Inactivation of NMB0419, Encoding aSel1-Like Repeat (SLR) Protein, inNeisseria meningitidis Is Associated withDifferential Expression of GenesBelonging to the Fur Regulon andReduced Intraepithelial Replication

Ming-Shi Li, Paul R. Langford, J. Simon KrollSection of Paediatrics, Department of Medicine, Imperial College London, London, United Kingdom

ABSTRACT Neisseria meningitidis is a commensal microbe that colonizes the humannasopharynx but occasionally invades the bloodstream to cause life-threateninginfection. N. meningitidis MC58 NMB0419 encodes a Sel1-like repeat (SLR)-containing protein, previously implicated in invasion of epithelial cells. A gene-regulatory function was revealed in Escherichia coli expressing plasmid-borneNMB0419 and showing significantly increased epithelial adherence compared tothe wild type, due to increased expression of mannose-sensitive type 1 pili.While a meningococcal NMB0419 mutant did not have altered epithelial adherence,in a transcriptome-wide comparison of the wild type and an NMB0419 mutant, alarge proportion of genes differentially regulated in the mutant were involved iniron acquisition and metabolism. Fifty-one percent and 38% of genes, respectively,up- and downregulated in the NMB0419 mutant had previously been identified asbeing induced and repressed by meningococcal Fur. An in vitro growth defect of theNMB0419 mutant under iron restriction was consistent with the downregulation oftbpAB and hmbR, while an intraepithelial replication defect was consistent with thedownregulation of tonB, exbB, and exbD, based on a known phenotype of a menin-gococcal tonB mutant. Disruption of the N-terminal NMB0419 signal peptide, pre-dicted to export the protein beyond the cytoplasmic membrane, resulted in loss offunctional traits in N. meningitidis and E. coli. Our study indicates that the expressionof NMB0419 is associated with transcriptional changes counterbalancing the regula-tory function of Fur, offering a new perspective on regulatory mechanisms involvedin meningococcal interaction with epithelial cells, and suggests new insights into theroles of SLR-containing genes in other bacteria.

KEYWORDS Fur regulon, NMB0419, Neisseria meningitidis, Sel1-like repeat, epithelialcells, intracellular replication, iron acquisition, transcriptome

Neisseria meningitidis is a human commensal bacterium that colonizes the nasopha-ryngeal mucosa of around 10% of healthy individuals, from where it occasionally

invades the bloodstream to cause life-threatening infections such as septicemia andmeningitis (1). As well as colonizing the mucosal surface superficially, meningococcisurvive and replicate within epithelial cells, establishing a basis for invasion of deepertissues to cause disease (2) and prolonged persistence on the nasopharyngeal mucosaas microbes exocytose to the surface again (3).

An important factor in meningococcal survival and replication in the human host isthe ability of the microbe to assimilate iron in the face of avid sequestration by the hostof all available iron in iron binding proteins such as transferrin, lactoferrin, and

Received 20 July 2016 Returned formodification 21 August 2016 Accepted 28February 2017

Accepted manuscript posted online 6March 2017

Citation Li M-S, Langford PR, Kroll JS. 2017.Inactivation of NMB0419, encoding a Sel1-likerepeat (SLR) protein, in Neisseria meningitidisis associated with differential expression ofgenes belonging to the Fur regulon andreduced intraepithelial replication. InfectImmun 85:e00574-16. https://doi.org/10.1128/IAI.00574-16.

Editor Craig R. Roy, Yale University School ofMedicine

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Paul R. Langford,p.langford@imperial.ac.uk, or J. Simon Kroll,s.kroll@imperial.ac.uk.

MOLECULAR PATHOGENESIS

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hemoglobin (4). In blood or other extracellular environments, iron acquisition by themeningococcus is achieved through surface expression of receptors such as TbpAB(transferrin binding proteins), LbpAB (lactoferrin binding proteins), and HmbR (hemo-globin binding protein), which interact with and mediate extraction of iron from theircognate host iron binding proteins. Within epithelial cells, iron is mostly stored inferritin, accessed by intracellular meningococci through ferritin degradation into low-molecular-weight iron complexes, which are then utilized via a TonB-dependent re-ceptor of unknown identity (5, 6). The expression of genes encoding all known ironreceptors is regulated by the ferric uptake regulator (Fur) (7), a multifunctional proteinalso regulating the expression of genes involved in energy metabolism, electrontransfer, cytochrome metabolism, stress responses, and production of secreted proteinsrelated to the RTX family of cytotoxins. The Fur regulon has been studied in N.meningitidis using DNA microarrays (8, 9).

In relation to microbial iron uptake, Fur typically acts as a repressor of iron receptorgenes to control the level of intracellular iron and to avoid cell damage mediated byreactive oxygen species. In an iron-restricted rather than a replete environment, astypically encountered by the meningococcus in vivo, the expression of these receptorsis increased (4, 10). However, there is very little knowledge about microbial cellularcomponents involved in the positive regulation of these iron receptors.

NMB0419 encodes a Sel1-like repeat (SLR)-containing protein located within agenomic locus of 18 open reading frames termed the division cell wall (dcw) cluster,involved in bacterial division and cell wall synthesis (11, 12). A functional promoter hasbeen located immediately upstream of NMB0419 (12). Sel1 (suppressor-enhancer of lin)was originally identified in Caenorhabditis elegans as a negative regulator of lin-12,encoding a membrane protein involved in the specification of cell fates during devel-opment (13). SLR proteins belong to the solenoid protein family and apparently playdiverse biological roles as components of macromolecular complexes. SLR proteinshave been described in the human pathogens Helicobacter pylori, Legionella pneumo-phila, and Pseudomonas aeruginosa as important for virulence (reviewed in reference14) but remain largely uncharacterized in Neisseria.

In previous work, we established that NMB0419 was involved— by an unknownmechanism—in meningococcal interaction with epithelial cells (15). In a study de-signed to investigate the involvement of the product of NMB0419 in this phenotype, weinitiated experiments with Escherichia coli expressing recombinant meningococcalNMB0419 (for which there is no E. coli homologue). We found, unexpectedly, that theresult was upregulation of genes involved in biosynthesis of type 1 pilus (T1P) (anadhesin for which there is no homologue in N. meningitidis). Thus, NMB0419 wasassociated with transcriptional changes in E. coli, rather than acting as an adhesin. Wehave gone on to compare the transcriptomes of the wild type (WT) and an NMB0419-knockout meningococcus, which revealed that the SLR-containing protein functions asa counterbalance to Fur in regulation of genes of the Fur regulon.

RESULTSExpression of NMB0419 in E. coli was associated with transcriptional changes,

including increased expression of genes for T1P biogenesis. Expression of NMB0419was detected in E. coli DH5� transformed with recombinant plasmid pET0419 but notin DH5�(pET) alone (see Fig. S1 in the supplemental material). DH5�(pET0419) had anincreased ability to adhere to and invade human epithelial cells, compared toDH5�(pET) (adherent, 14.3% versus 2.1%, with P � 0.019, and intracellular, 0.004%versus 0.0007%, with P � 0.025) (Fig. 1A and B). Transcriptome analysis showed a setof genes being differentially regulated in DH5�(pET0419) in comparison withDH5�(pET), and among the top 15 upregulated genes, eight were constituents of thefim operon encoding the T1P (Table S1). Reverse transcription-PCR (RT-PCR) confirmedupregulation of fimA (encoding the major pilin) and fimC (encoding a minor pilin) inDH5�(pET0419) (Fig. S2). Light microscopy showed a pattern of aggregative adherence(AA) (16) for strain DH5�(pET0419) on epithelial cells (Fig. S3), consistent with T1P

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expression being a major contributor to AA (17). Transmission electron microscopy(TEM) showed numerous T1P-like structures (18) on the surface of DH5�(pET0419) (Fig.S4), which were rarely seen on DH5�(pET) (data not shown). The addition of D-mannosewas shown to reduce DH5�(pET0419) adherence to epithelial cells to 0.29% in com-parison with the control (12.5%, without mannose) (Fig. 1C), confirming the role of T1P(19). The results showed that expression of NMB0419 in E. coli was associated withincreased expression in genes for T1P biogenesis, which resulted in increased adher-ence to and invasion of epithelial cells.

T4P in the meningococcal NMB0419 mutant are expressed at WT level. Menin-gococci express type 4 pili (T4P) rather than T1P, the major pilin subunit being encodedby pilE (NMB0018) (11). We compared transcription of pilE in the NMB0419 mutant andthat in WT MC58 using quantitative RT-PCR (qRT-PCR). Expression of pilE was notsignificantly different between the strains (P � 0.83), with an expression ratio of 1.19(mutant/WT) (Fig. S5). TEM inspection of WT and mutant cells revealed that T4P were

FIG 1 Increased epithelial adherence and internalization of E. coli due to the expression of NMB0419 andT1P. (A and B) Adherent (A) and internalized (B) CFU of DH5�(pET) and DH5�(pET0419) recovered at 4h post-infection of cultured 16HBE14 cells is expressed as percentage of total bacteria. (C) Adherence ofDH5�(pET0419) and DH5�(pET) was tested with (�) or without (-) mannose in the growth medium. Errorbars indicate standard errors of the means of three biological replicates. Asterisks indicate CFU with asignificant difference (P � 0.025) between the corresponding strains.

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expressed on the surface very similarly in each, in the form of single and bundled pili(Fig. S6).

Many genes differentially regulated in the NMB0419 mutant belong to thepreviously identified meningococcal Fur regulon. The transcriptional profiles of theNMB0419 mutant and its parental WT MC58 were compared using RNA sequencing.Forty-four and 47 genes (comprising 1.8% and 1.9% of the genome), respectively, wereup- and downregulated in the NMB0419 mutant compared to WT using a 1.5-foldchange (log2 ratio of 0.5849) and false discovery rate (FDR) of �0.01 as the cutoff in allthree pairs of mutant-WT comparisons. Most of the genes encoding productsknown to be important for iron uptake were downregulated in the NMB0419mutant. These were tbpA (NMB0461) and tbpB (NMB0460), encoding transferrinbinding proteins; lbpA (NMB1540) and lbpB (NMB1541), encoding lactoferrin bindingproteins; hmbR (NMB1668), encoding hemoglobin binding protein; frpB (also fetA,NMB1988), encoding a siderophore receptor; and exbB (NMB1729), exbD (NMB1728), andtonB (NMB1730), involved in providing energy for these receptors. In contrast, bfrA(NMB1207), encoding the bacterial iron storage protein bacterioferritin, was upregu-lated in the NMB0419 mutant (Fig. 2).

We compared our list of differentially regulated genes with those previously de-scribed as being regulated by Fur in meningococci (9). Twenty-three out of 44 genes(52%) upregulated in the NMB0419 mutant were coidentified as being upregulated(induced) by Fur (9). These included genes involved in energy metabolism and electrontransfer, including 19 genes of the entire nuo operon (NMB0241 to NMB0259) and 3genes of the pet operon (NMB2051 to NMB2053), as well as the above-mentioned bfrA(Table S2). Eighteen of 47 genes (38%) downregulated in the NMB0419 mutant werecoidentified as being downregulated (repressed) by Fur (Fig. 3). These included theabove-mentioned iron receptor genes, those encoding hypothetical iron receptors

FIG 2 Differential expression of genes encoding iron uptake and storage in the NMB0419 mutantcompared with WT MC58 measured by qRT-PCR. The average ratio from three biological comparisons foreach gene is expressed as log2 fold change. Error bars indicate standard errors of the means from threebiological replicates.

FIG 3 Venn diagram showing numbers of differentially regulated genes coidentified in the NMB0419mutant and in the previously reported Fur regulon (9).

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[such as NMB0034 and NMB0035 (efeO)], and frpA and frpC encoding RTX-like secretedcytotoxins (Table S3). In addition to the coidentified genes, NMB1989 to NMB1991(encoding a hypothetical iron ABC transporter system) were significantly downregu-lated in the NMB0419 mutant (Table S3).

The results indicate an apparent association between the genes differentially reg-ulated in the NMB0419 mutant and those previously identified as being part of the Furregulon (9). Relevant experiments were carried out to ascertain whether the transcrip-tional changes in the NMB0419 mutant would result in corresponding phenotypicchanges.

The NMB0419 mutant is deficient in utilizing transferrin and hemoglobin underiron-restricted conditions, and the signal peptide domain is important for thefunction of NMB0419. Fetal bovine serum (FBS) (providing 15 �M hemoglobin)containing additional deferoxamine mesylate (Desferal) (100 �M) was able to supportthe growth of WT MC58 and MC58 (ΔNMB0419::Km) (pMIDG0419) but not the growthof the MC58 (ΔNMB0419::Km) and MC58 (ΔNMB0419::Km) (pMIDG0419ΔSIGL) mutants(Fig. 4A). Further growth tests using the same agar plates with iron supplementation onfilter discs showed that the NMB0419 mutant was able to grow around a filter discloaded with high concentrations of hemoglobin (1.25 mM) or iron(III) chloride (100 mM)but not with apo- or holotransferrin (Fig. 4B). These results provided phenotypicvalidation that the level of downregulation achieved of genes involved in iron uptake(tbpAB and hmbR) impaired the ability of the NMB0419 mutant to utilize transferrin andhemoglobin for growth.

Functional characterization using MC58 (ΔNMB0419::Km) (pMIDG0419ΔSIGL) indi-cated that NMB0419 is secreted and acts beyond the bacterial inner membrane

FIG 4 Growth of N. meningitidis on BHI agar containing Desferal and 10% FBS. (A) From each menin-gococcal strain as indicated, 106 CFU was streaked out on agar plates for overnight growth. (B) Growthof the NMB0419 mutant around discs impregnated with iron chloride or hemoglobin but no growth withapotransferrin or holotransferrin.

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(Fig. 4A). Exploiting the effect of NMB0419 on epithelial adherence of E. coli as areporter system, strains bearing pET0419ΔSIGL, a recombinant plasmid carryingNMB0419 with an in-frame deletion of the signal peptide coding sequence,DH5�(pET0419ΔSIGL) and DH5�(pET) (negative control) showed similar levels of ad-herence to 16HBE14 cells, at 3.0% and 2.3%, respectively, while DH5�(pET0419) showeda significantly higher level of adherence (8.1%) (P � 0.014) (Fig. S7). These results showthat the signal peptide is required for function both in N. meningitidis MC58 and in E.coli.

Reduced intraepithelial replication by the NMB0419 mutant. Meningococcalmutants with mutations in tonB, but not in hmbR or fbpA, replicate inside epithelial cellsmuch more slowly than WT strains (5). We explored intraepithelial meningococcalreplication using the COR-L23 respiratory epithelial cell line, in which we have previ-ously shown that the NMB0419 mutant is less invasive than WT MC58. After an initial24-hour infection period, gentamicin killing was performed to remove extracellularmeningococci. The intracellular meningococci within COR-L23 respiratory cells wereallowed to grow for another 4 h under different conditions (iron-replete and restrictedmedium), at which point the intracellular CFU count was assessed. With incubation inRPMI 1640 medium, the NMB0419 mutant replicated less efficiently in COR-L23 cellsthan WT MC58 (to 45% of WT level, P � 0.0042). When an iron-restricted growthcondition was imposed by addition of Desferal to the external medium, the replicationefficiency of the NMB0419 mutant was further reduced (to 25% of WT level, P � 0.0089).However, when free iron [iron(III) chloride] was added to the growth medium, thereplication efficiency of the NMB0419 mutant was comparable to that of WT (P � 0.9)(Fig. 5).

Homologues of NMB0419 with differing numbers of SLR domains are foundthroughout the genus Neisseria. By PCR and sequencing using NMB0419-specificprimers, NMB0419 was shown to be widespread in the genus Neisseria (Fig. 6). NMB0419homologues were found in every N. meningitidis strain examined (n � 59), includingstrains of serogroups A (n � 3), B (n � 42), and C (n � 10) (Table 1) and of serogroupsW, X, Y, and Z (four and one each, respectively; see Table S4) as well as in 28/28 Neisserialactamica strains (Table 1) and 2/2 Neisseria gonorrhoeae strains (see Table S4). Thenumber of SLR domains encoded by different homologues varied strikingly, from 1 to11 (Table 1). The NMB0419 homologues from all 59 N. meningitidis strains encoded 1 to8 SLR domains, whereas the NMB0419 homologues from most (23/28) N. lactamicastrains encoded 9 to 11 SLR domains. This variation mirrors the situation in H. pylori(reviewed in reference 14) and Haemophilus influenzae (20). The functional conse-quences are unknown but will be the subject of further study. It is intriguing that thehighest numbers of SLRs (�9) are in genes within commensal N. lactamica compared

FIG 5 Replication efficiency of intraepithelial (COR-L23) meningococci under various medium conditions.The intracellular meningococcal CFU taken at the 4-h point after gentamicin killing was compared withthe CFU taken immediately after gentamicin killing to give a ratio. The ratio obtained for the NMB0419mutant (NMB0419) was further normalized to the ratio obtained for the MC58 (WT) under each conditionexpressed as percentage of WT replication. Medium conditions: RPMI, RPMI 1640; Desferal�, addition ofDesferal; Iron�, addition of iron chloride to RPMI. Asterisks indicate CFU with a significant difference (P �0.009) between the WT and NMB0419 mutant.

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to the lower numbers within pathogenic N. meningitidis. In addition, we interrogatedthe information available at EMBL (http://smart.embl-heidelberg.de), which showedthat higher numbers of SLR domains were common among SLR proteins encoded onthe genomes of other commensal Neisseria spp. (N. bacilliformis, N. cinerea, N. polysac-charea, N. sicca, and N. subflava) (Table S5).

DISCUSSION

In a previous study, we have found the product of NMB0419 of N. meningitidis MC58to be involved in meningococcal interaction with epithelial cells (15), although it wasnot clear whether its functional role was as an adhesin, invasin, or other component. Inthe present study, we started our investigation characterizing the effect of transforma-tion of the nonadhesive and noninvasive E. coli strain DH5� with the recombinantexpression plasmid pET0419. DH5�(pET0419) was significantly increased in its ability toadhere to epithelial cells, through upregulation of genes involved in T1P biosynthesisand regulation. The adherence of DH5�(pET0419) to the monolayer of epithelial cellsformed a unique AA pattern on epithelial cells, which has been associated with theexpression of T1P (17). The expression of T1P on the surface of DH5�(pET0419) wasfurther confirmed using electron microscopy. The importance of T1P in epithelialadherence by DH5�(pET0419) was also confirmed using mannose competition assays.NMB0419 is predicted to contain an N-terminal signal peptide followed by four SLRdomains. Using E. coli strain DH5�(pET0419SIGL) with an in-frame deletion of the signal

FIG 6 Representative PCR products of NMB0419 homologues amplified from genomic DNA of differentN. meningitidis strains detected by gel electrophoresis. The corresponding number of SLR domainsencoded by the strain-specific NMB0419 homologue (confirmed by sequencing) is indicated on top ofeach sample lane. Right lane, lambda DNA/HindIII marker (Thermo Fisher).

TABLE 1 Number of SLRs encoded by NMB0419 homologues from different N.meningitidis and N. lactamica strains

No. of SLRs

No. of strains

N. meningitidis serogroup:

N. lactamicaA B C

1 2 21 22 1 10 2 14 10 6 27 18 1 19 410 711 12

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peptide coding sequence in pET0419, we showed that the signal peptide was necessaryfor NMB0419 to modulate epithelial adherence, indicating that its functional location isbeyond the cytoplasmic membrane. PSORTb 3.0.2 (www.psort.org/psortb/) and SignalP4.1 (www.cbs.dtu.dk/services/SignalP/) predict that NMB0419 is exported beyond thecytoplasmic membrane and is extracellular, respectively. There is no prediction of amembrane location.

N. meningitidis strains express T4P rather than T1P, and in contrast to the situationdescribed in E. coli with T1P, we could show no influence of NMB0419 on pilus-mediated epithelial adherence. The meningococcal major pilin gene pilE was expressedat similar levels, as determined by qRT-PCR, in the WT and NMB0419 mutant, and TEMshowed normal surface expression of T4P by the NMB0419 mutant.

Genome-wide RNA analysis demonstrated that, compared with WT, many genesencoding receptors involved in iron acquisition were downregulated in the meningo-coccal NMB0419 mutant. Since these genes are known to be regulated by Fur, wecompared our list of differentially regulated genes with those previously identified aspart of the meningococcal Fur regulon (9) and found a high degree of coidentificationof genes downregulated in the NMB0419 mutant being repressed by Fur (38%) andgenes upregulated in the NMB0419 mutant being induced by Fur (51%). The resultssuggest that the expression of NMB0419 is associated with the upregulation of a set ofgenes negatively regulated by Fur and the downregulation of a set of genes positivelyregulated by Fur. The inference is that the biological role of NMB0419 expression is tocounterbalance the regulatory function of Fur.

To determine whether there was a correlation between transcriptional changes andphenotype, we tested whether the downregulation of iron receptor genes would affectthe growth of the NMB0419 mutant under iron-restricted conditions. The resultsshowed that acquisition of hemoglobin and transferrin by the mutant was affected.Acknowledging that the initial meningococcal phenotype of interest was epithelialintracellular multiplication, we went on to investigate whether the downregulation oftonB, exbB, and exbD would be enough to impair the NMB0419 mutant’s intracellularreplication rate (mutations in meningococcal tonB lead to impaired intracellular repli-cation, which is considered to result from blockade of iron uptake from degradedferritin iron complexes that occurs via a TonB-dependent receptor of unknown identity[6]). In our experiments, the NMB0419 mutant replicated less efficiently within epithelialcells than the WT, and the effect was reversible by the addition of free iron, consistentwith the NMB0419 mutant lacking the ability to acquire transferrin degradation prod-ucts.

Hooda et al. (21) have determined that the surface lipoprotein assembly modulator(Slam) protein (encoded by NMB0313) has an ancillary role in transporting lipoproteins,such as TbpB and LbpB, to the surface of N. meningitidis. NMB0313 contains anN-terminal single tetratricopeptide repeat (TPR) domain which is similar in structure toSel1-like domains. NMB0313 is downregulated in the NMB0419 mutant together withother iron receptor genes (including tbpB and lbpB). The downregulation of NMB0313in the NMB0419 mutant is consistent with the proposition that the expression ofNMB0419 is associated with not only upregulation of genes encoding iron receptors butalso their surface expression.

SLR proteins, like NMB0419, mediate their biological roles through their interactionwith other proteins, forming protein complexes (14). While we have not determined theprecise cellular location of NMB0419, as discussed above the most likely location iseither periplasmic or extracellular. Where investigated previously in Gram-negativebacteria, SLR proteins have been found to be periplasmic. For example, the SLR proteinExoR from Sinorhizobium meliloti has a similar domain structure as NMB0419 and hasbeen demonstrated to interact in the bacterial periplasm with the ExoS/ChvI two-component system (TCS) (22). Our characterization of NMB0419 in the meningococcus(using pMIDG0419ΔSIGL) and E. coli (using pET0419ΔSIGL) indicates a possible periplas-mic functional location for NMB0419, similar to ExoR, which supports the possibilitythat NMB0419 is involved in sensing an environmental signal(s) in the periplasm and

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exerts its regulatory role through interactions with a TCS(s) in the meningococcusand E. coli. If correct, it is likely that NMB0419 interacts with a different TCS in themeningococcus than in E. coli, resulting in the phenotypic changes in different biolog-ical processes, i.e., iron uptake and pilus biosynthesis, respectively. Four sets of TCSshave been annotated in meningococcal genomes (11). Of these, only the MisR/S(NMB0595/NMB0594) system has been extensively studied. However, MisR/S is known toregulate only hmbR but not other iron receptors (23), and microarray analysis did notshow any evidence that the MisR/S and Fur regulons overlap (24, 25). In contrast, theMisR/S system in N. gonorrhoeae has been reported to regulate tbpB/A through directbinding to the upstream region of the tbpB gene (26). The authors also determined thatthe DNA sequence in the cognate region upstream of the meningococcal tbpB gene isdivergent from that found in N. gonorrhoeae, consistent with previous findings in N.meningitidis (24, 25). Alternatively, NMB0419 could be extracellular or may have func-tions related to transport of lipoproteins involved in iron regulation, possibly similar tothat described for the meningococcal outer membrane protein Slam (21). Our experi-mental data and the predictions that NMB0419 is exported beyond the cytoplasmicmembrane make it unlikely that NMB0419 is acting as a transcriptional regulator andbinding directly or indirectly to classical Fur motifs present in the promoters ofFur-regulated genes. Further investigation is required to identify the interaction part-ner(s) for NMB0419, the cellular location of these interactions, and how these influencethe expression of genes of the Fur regulon.

Fur is a well-studied global transcriptional repressor and activator for the genesinvolved not only in iron homeostasis but also in the stress response and otherfunctions related to virulence and energy metabolism, enabling bacteria to survive inadverse environments (7, 27). Our finding that the expression of NMB0419 was associ-ated with transcriptional changes involving genes not only for iron uptake but also forvirulence functions and energy metabolism, in a manner counterbalancing the regu-latory function of Fur, is significant and novel. Factors associated with positive regula-tion of meningococcal iron receptors under iron-restricted conditions have not beendescribed before and may be important determinants for meningococcal survival in thehost environment.

MATERIALS AND METHODSBacterial strains and growth conditions. E. coli DH5� (Invitrogen) and derivatives (Table 2) were

grown on Luria-Bertani (LB) agar plates or in LB broth (Oxoid). N. meningitidis strain MC58 (B:15:P1.7,16b;ST-74) and derivatives (Table 2) were routinely grown on brain heart infusion (BHI) (BD Biosciences) agarplates supplemented with 10% FBS (Invitrogen) or in BHI broth (BD Biosciences). In some experiments,N. meningitidis was cultured in RPMI 1640 (Invitrogen) with or without 10% FBS supplementation.Bacteria grown on agar plates were incubated at 37°C in an atmosphere containing 5% CO2 or in brothat 37°C with shaking at 200 rpm. Where appropriate, kanamycin (Km) or chloramphenicol was added tothe growth medium to a final concentration of 50 and 20 �g/ml, respectively.

General molecular biology techniques. Unless otherwise stated, standard recombinant DNAtechniques, including DNA restriction digestion, agarose gel electrophoresis, DNA band recovery, andDNA ligation, were carried out as described elsewhere (28). Chromosomal and plasmid DNA wasprepared using the QIAprep Spin miniprep kit and QIAamp DNA minikits (Qiagen), respectively.

TABLE 2 Bacterial strains used in this study

Strain name Modification/plasmid Reference

E. coliDH5� WT 28DH5�(pET) pET This studyDH5�(pET0419) pET0419 This studyDH5�(pET0419ΔSIGL) pET0419ΔSIGL This study

N. meningitidisMC58 WT 11MC58 (ΔNMB0419::Km) pMIDG0419 15MC58 (ΔNMB0419::Km) (pMIDG0419) pMIDG0419 This studyMC58 (ΔNMB0419::Km) (pMIDG0419ΔSIGL) pMIDG0419ΔSIGL This study

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Construction of pET-based recombinant plasmids and generation of E. coli transformants.NMB0419 was amplified by PCR from genomic DNA prepared from N. meningitidis strain MC58 usingprimers 0419PETF and 0419PETR (see Table S6 in the supplemental material) and HotStar DNA poly-merase (Qiagen) with Taq Extender PCR additive (Stratagene) according to the manufacturers’ protocols,with the addition of 5% dimethyl sulfoxide (DMSO) (Sigma). The product was cloned into the NcoI andXhoI sites of the expression vector pET-29a (Novagen), generating the recombinant plasmid pET0419. E.coli DH5� (Invitrogen) was transformed with pET0419 and pET, generating transformants DH5�(pET0419)and DH5�(pET), respectively. The coding sequence for the N-terminal signal peptide (identified usingSignalP [29]) was removed from pET0419 using PCR primers SIGL0419F and SIGL0419R (Table S6) tocreate plasmid pET0419ΔSIGL, in which the remaining coding sequence lies in frame with the start codon(verified by sequencing). The resulting E. coli transformant was named DH5�(pET0419SIGL).

Construction of recombinant plasmids for genetic complementation of MC58 NMB0419 mu-tants. Plasmid pMIDG0419 carrying NMB0419 was created by insertion of an NMB0419 ampliconprepared using primers pMIDG0419F and pMIDG0419R (Table S6) into BamHI and NheI sites of thebroad-host-range vector pMIDG201 (30), and the aphA-3 gene was subsequently replaced at EcoRV siteswith a chloramphenicol acetyltransferase (cat) gene amplified from plasmid pMC-Express (31) usingprimers EcoRVcatF and EcoRVcatR (Table S6). The resulting construct pMIDG0419 was transformed into E.coli S17.1 �pir and into the MC58 NMB0419 mutant through conjugation, generating MC58 (ΔNMB0419::Km) (pMIDG0419) selected using chloramphenicol (5 �g/ml).

In addition, an in-frame deletion of the signal peptide coding sequence from NMB0419 was made inpMIDG0419 by PCR amplification using primers SIGL0419F and SIGL0419R2 and recircularization of theplasmid using T4 ligase, creating plasmid pMIDG0419ΔSIGL, which was also conjugated into MC58(ΔNMB0419::Km) to create MC58 (ΔNMB0419::Km) (pMIDG0419ΔSIGL). The desired NMB0419 codingsequence was confirmed by sequencing.

The expression of NMB0419 was detected in both complemented strains in log-phase growth (in BHI)meningococci as determined by RT-PCR (Fig. S8), using primers NMB0419RTF and NMB0419RTR posi-tioned downstream of the coding sequence for the N-terminal signal peptide domain (Table S6).

RT-PCR. E. coli RNA samples were prepared from DH5�(pET0419) and DH5�(pET) grown in LB broth(Oxoid) to an optical density at 600 nm (OD600) of 0.5 using the RNeasy kit (Qiagen). RNA samples weretreated with DNase I (Invitrogen). As a negative control, RNA was incubated with RNase A (Sigma) (1�g/�l) at 37°C for 20 min prior to reverse transcription. RT-PCR and verification of PCR products wereperformed as previously described (15). Primers are listed in Table S6.

Quantitative assessment of epithelial adherent and intracellular E. coli. Cells of the transformedhuman bronchial epithelial cell line 16HBE14 (32) were cultured at 37°C in 5% CO2 in Dulbecco modifiedEagle medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine (all from Invitrogen). Bacteriawere suspended in culture medium and added to confluent cell monolayers grown in 24-well tissueculture plates, at a multiplicity of infection (MOI) of 50 to 80. After incubation for 4 h at 37°C, cellmonolayers were washed vigorously three times with phosphate-buffered saline (PBS) and adherentbacteria were released from cells by treatment with 1% saponin (VWR) for 10 min at 37°C. Serial dilutionswere plated out for CFU determination. Adherent bacterial CFU were expressed as percentage of totalgrowth. Inhibition of T1P-mediated adherence was achieved by adding D-mannose (Sigma) at 2% to theDMEM prior to inoculation of bacteria. The number of internalized E. coli bacteria was assessed using thegentamicin protection assay as previously described (15). In each experiment, three independentbiological replicates were performed.

Light microscopy. 16HBE14 cells were seeded (5 � 104 cells per well) onto glass coverslips (VWR)laid at the bottom of wells in 24-well tissue culture plates. Adherence assays were carried out asdescribed above. After washing with PBS, samples were fixed onto the slides using 3.7% formaldehydeand stained with Giemsa stain (Sigma). Samples were visualized with an Olympus BH2-RFC lightmicroscope, and photographs were taken at �100 magnification.

TEM. Copper palladium grids (300 mesh; TAAB Laboratories Equipment Ltd., United Kingdom) weregently floated onto 5 �l of bacterial suspension for 5 min and air dried. The bacteria were fixed on gridswith 4% glutaraldehyde (Sigma) and stained with 1% uranyl acetate. Resulting samples were visualizedusing a FEI Tecnai G2 transmission electron microscope.

qRT-PCR. RNA samples were prepared from MC58 (WT) and MC58 (ΔNMB0419::Km) (15) grown to anOD600 of 0.75 using the FastRNA Pro Blue kit and FastPrep FP120 system (MP Biochemicals). Threeindependently prepared biological samples without detectable genomic DNA contamination as ad-judged by Bioanalyzer (Agilent) analysis were used. RNA samples were reverse transcribed to first-strandcDNA by using Superscript III and random primers (Invitrogen). qRT-PCR was performed using TaqMantechnologies, Universal PCR master mix, and the StepOnePlus system (Applied Biosystems). Primer andreporter (6-carboxyfluorescein [FAM]- and nonfluorescent quencher [NFQ]-labeled) sequences are listedin Table S6. Statistical analysis was carried out using the threshold cycle (2�ΔΔCT) method (33) with 16SrRNA as the endogenous reference. Relative expression of pilE in MC58 (ΔNMB0419::Km) was comparedto that in MC58 (WT) and expressed as fold change.

Transcriptome analysis. E. coli microarray analysis was carried out using RNA samples preparedfrom DH5�(pET0419) and DH5�(pET) cultured in LB broth to an OD600 of 0.5 and fluorescently labeledas described previously (34). Labeled RNA samples of three biological replicates and each with a pair ofdye swaps for each strain were hybridized on ShEcoli arrays, which were obtained from the Institute ofFood Research (Norwich, United Kingdom) and have been described previously (35). The hybridizationand washing conditions were as previously described (34). Slides were scanned on a GenePix 4000B

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scanner (Axon), and data analysis was performed in GenePix Pro6.0 and GeneSpring GX7.3 (Agilent) aspreviously described (34). Genes differentially expressed in DH5�(pET0419) compared to DH5�(pET)(Table S1) were selected using a t test P value with multiple testing using the Benjamini and Hochbergfalse discovery rate (FDR) (36) at a cutoff of 0.05.

Meningococcal RNA samples of three biological replicates were prepared for WT and mutant(ΔNMB0419::Km) strains grown in supplemented RPMI 1640 to an OD600 of 0.5 to 0.7 using the FastRNAPro Blue kit and FastPrep-24 (MP Biochemicals). All RNA samples were treated with DNase I (Invitrogen).RNA samples with an RNA integrity number (RIN) of �9.5, as adjudged by Bioanalyzer analysis, weresubmitted to the Beijing Genome Institute (Hong Kong) for RNA sequencing analysis, which includedremoval of rRNA, RNA fragmentation, cDNA synthesis by random priming, size selection, PCR amplifi-cation, Illumina sequencing (HiSeq 2000), quality control (QC) of raw data, alignment with MC58 genome,QC of alignment, calculating expression level (expressed as reads per kilobase per million [RPKM] [37]),and expression level comparison for each pair (mutant versus WT) of biological replicates. We furtherfiltered results of comparisons using an FDR (described by Benjamini and Yekutieli [38]) of �0.01 and log2

ratio ��0.5849 or �0.5849 (fold change of �1.5). The genes that passed filters in all three sets ofcomparison were listed as up- and downregulated in the NMB0419 mutant (Tables S2 and S3).

Iron utilization bioassay. N. meningitidis (107 CFU) was streaked out onto BHI agar plates containing100 �M deferoxamine mesylate (Desferal) (Sigma) and supplemented with 10% FBS, providing 15 �Mbovine hemoglobin (5), an iron source that can be utilized by the meningococcus (39). Further tests weredone by placing filter discs impregnated with 10 �l iron supplements over the 107 CFU spread on theBHI agar plates containing Desferal and FBS (as described above). Iron supplements were hemoglobin(1.25 mM), iron(III) chloride (100 mM), apotransferrin (40 mg/ml), and holotransferrin (40 mg/ml). All agarplates were incubated at 37°C in 5% CO2 for 17 h.

Meningococcal intraepithelial replication assay. Cells of the respiratory epithelial cell line COR-L23(ATCC) were cultivated in RPMI 1640 with 10% FBS and 1% nonessential amino acids (all from Invitrogen)and used in 24-well plates for assays. Gentamicin killing of extracellular meningococci was performed aspreviously described (15). After gentamicin killing, formulated media were added to designated wells toassess meningococcal intracellular replication. Formulated media were RPMI 1640 alone and RPMI 1640with added Desferal (100 �M) or iron(III) chloride (25 �M). CFU were enumerated immediately (T0) and4 h (T4) after gentamicin killing. For each strain, CFU at T4 was divided by CFU at T0 to give the replicationrate, which was further normalized to the value obtained for MC58 (WT) for each medium condition andexpressed as percentage of WT replication.

PCR amplification of the NMB0419 homologue from neisserial strains. Genomic DNA from N.meningitidis and N. lactamica strains (grown on BHI agar) was prepared using a DNA minikit (Qiagen). PCRwas performed using primers 0419PETF and 0419PETR (Table S6), HotStar DNA polymerase (Qiagen), andTaq Extender PCR additive (Stratagene) plus 5% DMSO (Sigma). PCR products were purified using theQIAquick gel extraction kit (Qiagen) and sequenced.

Accession number(s). The array design is available in ArrayExpress (accession no. A-MEXP-1748;http://www.ebi.ac.uk/arrayexpress). Fully annotated microarray data have been deposited in Array-Express (accession number E-MEXP-2485). The raw data and processed data with description have beendeposited in Gene Expression Omnibus under accession number GSE81572.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00574-16.

SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.

ACKNOWLEDGMENTSThis study was supported by the George John and Sheilah Livanos Charitable Trust.We thank Asa Hedman and Sunita Sinha for their technical assistance in preparing

RNA samples for E. coli microarray analysis and constructing pET0419ΔSIGL. We thankMichael Hollinshead at Investigative Science, Imperial College London at St. Mary’sCampus (present affiliation, Department of Pathology, University of Cambridge), forassistance in electron microscopy.

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