growth under Zn-restricted conditions.Likewise, we expected that upon supplementation with Zn,growth of wild-type gonococci would recover, whereas strainslacking the receptors would remain suppressed for growth. Totest these hypotheses, wild-type or tdfH, tdfJ, and tonB mutantstrains (FA19, MCV927, MCV928, and MCV650, respectively)were grown in GC broth medium treated with TPEN in the pres-ence or absence of additional Zn. After growth for 6 h, viablebacteria were enumerated. As expected, significantly more viablebacteria were recovered from all bacterial strains tested whengrown under Zn-replete versus Zn-depleted conditions, exceptfor the tdfH mutant, where addition of Zn did not increase thenumbers of recovered bacteria (Fig. 2). Moreover, significantlyfewer viable tdfH mutant than wild-type bacteria were recoveredafter growth in the presence of Zn. These data are consistent with

FIG 1 Zn regulation of TdfH production. Whole-cell lysates of equal culturedensity were analyzed by SDS-PAGE followed by immunoblotting with anti-sera against TdfH, TdfJ, or TbpB as indicated. (A) Wild-type strain FA1090was grown for 4 h in CDM supplemented with Fe(NO3)3, ZnSO4, or Zn chela-tor TPEN at the given concentrations. (B) Parental strains FA1090 and FA19and the respective zur mutant derivatives were grown under Zn-replete and-depleted conditions. (C) Parental strains FA1090 and FA19 and the respectivemisR mutant derivatives were grown under Zn-replete and -depleted condi-tions.

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a function for TdfH in Zn acquisition. The tdfJ mutant was themost impaired for growth under Zn-replete or -depleted condi-tions; however, growth was partially restored by addition of Zn(Fig. 2). This finding suggests that while TdfJ is important forgrowth under Zn restriction, gonococci have additional mecha-nisms of Zn acquisition independent of TdfJ. In agreement withthe effects of the TdTs TdfH and TdfJ on Zn acquisition, signifi-cantly fewer tonB mutant bacteria were also recovered, comparedto wild-type bacteria.

TdfH is required for growth in the presence of calprotectin.Since both TdfH and TdfJ contribute to growth under Zn-re-stricted conditions and the meningococcal homologue of TdfHbinds to the host Zn-binding protein calprotectin (CP) (34, 35),we tested whether these putative transporters are required for theassimilation of Zn from CP. We tested the ability of wild-type ortdfH and tdfJ mutant strains to grow in the presence of CP supple-mented with Zn under Zn-restricted conditions. Contrary to theantimicrobial activity reported against other pathogens (26, 27,30, 31, 52), we found that the growth of wild-type gonococci wasenhanced in the presence of Zn plus CP (Fig. 3A). Addition of CPalso suppressed growth of the tdfH mutant, which is particularlyevident at 8 h (Fig. 3A and C). These data suggest that N. gonor-rhoeae is capable of overcoming the antimicrobial effects of CP byproduction of TdfH. Growth of the tdfJ mutant was similar to thatof the wild type (Fig. 3A). To confirm that TdfH was indeed re-sponsible for the observed resistance of gonococci to CP, we gen-erated a complemented derivative of the tdfH mutant strain,MCV956 (tdfHC), which we confirmed produced TdfH in thepresence of IPTG (Fig. 3B). Growth in the presence of CP wasrestored in the tdfHC strain (Fig. 3C), demonstrating that growthin the presence of CP specifically requires TdfH production. Ad-ditionally, the tdfH growth defect was specific to CP treatment, astdfH, tdfHC, and wild-type bacteria all grew similarly when sup-plemented with Zn alone (Fig. 3C).

TdfH enables binding of CP by N. gonorrhoeae. Given theability of N. gonorrhoeae to grow in the presence of CP, we nextsought to determine whether the gonococcus was capable of bind-ing to CP. Whole gonococcal cells, grown in CDM with Fe(NO3)3

and TPEN, were applied to nitrocellulose paper, presenting cellsurface proteins in their native conformation within an intact bac-terial outer membrane. After blocking, membranes were incu-bated with Zn plus CP, before being washed and probed for CP.CP was specifically detected in association with wild-type FA1090,indicating that N. gonorrhoeae is indeed capable of binding CP(Fig. 4A). CP binding was reduced by 80% in both tdfH single andtdfH tdfJ double mutants in the FA1090 background, but bindingwas unaffected in the tdfJ mutant. These findings indicate thatTdfH mediates the gonococcus-CP interaction. Consistent withthe reduced level of TdfH detected in FA19 versus FA1090 whole-cell lysates (Fig. 1B and C), CP binding by FA19 was low and notsignificantly different between wild-type and tdfH mutant bacteria(Fig. 4B). However, CP binding was enhanced in FA19 tdfHC

upon IPTG induction (Fig. 4B). These results demonstrate thatTdfH specifically facilitates the binding of CP to gonococci in bothFA1090 and FA19 strain backgrounds.

TdfH and TonB are important for Zn accumulation in N.gonorrhoeae. Our Zn regulatory and growth data led us to hy-pothesize that TdfH, a TonB-dependent transporter, and TonBwould contribute to Zn internalization by gonococci. While me-ningococcal CbpA was shown to contribute to growth under Znlimitation, Zn accumulation inside N. meningitis cells was notdirectly demonstrated (35). To address whether N. gonorrhoeaeaccumulated intracellular Zn in a TdfH-dependent manner, wegrew the wild-type or tdfH and tonB mutants under Zn-repleteand Zn-depleted conditions and measured the amount of inter-nalized Zn using inductively coupled plasma optical emissionspectrometry (ICP-OES). All of the gonococcal strains tested ac-cumulated significantly more Zn when grown under Zn-repleteversus Zn-depleted conditions (P � 0.05), indicating that thegrowth conditions effectively modulated Zn availability (Fig. 5A).The tdfH and tonB mutants internalized significantly less Zn whengrown under both Zn-replete and Zn-depleted conditions com-pared to the wild type (P � 0.05). Thus, TdfH and TonB contrib-ute to Zn accumulation.

Given the demonstrated interaction between TdfH and CP(Fig. 3 and 4) and the role of TdfH and TonB in Zn accumulation(Fig. 5A), we assessed whether TdfH contributed to Zn accumu-lation in the presence of CP. Wild-type and tdfH mutant bacteriawere grown under Zn-depleted conditions in the presence of Znplus CP, and the level of internalized Zn was assessed via ICP-OES.We found that the tdfH mutant accumulated less Zn than the wildtype in the presence of CP (Fig. 5B). These results show that TdfHenables Zn assimilation from CP.

Calprotectin colocalizes with N. gonorrhoeae in NETs.Gonococcal infection is characterized by a robust recruitment ofneutrophils, which migrate to the site of infection and attempt toeliminate the invading microbes via phagocytosis and/or NET for-mation (17). CP is a major component of the neutrophil cytosoland is released during NET formation. CP remains associated withNETs, where it has been demonstrated to be the active componentin NET-mediated killing of Candida albicans (27). Given our find-ings that TdfH can bind and utilize CP as a Zn source, we hypoth-esized that gonococci can utilize CP in NETs to enhance its sur-vival in association with NETs. To test this hypothesis, we first

FIG 2 Gonococcal growth under Zn-replete and Zn-restricted conditions.Wild-type (WT) (FA19) or tdfH (MCV927), tdfJ (MCV928), and tonB(MCV650) gonococcal strains were grown in GCB treated with Kellogg’ssupplement I, 12 �M Fe(NO3)3, and 12.5 �M TPEN in the presence or absenceof ZnSO4 (18.75 �M) for 6 h before being serially diluted and spot plated onGCB-VCN plates for CFU determination. Means and standard errors from arepresentative of n � 3 independent experiments are plotted. An unpairedStudent’s t test was used to determine significance. *, P � 0.05; **, P � 0.005;n.d., no difference.

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sought to determine if CP is accessible to gonococci in the pres-ence of neutrophils and NETs. Gonococcal opacity (Opa) pro-teins, which are subject to phase variation, have been shown todifferentially affect gonococcal survival in association with neu-trophils (16). Therefore, all neutrophil experiments were con-ducted in the FA1090 Opa-deficient (Opaless) background (43).Primary human neutrophils seeded onto chambered cover glassesand induced to form NETs with phorbol myristate acetate (PMA)(53) were stained with antibodies against CP and gonococci. CPdemonstrated a punctate staining pattern throughout the NETsand was detected in proximity to gonococci in NETs (Fig. 6). Thisobservation indicates that CP is accessible to N. gonorrhoeae inNETs.

TdfH contributes to gonococcal NET survival. We nextsought to determine if gonococcal CP utilization, as mediated by

TdfH, conferred a bacterial survival advantage in the presence ofNETs. We constructed a tdfH mutant (MCV955) in the FA1090Opaless background, demonstrated that this strain produced noTdfH (Fig. 7A), and then assessed this mutant’s survival in NETscompared to that of the Opaless parent. Primary human neutro-phils were treated with PMA and cytochalasin D to activate neu-trophils and stimulate NET production while inhibiting phagocy-tosis, thereby selecting for only extracellular mechanisms ofneutrophil killing. DNase I was added under some conditions toassess the role of intact DNA fibers on NET-mediated killing.Neutrophils were then infected with the Opaless parent and iso-genic tdfH mutant and tdfHC complement. The Opaless �nucstrain was used as a positive control for DNase I-reversible sensi-tivity to NET-mediated killing (21). We found that while the pa-rental strain demonstrated little to no survival defect in the pres-

FIG 3 Gonococcal growth in the presence of calprotectin requires tdfH. (A) Gonococcal strains were grown under Zn-limited conditions (CDM with 1 �MTPEN) and further supplemented with a CP-Zn mixture (10 �M CP and 5 �M ZnSO4) or left untreated. Growth was monitored by OD600 readings every 2 h for8 h. Plotted are the means and standard deviations of the means from n � 3 independent experiments. An unpaired Student’s t test was used to determinesignificance at the 8-h time point. *, P � 0.05; **, P � 0.005. (B) Gonococcal strains FA19, MCV927 (tdfH), and MCV956 (tdfHC) were grown in CDMsupplemented with 24 �M Fe (NO3)3 � 2 mM IPTG. Whole-cell lysates of equal culture density were subjected to SDS-PAGE analysis followed by immuno-blotting with antiserum specific to TdfH. (C) Gonococcal strains were grown under Zn-limited conditions and further supplemented with CP plus Zn or 5 �MZn alone or were left untreated. Growth was monitored by OD600 readings every 2 h for 8 h. Plotted are the means and standard deviations of the means from n �3 independent experiments. An unpaired Student’s t test was used to determine significance at the 8-h time point. *, P � 0.05; **, P � 0.005.

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ence of NETs, with or without DNase I, only 40% of tdfH mutantbacteria survived exposure to neutrophils producing NETs (Fig.7B). The defect in survival exhibited by the tdfH mutant was re-versed by complementation (Fig. 7B). Additionally, treatmentwith DNase I significantly increased survival of the tdfH mutant,indicating that intact DNA fibers contribute to the observed NETsensitivity of the tdfH mutant. Thus, TdfH is critical for optimalgonococcal resistance to NET-mediated killing.

Zn supplementation abrogates sensitivity of tdfH to NET-mediated killing. Given our in vitro data that TdfH binds to andcan utilize Zn from calprotectin, we postulated that TdfH facili-tates gonococcal survival in NETs by overcoming CP-mediatedZn chelation. In support of this hypothesis, addition of Zn suc-cessfully restored survival of the tdfH mutant to wild-type levelsafter exposure to NETs (Fig. 7C). We conclude that NETs can killN. gonorrhoeae by sequestering the essential metal Zn, and TdfHenables gonococcal survival in the presence of neutrophils andNETs by overcoming CP-mediated Zn chelation.

DISCUSSION

Nutritional immunity, the host strategy of limiting essential nu-trients from invading pathogens, is a prevalent theme in host de-fense against microbes. While traditionally characterized as thesequestration of iron from pathogens, roles for other transitionmetals such as Zn and Mn are now being examined in the strugglefor nutrients within the context of infection (22, 23). N. gonor-rhoeae is particularly adept at overcoming host nutritional immu-nity strategies. Despite being unable to produce any siderophoresto scavenge iron or iron chelates from the iron-limited host envi-ronment, N. gonorrhoeae cells express TonB-dependent trans-porters that enable utilization of siderophores produced by otherbacteria as well as the host iron binding proteins transferrin andlactoferrin and the heme-binding protein hemoglobin (13, 54).Given the role of the transferrin and lactoferrin receptors in gono-coccal pathogenesis (14), we propose that the remaining TdTs,

FIG 4 Calprotectin binding to whole gonococcal cells. Gonococcal strains were grown in CDM supplemented with 24 �M Fe(NO3)3 for 4 h before equal culturedensities of bacteria were applied to nitrocellulose paper. Membranes were blocked and then incubated with CP. After washing, membranes were subjected toimmunoblotting using CP-specific antiserum. Densitometry was performed on replicate blots using NIH ImageJ. Signal intensities were normalized to that of thewild type and produced as a percentage of CP binding. Paired Student’s t test was used to compare the percentage of CP binding; P � 0.05 was consideredsignificant and is designated by an asterisk. (A) Representative dot blot with gonococcal strains FA1090 (wild type), MCV661 (tdfH), MCV662 (tdfJ), andMCV936 (tdfH tdfJ) applied to nitrocellulose before CP incubation and detection. Below, densitometry results from n � 3 replicate blots are shown. (B)Representative dot blot with gonococcal strains FA19 (wild type), MCV927 (tdfH), and MCV956 (tdfHC) applied to nitrocellulose. TdfH production inthe tdfHC strain was uninduced (UI) or induced (I) by the addition of IPTG (2 mM). Densitometry results from n � 3 replicate blots are shown below thedot blot.

FIG 5 TdfH and TonB contribute to gonococcal Zn accumulation. Gonococ-cal strains were grown in Zn-replete or -depleted medium before bacterial cellswere harvested and analyzed for metal levels via ICP-OES. Means and standarderrors from a representative of n � 3 independent experiments are plotted. Anunpaired Student’s t test was used to determine significance. *, P � 0.05; **,P � 0.005. Trends in statistical significance were consistent between biologicalreplicates. (A) Strains FA19 (WT), MCV927 (tdfH), and MCV650 (tonB) wereanalyzed for Zn levels. (B) Strains FA19 and MCV927 (tdfH) were grown inZn-depleted medium supplemented with CP/Zn (2 �M CP, 1 �M Zn) beforebacterial cells were harvested for metal analysis via ICP-OES. Means and stan-dard errors from a representative of n � 3 independent experiments areplotted.

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which have yet to be fully characterized, also have important rolesin the ability of N. gonorrhoeae to survive in its human host andcause disease. Moreover, these transporters also have the potentialto be manipulated for the development of anti-infective therapies.

In this report, we define for the first time the strategy used by N.gonorrhoeae to overcome nutritional immunity posed by Zn re-striction in the host. We demonstrate that N. gonorrhoeae uses theTdT TdfH to overcome growth restriction by the host Zn-chelat-ing immune effector protein CP. TdfH directly facilitates bacterialbinding of CP binding and growth in its presence. Due to its se-quence similarity to heme receptors, TdfH was initially thought tocontribute to heme acquisition; as such it is designated Hup, forheme utilization protein, in neisserial genome annotations. How-ever, a gonococcal mutant unable to express TdfH was not defec-tive for growth under heme-limiting conditions (45), placing indoubt TdfH’s role in heme utilization. Additionally, productionof TdfH along with the neisserial TonB system could not restoreheme utilization in an E. coli hemA mutant (45), as has been shownfor other heme receptors (55–57). Instead, our studies demon-strate that gonococcal TdfH binds to CP and enables Zn acquisi-tion. These findings are consistent with a report that CbpA, themeningococcal TdfH homologue, is required for N. meningitidisgrowth in the presence of CP and for CP binding (35). The presentreport extends these findings to N. gonorrhoeae and furthermoredemonstrates that TdfH contributes to intracellular Zn accumu-lation in the presence and absence of CP.

Consistent with its role in nutritional immunity, CP inhibitsthe growth of numerous microbial pathogens, including Candidaalbicans (58), Staphylococcus aureus (26), Helicobacter pylori (28),Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococ-cus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa,Escherichia coli, and Shigella flexneri (52) due to its Zn/Mn-chelat-ing activity. To overcome this growth restriction, pathogens, in-cluding Aspergillus fumigatus (59), Acinetobacter baumannii (29),Pseudomonas aeruginosa (60), and Salmonella enterica serovar Ty-phimurium (33), use Zn acquisition systems to promote resis-tance to CP. For Gram-negative bacteria, our understanding ofthe bacterial factors contributing to CP resistance have mostly

been limited to the high-affinity Zn uptake system ZnuABC, amember of the ATP-binding cassette-type transporters located atthe inner membrane (29, 32, 60). However, the mechanism of Znentry through the outer membrane has yet to be fully resolved,although TonB-dependent transporters such as ZnuD in N. men-ingitidis have been implicated (34). Our finding that TdfH notonly binds CP but is also required for Zn accumulation from CPidentifies a bacterial surface protein that can directly interact withCP to overcome CP-mediated Zn sequestration.

We found here that gonococcal TdfH is Zn and Zur repressed.In agreement with previous reports (45, 61), we confirmed thatproduction of TdfH is independent of iron availability, consistentwith a function distinct from iron acquisition. Expression of me-ningococcal tdfH was found to be increased under low-Zn condi-tions in microarray studies (62), and production of the homolo-gous protein, CbpA, was also increased in a zur mutant derivative(35). Gonococcal tdfH was found to be differentially expressed inmicroarray studies of PerR, which is described as a Mn-dependentrepressor (63). However, gonococcal and meningococcal Zurshare 96% protein identity, and the gonococcal PerR and menin-gococcal Zn-responsive regulons overlap significantly. Moreover,TdfH production was not altered by Mn availability (data notshown). Our data thus support the contention that gonococcalPerR functions more like Zur, responding to Zn as opposed to Mn(64), and agree with our finding that the Zn acquisition proteinTdfH is regulated by Zn in a Zur-dependent manner.

TdfH has been reported to be a member of the regulon of thetwo-component regulatory system MisRS in N. meningitidis (49).A mutant derivative unable to express the response regulator,MisR, had reduced levels of tdfH in microarray studies, confirmedby quantitative reverse transcription (RT)-PCR (49). CbpA pro-duction was also reduced in a meningococcal �misRS mutant(35). Our data demonstrate that while TdfH production was un-affected by the presence of MisR in N. gonorrhoeae strain FA1090,it was reduced in the misR mutant in gonococcal strain FA19,indicating that TdfH regulation is distinct in these two gonococcalstrains. We also report an increase in basal levels of TdfH produc-tion in strain FA1090 versus FA19, which is retained in both zur

FIG 6 Gonococci colocalize with calprotectin in NETs from primary human neutrophils. Shown are representative confocal microscopy images of PMA-treatedprimary human neutrophils exposed to Opaless parent gonococci (Gc). Samples were fixed and stained with antibodies against N. gonorrhoeae (red) andcalprotectin (blue) and Sytox green to visualize DNA. Individual panels from each fluorophore are shown as both gray scale and color images for clarity. Themerged image shows calprotectin closely associated with DNA in NETs and colocalized (yellow) with gonococcal microcolonies.

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and misR mutants, indicating that the different regulation pat-terns between strains are independent of the transcriptional reg-ulators Zur and MisR. This is the first study to report that TdfHproduction is distinct in different gonococcal strains and may alsobe controlled by additional factors beyond transcriptional regula-tors Zur and MisR. However, despite the differences in TdfH pro-duction, both the FA1090 (data not shown) and FA19 gonococcalstrains demonstrated TdfH-dependent CP resistance.

Our novel observation that TdfH contributes to gonococcalsurvival within NETs is yet another example of the many ways inwhich N. gonorrhoeae manipulates host-pathogen interactions toits advantage (9, 16). NETs are comprised of DNA and histones, aswell as neutrophil granule and cytosolic proteins. CP has beendetected as a major component of NETs (27) (Fig. 6). NET-medi-ated killing is thought to occur by trapping microbes in chromatinfibers in a microenvironment with high levels of antimicrobialproteins and molecules like CP and cathepsin G (53). DNase treat-ment degrades NETs and destroys the NET microenvironmentand can thus abrogate NET-mediated killing of bacteria, includingNeisseria spp. (21, 53, 65). CP, through its Zn-chelating activity,was found to be the major antimicrobial component in NETsagainst Candida albicans, Klebsiella pneumoniae, and Aspergillusnidulans (27, 30, 66). Antimicrobial metal chelation in NETs canalso be mediated by DNA itself (67). As such, a mechanism tooutcompete or overcome host nutrient sequestration strategiesprovides a significant survival advantage in NETs. Indeed, in N.meningitidis, the Zn outer membrane transporter ZnuD wasfound to contribute to survival within NETs (65). The data re-ported in this study demonstrate that CP is in close proximity andtherefore accessible to gonococci in NETs. Additionally, this studyis the first to demonstrate that TdfH is critical for N. gonorrhoeaesurvival of NET-mediated killing, where CP has been shown to bea key antimicrobial component (27, 30). We also show that Znsupplementation abrogates sensitivity of the tdfH mutant toNETs, demonstrating that Zn sequestration is a critical compo-nent of NET-mediated killing and that TdfH contributes to over-coming Zn sequestration in neutrophil NETs. Our findings rep-resent a novel gonococcal neutrophil resistance strategy, whereinTdfH assimilates Zn from CP, neutralizing its antimicrobial activ-ity, to contribute to survival in NETs.

This report describes TdfH as a TonB-dependent transporterthat mediates CP binding and Zn internalization. While at thistime we do not know whether TdfH preferentially binds to Zn-laden CP as opposed to apo-CP, the data presented in this reportclearly demonstrate that TdfH enables Zn transport into gono-cocci whether CP is present or not, suggesting that both TdfH andTdfJ have the capacity to import Zn that is not chelated to protein.It is also currently unclear why the tonB mutant is not impairedmore than either of the single tdfH or tdfJ mutants for growth withor internalization of unchelated Zn. However, TonB-dependentfunctions in N. gonorrhoeae have to date been exclusive to protein-bound metals such as transferrin and lactoferrin, whereas sidero-phore-iron internalization can be Ton independent, dependingon the strain tested (39). The data presented here suggest that Zninternalization by the gonococcus occurs in both a TonB-depen-dent manner and a TonB-independent manner. The growth ex-periments presented here also suggest that growth stimulationwith unchelated Zn is more efficient at employing the TdfJ path-way and that TonB-dependent growth with unchelated Zn is pri-marily accomplished by TdfJ. Explanations for these observationscould be related to relative affinities of TdfH and TdfJ for Zn ortheir respective copy numbers in the gonococcal outer membrane.

We propose that TdfH is an attractive potential vaccine targetfor several reasons. First, TdfH is surface exposed and not subjectto phase variation. Second, it is produced by most pathogenicNeisseria strains. TdfH production has been detected in all menin-gococcal strains tested (35, 45) and 81% of gonococcal strains(45), but only a single isolate of Neisseria lactamica (45), a com-

FIG 7 TdfH enhances bacterial survival in NETs. (A) Immunoblot of TdfHproduction in the Opaless (opa-) parent strain, MCV661 (tdfH), and MCV955(Opaless tdfH). (B) Human neutrophils were stimulated to induce NET for-mation and infected with the Opaless, MCV955 (tdfH), MCV956A (tdfHC), or�nuc strain at an MOI of 1 in the presence of cytochalasin D � DNase I for 1h. Bacterial survival is produced as a percentage of the initial inocula. Meansand standard errors from n � 6 independent experiments are shown. Signifi-cance was determined by an unpaired Student’s t test. *, P � 0.05; **, P �0.005. (C) PMA- and cytochalasin D-treated human neutrophils were infectedwith the Opaless and MCV955 (tdfH) strains at an MOI of 1 in the presence orabsence of 0.5 �M ZnSO4 for 1 h. Bacterial survival is produced as a percent-age of initial inocula. Means and standard errors from n � 3 independentexperiments are shown. Significance was determined by paired Student’s ttest. **, P � 0.005; n.d., no difference.

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mensal that can occasionally cause infection (54). Third, it exhib-its a high degree of sequence conservation among gonococcalstrains. Fourth, TdfH is produced by gonococci recovered fromwomen with lower genital tract infection (58). Finally, it is likely tobe important for gonococcal survival in vivo by mediating acqui-sition of Zn via CP binding.

In summary, this study exposes a novel gonococcal strategy,centered on production of TdfH, to evade CP-mediated nutri-tional immunity and thereby contribute to survival in NETs.Given the role we demonstrate for TdfH in gonococcal growthand survival and its potential as a vaccine candidate, we anticipatefuture studies will fully discern the contributions of TdfH in N.gonorrhoeae pathogenesis and infection of its obligate humanhost.

ACKNOWLEDGMENTS

We thank Alastair G. McEwan and Karrera Djoko for the zur::kan mutantconstruct used to generate the FA1090 and FA19 zur mutants. We are alsograteful to William M. Shafer for providing the FA1090 misR and FA19misR strains. We also thank Eric P. Skaar, Walter J. Chazin, and BenjaminGilston for providing the purified, heterodimeric calprotectin used in thisstudy. We thank Frederick Sparling and James Anderson for providing theTdfH-specific antibody. Finally, we thank Joseph Turner for assistancewith ICP-OES and Donna Woodburn who helped with initial regulationstudies.

The funders of this study had no role in study design, data collectionand interpretation, or the decision to submit the work for publication.

FUNDING INFORMATIONThis work, including the efforts of Cynthia Nau Cornelissen, was fundedby HHS | National Institutes of Health (NIH) (R01 AI047141, R21AI065555, R01 AI084400, and U19 AI31496). This work, including theefforts of Alison K. Criss, was funded by HHS | National Institutes ofHealth (NIH) (R01 AI097312 and R21 AI110889). This work, includingthe efforts of Richard A. Juneau, was funded by HHS | National Institutesof Health (NIH) (R01 AI097312-S1). This work, including the efforts ofSophonie Jean, was funded by American Society for Microbiology (ASM)(Robert D. Watkins fellowship).

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

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