A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae

Abstract

Non-typeable Haemophilus influenzae contains an N⁶ -adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

ARTICLE

Received 26 Aug 2014 | Accepted 16 Jun 2015 | Published 28 Jul 2015

DOI: 10.1038/ncomms8828 OPEN

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus inuenzae

John M. Atack1, Yogitha N. Srikhanta1,w, Kate L. Fox2, Joseph A. Jurcisek3, Kenneth L. Brockman3, Tyson A. Clark4, Matthew Boitano4, Peter M. Power1, Freda E.-C. Jen1, Alastair G. McEwan2, Sean M. Grimmond5,w,Arnold L. Smith6, Stephen J. Barenkamp7, Jonas Korlach4, Lauren O. Bakaletz3 & Michael P. Jennings1

Non-typeable Haemophilus inuenzae contains an N6-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all ve of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biolm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

1 Institute for Glycomics, Grifth University, Gold Coast, Queensland 4222, Australia. 2 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. 3 Center for Microbial Pathogenesis, The Research Institute at Nationwide Childrens Hospital and The Ohio State University College of Medicine, Columbus, Ohio 43205, USA. 4 Pacic Biosciences, 1380 Willow Road, Menlo Park, California 94025, USA. 5 Institute of Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. 6 Center for Global Infectious Disease Research, Seattle Childrens Research Institute, Seattle, Washington 98105, USA. 7 Department of Pediatrics, Saint Louis University School of Medicine, and the Pediatric Research Institute, Cardinal Glennon Childrens Medical Center, Saint Louis, Missouri 63104, USA. w Present addresses: School of Biomedical

Science, Monash University, Melbourne, Victoria 3800, Australia (Y.N.S.); Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden, Glasgow, Scotland G61 1BD, UK (S.M.G.). Correspondence and requests for materials should be addressed to L.O.B. (email: mailto:[email protected]

Web End [email protected] ) or to M.P.J. (email: mailto:[email protected]

Web End [email protected] ).

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Non-typeable Haemophilus inuenzae (NTHi) is a signicant bacterial pathogen commonly associated with paediatric infection as a predominant cause of otitis

media (OM) or middle ear infection. While NTHi contributes to B40% of all cases of acute OM, it remains the major aetiological agent of chronic OM, recurrent acute OM and OM treatment failure1. It is also a major cause of community-acquired pneumonia and exacerbations of chronic obstructive pulmonary disease. With the rising prevalence of antimicrobial resistance, the success rate in the treatment of NTHi has recently declined. There is no vaccine to prevent infection by NTHi.

Phase variation is the high frequency reversible ON/OFF switching of gene expression, and is a common feature of many virulence determinants expressed by bacterial pathogens25. While phase variation is typically associated with genes that encode surface structures, several host-adapted bacterial pathogens, including NTHi, have DNA methyltransferases (mod genes) associated with type III restriction modication systems that are subject to phase-variable expression. Simple tandem DNA repeats have been shown to mediate phase variation of mod genes by rapid reversible loss or gain of a repeat unit leading to frame shifts and ON/OFF switching of expression610.

The variable central region of Mod methyltransferases contains the DNA-recognition domain (DRD; see Fig. 1e) that dictates methylation-sequence specicity1113. In H. inuenzae strain Rd, our previous work has demonstrated that random switching of the modA1 gene controls expression of multiple genes via differential methylation of the genome in the modA1 ON and OFF states10. This novel genetic system, termed the phasevarion (phase-variable regulon) regulates gene expression in four other important human pathogens: Neisseria gonorrhoeae, Neisseria meningitidis7, Helicobacter pylori9 and Moraxella catarrhalis14. Differences in the DRD have previously been observed in a genetically diverse collection of H. inuenzae strains and these differences dene 20 distinct mod alleles (modA1 to modA20)6,15. Different DRDs are proposed to recognize and methylate different target sequences. Our studies with pathogenic Neisseria, which also contains modA, conrmed that different modA alleles methylate different target sequences and thereby regulate different sets of genes, that is, have distinct phasevarions. Conversely, strains that harbour the same modA allele regulate the same phasevarion of genes7,16.

In this study, we sought to assess the role that candidate NTHi phasevarions may play in gene expression, immunoevasion and in the pathogenesis of NTHi during experimental OM. Five modA alleles are present in over two-thirds of all isolates of NTHI. We demonstrate that phase variation of all ve of these modA alleles controls gene expression differences in all the strains studied. These expression differences include outer-membrane proteins and key vaccine candidates. Biphasic switching of specic modA alleles also inuences tness in opsonophagocytic killing assays and in susceptibility to antibiotics. In an in vivo animal model, we demonstrate, for the rst time, direct selection for a particular state of modA expression in the major phase variable modA allele present in NTHi, modA2.

ResultsDistribution of modA alleles in NTHi isolates. We conducted a detailed analysis examining the modA allele frequency in a diverse set of NTHi isolates taken from healthy individuals and OM patients (Fig. 1). The modA alleles modA3, modA6, modA7, modA8 and modA14 do not contain tetranucleotide repeats, and thereby do not phase vary in expression. In a previous survey6, we reported that B70% of NTHi strains contain a phase variable modA allele (n 41; Fig. 1a). Of all the modA alleles,

modA2 was the most prevalent, being found in almost a quarter of all isolates (24%).

We then analysed modA allele distribution in three further strain collections (Fig. 1). The rst collection contained paired isolates, isolated from the nasopharynx and the middle ear, from children presenting with chronic and/or recurrent OM (Fig. 1b)17. This collection contained 27 pairs of isolates: 16 pairs were collected from 1982 to 1986 and 11 pairs were collected from 2004 to 2006. Almost two-thirds of strains (59.3%) within this particular collection contain a phase variable modA allele (n 33), with modA2 most prevalent (22% of all isolates).

The second strain collection contained clinical isolates from OM-prone children (Fig. 1c)18. Of these 34 separate isolates (25 recovered from nasopharyngeal swabs and 9 recovered from middle ear effusions), 65% contained a phase variable modA allele. The most common was modA10 (19%). As NTHi is frequently isolated from the upper respiratory tract, we collected samples and analysed modA allele distribution in isolates from the nasopharynges of healthy children (Fig. 1d), with a reported colonization rate of B80% (ref. 19). In addition, a correlation between NTHi colonization frequency and the incidence of recurrent OM in children has been suggested20. We observed that the predominant modA alleles in healthy nasopharyngeal isolates were modA10 (24%), modA4 (16%), modA2 (14%) and modA9 (14%) (Fig. 1d). These four modA alleles are all phase variable, and alone comprise over two-thirds of all alleles from this collection. One new modA allele was also identied in this study in strain 515 (Supplementary Table 1). We have designated this new allele as modA21.

Taken together, this analysis of four distinct strain collections shows that not only are phase variable modA alleles frequently associated with disease, they are also prevalent in NTHi isolated from healthy individuals. From all the four studies, phase variable modA alleles are present in approximately two-thirds of all the isolates (n 135), with the modA2 and modA10 alleles, the two

most common overall, at 17.2 and 15.4%, respectively. Based on these surveys, we selected the ve modA alleles most commonly associated with OMmodA2, 4, 5, 9 and 10for further analysis in this study (Fig. 1e).

Generation of natural modA ON and OFF strains. We generated modA natural ON- and OFF-enriched populations (490%

ON or OFF) from the ve NTHi strains containing the modA2, 4, 5, 9 and 10 alleles (Fig. 1e). All ON strains contain a number of AGCC repeats that places the modA open reading frame (ORF) in frame, and therefore express a full-length functional protein, as conrmed by western blot using an anti-ModA antibody (Fig. 2a). All the natural ON and OFF strains were continually veried using our well-established FAM-labelled primer PCR screen coupled to fragment length analysis (an example of the methodology and results is given in Fig. 2b,c)6,10. Natural modAOFF strains contain a number of AGCC repeats that place the modA ORF out of frame, leading to a frame shift mutation and premature termination at stop codons in the alternate reading frames (Fig. 2a). Kanamycin knockout modA::kan mutants were also generated in all the ve NTHi strains bearing these alleles (Fig. 1e) by disruption of the modA gene via insertion of a kanamycin resistance cassette, as described previously6,10.

Methylome analysis. Single molecule, real-time (SMRT) sequencing21 was carried out to determine the complete genomes and identify the methylation recognition sequence of these ve distinct ModA N6-adenine methyltransferases. DNA isolated from each modAON and modA::kan pair was sequenced and analysed. By comparing the methylomes of the modAON with the modA::kan

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Fox et al., 2007 (ref. 6); samples of NTHi from a variety of sources

Fox et al., 2014 (ref. 17); samples from patients with chronic/recurrent OM

Novotny et al., 2009 (ref. 18); samples from OM-prone individuals

Nasopharyngeal samples from healthy individuals (this work)

(AGCC)16ON modA2

(AGCC)22ON modA4

Major PV

NTHi strain

Repeats DNA recognition

domain

723

C486

477

1209

R2866

modA2 modA4 modA5 modA9 modA10

Minor PV

modA13

modA11

modA20

(AGCC)13ON modA5

(AGCC)19ON modA9

(AGCC)16ON modA10

modA21

Non-PV

modA3 modA6 modA7

modA14

modA8

500 bp

Figure 1 | Analysis of NTHi strain collections to ascertain the proportion of isolates containing phase variable modA alleles. (ad) Presents the four separate strain collections that were analysed to ascertain the distribution of phase variable modA alleles in NTHi populations; (a) collection containing a broad selection of NTHi isolates from a variety of sources (for example, culture collections, OM-prone individuals)6; (b) samples of paired isolates of NTHi isolated from patients presenting with chronic/recurrent OM17; (c) sample collection of NTHi isolates taken from OM-prone individuals18; (d) NTHi isolates from the nasopharynx of healthy children (collected as part of this study); and (e) the ve modA alleles most commonly associated with OMmodA2, modA4, modA5, modA9 and modA10. Each modA gene is represented as a white arrow, with the DRD represented by a coloured box that matches the colour in panels ad. The black box to the right of each gene represents the 50 region of the downstream gene (an inactive restriction endonuclease). The blue arrow to the left of each modA gene represents the 30 end of the gene upstream of each modA (in all the cases, a ribonuclease, rnhB). Details of the strain collections used in c and d are presented in Supplementary Table 1.

knockout mutants, we were able to identify the motif methylated by the ModA methyltransferases of ModA4, 5, 9, 10 (Table 1). The ModA2 motif was identied by heterologous expression of ModA2 in Escherichia coli, as described previously22. ModA2, ModA5, ModA9 and ModA10 all have prototypical type III methyltransferase recognition sequences (Table 1), in that they methylate an adenine residue on the N6 position in a 5 base pair (5 bp) non-palindromic sequence23,24. The recognition sequence of ModA4 was different, instead recognizing a four base sequence: 5-CG(m6A)G-30 (Table 1). In accordance with REBASE25 naming conventions, we have given the ModA2, ModA4, ModA5, ModA9 and ModA10 methyltransferases the designationsM.Hin723I, M.HinC486I, M.Hin477I, M.Hin1209I and

M.Hin2866I, respectively. Summary data from the SMRT methylome analysis of the ve modA ON/OFF pairs is shown in Supplementary Fig. 1. Complete closed genome sequences for each of the ve strains containing these modA alleles were also generated (NTHi strains 723, C486, 477, 1209 and R2866; the R2866 genome sequence had already been deposited in NCBI Genbank; accession number CP002277). Accession codes for the complete annotated genomes of NTHi strains 723, C486, 477 and 1209 are listed in the Accession codes section.

Distribution of ModA sites in NTHi genomes. Bioinformatic analysis of the recognition sequences of ModA2, 4, 5, 9, 10

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ON OFF Kan

723 modA2

(AGCC)n DRD

90% on 16 reps

91% off 14 reps

NA

0.5 1.0 1.5 2.0 kb

C486 modA4

477 modA5

1209 modA9

R2866 modA10

86% on 22 reps

88% off 23 reps

NA

95% on 13 reps

100% off 12 reps

NA

87% on 19 reps

89% off 20 reps

NA

88% on 16 reps

95% off 15 reps

NA

Length Peak area % of sample

Repeats

195 6,5838.1% 15 off

199 72,66889.5% 16 on

203 1,9392.4% 17 off

Figure 2 | Western blots showing ModA presence in modA ON OFF and kanamycin knockout strains and an example of the fragment analysis methodology. (a) Western blot analysis using ON/OFF/kan cells from all the ve modA alleles; anti-modA primary antibody at 1:5,000; AP conjugated anti-rabbit secondary at 1:20,000. A modA band is only present in those cells possessing the ON variant of each strain. The number of repeats (reps) represents the major number of repeats present in that particular isolate, and the percentage ON of that particular strain is noted underneath each relevant blot. Full western blots are presented in Supplementary Fig. 4; (b) an illustration of the methodology used during fragment analysis to ascertain modA ON/OFF state and percentages of cell populations. A 6-carboxyourescein (FAM)-labelled forward primer (green hexagon) and unlabelled reverse primer was used to generate a PCR product over the repeat tract (AGCCn; grey box) that was analysed using GeneScan technology; and (c) an example trace produced by

GeneScan analysis on a representative genomic sample from an NTHi strain containing a phase variable modA gene.

Table 1 | Summary of SMRT sequencing and methylome analysis of representative strains containing the ve modA alleles under study.

modAallele NTHi strain

Predicted ORFs

modA2 723 OM CP007472 5-CCGA(m6A)-3 M.Hin723I 2,270 1,887,620 1,868 modA4 C486 OM CP007471 5-CG(m6A)G-3 M.HinC486I 6,203 1,846,507 1,783 modA5 477 OM CP007470 5-AC(m6A)GC-3 M.Hin477I 2,548 1,846,259 1,813 modA9 1209 OM JMQP01000000 5-CCTG(m6A)-3 M.Hin1209I 2,504 1,895,979 2,247 modA10 R2866 Blood CP002277* 5-CCT(m6A)C-3 M.Hin2866I 1,244 1,932,238 1,905

*Strain already annotated and submitted (October 2010) to the EMBL/GenBank/DDBJ databases. A full summary of SMRT sequencing/methylome analysis derived data is presented in Supplementary Fig. 1.

Clinical symptoms

Accession number

Methylation sequence

Systematic name

Number of sites in genome

Genome size (bp)

showed that these sites are widely and evenly distributed in their respective genomes (see Supplementary Fig. 1). ModA4 occurs much more frequently than ModA2, 5, 9 or 10, as is expected for a 4 bp recognition sequence compared with 5 bp recognition sequences. For ModA2, A4, A5 and A9, sites appear to be equally distributed between coding and non-coding regions of the genome. For ModA10, 69.1% of sites are present in non-coding regions, compared with only 30.9% of ModA10 sites in coding regions. Further, there are far fewer ModA10 sites in the R2866 genome than would be expected by chance (1,244 observed/2,714 expected 45.8%; Supplementary Fig. 1). Other ModA recogni

tion sequences occurred at frequencies close to that predicted by chance: ModA2 (91.2%), ModA4 (79%) ModA5 (104.7%), ModA9 (94%).

modA ON/OFF pairs show outer-membrane protein expression differences. To determine whether modA switching altered the expression of outer-membrane proteins (OMPs), outer-membrane samples were separated using Bis-Tris PAGE gels and silver stained (Fig. 3a) to explore whether any gross protein differences

existed within each modA ON/OFF strain pair. Clear differences could be visualized within the OMP prole of each modA ON/ OFF pair (noted by arrows in Fig. 3a), indicating that modA phase variation inuences the OMP prole, and may therefore inuence immunoevasion, pathogenesis and virulence.

Vaccine candidate expression in modA ON/OFF strain pairs. OMP preparations from modA ON and OFF strains were studied using western blot with primary antibodies raised against several well-studied vaccine candidates (Fig. 3b): major outer membrane proteins P2 (ref. 26), P5 (refs 27,28) and P6 (refs 29,30); lipoprotein D31 and its deacylated derivative PDM31,32; the high molecular weight (HMW) proteins33,34; and the adhesin Hia35. Three different antisera were used to probe for relative expression of OMP P5 (anti-P5, anti-LB1 and anti-chimV4), as these sera contained antibodies specic to unique epitopes of the protein27,28,36 (Fig. 3b).

We saw an expression difference of OMP P6 in the modA9 ON/OFF pair, with a greater level of OMP P6 seen in modA9ON relative to modA9OFF cells (Fig. 3b). Lipoprotein D showed

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a

723 modA2

723 modA2

C486 modA4

477 modA5

1209 modA9

R2866 modA10

ON OFF

ON OFF

ON OFF

ON OFF

b

C486 modA4

477 modA5

1209 modA9

R2866 modA10

ON OFF

ON OFF

ON OFF

ON OFF

ON OFF

ON OFF

OMP P2

OMP P5

OMP P6

LPD

PDM

HMW

Hia

LB1

chimV4

Titre modA2modA4

modA5

c

ON OFF 1:40,0001:8001:800

1.0270.027 0.2660.004**0.4570.041 0.2430.038*0.3600.022 0.8160.089**

Figure 3 | Analysis of outer-membrane protein (OMP) preparations.(a) Silver-stained gel of each modA ON/OFF pair; (b) western blots using antibodies against currently investigated vaccine candidates. Full western blots are presented in Supplementary Fig. 5; (c) ELISA results for HMW in modA2, modA4 and modA5 ON/OFF strain pairs using primary antibody AD6. Values presented are mean valuess.d. Each experiment was carried out in triplicate. P values were calculated using Students t-test based on specic titres in the linear range of the response curve (* o0.01;

** o0.001). Full ELISA data are presented in Supplementary Data 1.

stable expression independent of modA status in NTHi strains 723 (modA2), C486 (modA4) and 477 (modA5) (Fig. 3b). We could not detect the presence of lipoprotein D in NTHi strains 1209 or R2866, regardless of modA status (Fig. 3b). The analysis of our NTHi strain 1209 SMRT-generated genome revealed that the gene-encoding lipoprotein D (glpQ) is truncated in this strain (1209_00054) to just the carboxy (C)-terminal portion (B65 residues). In strain R2866, a full-length gene is present

(R2866_1787), but it appears to not be expressed at a level detectable by western blotting.

Expression of HMW1/2A was greater in OMPs prepared from modA2ON versus modA2OFF; modA4ON versus modA4OFF; and modA5OFF versus modA5ON (Fig. 3b). In these latter two examples, a single protein band was clearly more prevalent in modA4ON and modA5OFF, when compared with their respective partners (Fig. 3b). However, in modA2ON, a higher MW band was more abundant in modA2ON compared with modA2-OFF, and a lower MW band was visible only in modA2ON, implying that both HMW1A and HMW2A are present in greater amounts in OMP preparations from NTHi modA2ON when compared with modA2OFF (Fig. 3b). Apparent differences in HMW expression in western blot results were veried using whole-cell enzyme-linked immunosorbent assays (ELISAs) to quantitate the relative expression of HMW1/2A. Statistical signicance was calculated using Students t-test, and signicant differences were observed for these three ON/OFF strain pairs (Fig. 3c).

As the genes encoding HMW1A and HMW2A proteins contain variable heptanucleotide repeats in their promoter region that are also reported to inuence gene expression4,37, we sequenced across this repeat tract in all the three strain pairs where a difference in HMW expression was evident. This analysis showed that the differences in repeat tract length in each ON/ OFF pair were not responsible for the expression changes observed (Supplementary Fig. 2). The literature states that the longer the repeat tract, the lesser the expression level of HMW37. As the repeat tracts of HMW1 and HMW2 are of identical or similar length within each strain pair where differences are seen, it is likely that expression differences are ModA-dependent, and not due to HMW phase variation.

Finally, our analysis of the adhesin Hia revealed that the hia gene was only present in the genome of NTHi strain R2866 (R2866_0725) containing the modA10 allele (Fig. 3b). Hia was expressed at a greater level in modA10ON relative to modA10OFF (Fig. 3b). However, our subsequent analysis showed that this difference was owing to modA10-independent phase variation event in a poly-T tract in the Hia promoter region38.

iTRAQ proteomics analysis of modA ON/OFF strain pairs. To further dene the impact of modA ON/OFF status on the expression of existing and potential vaccine candidates, as well as the OMP prole as a whole, preparations of OMPs used in silver staining and western blotting (Fig. 3) were characterized using iTRAQ 1D nanoLC ESI MS/MS. All the data have been deposited to the ProteomeXchange Consortium39 via the PRIDE partner repository, with the data set identier PXD002210. Proteins with either 41.5- or o0.65-fold expression differences between ON/

OFF pairs when comparing two biological replicates of each OMP preparation are reported (Table 2). Expression changes in OMPs were seen in the modA2, modA4, modA5 and modA10 ON/OFF strain pairs.

Several OMPs involved in the sequestration of iron and haem were downregulated in modA2ON (Table 2). This included HxuC and HxuB (723_01435 and 723_01434; a haem/haemopexin utilization and haem/haemopexin-binding OMP, respectively), transferrin-binding protein 1 (723_00620), a hemin receptor (HemR, also annotated as HxuC2; 723_01596); and a major ferric iron-binding protein (HitA, 723_01615). This analysis also revealed OMP P6 to be differentially regulated in the modA2 pair (Table 2), a nding not evident from our western blotting analysis (Fig. 3b), but having implications for its use as a suitable vaccine antigen.

HMW2B is an outer membrane-associated protein responsible for the translocation of the adhesins HMW1/2A to the cell

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Table 2 | iTRAQ quantitative mass spectrometry analysis using OMPs from each ON/OFF pair.

Gene ID Gene Ratio ON:OFF* Stouffers P-value Stouffer Adj Increased expression in 723 modA2 ON723_01555 N-acetylneuraminate epimerase NanM 2.36 5.40E 14 6.32E 13

723_01788 Adhesin translocation protein HMW2B 2.05 0 0

Decreased expression in 723 modA2 ON723_01435 Haem/haemopexin utilization protein C HxuC1 0.28 0 0 723_00620 Transferrin-binding protein 1 0.32 4.74E 08 3.45E 07

723_01615 Major ferric iron-binding protein HitAw 0.37 0 0 723_01434 Haem/haemopexin transporter protein HxuBw 0.41 0 0 723_01596 Haemin receptor, HemR 0.42 0 0 723_01306 15 kDa peptidoglycan-associated lipoprotein OMP P6 0.6 2.57E 04 1.12E 03

Increased expression in C486 modA4 ONC486_00892 OMP P2 1.52 0 0

Increased expression in 477 modA5 ON477_00572 OMP P5 1.65 0 0

Increased expression in R2866 modA10 ONR2866_0725 Adhesin Hiaz,y 11.53 0 0 R2866_0192 OMP P6 1.85 0.046 0.17

R2866_1237 OMP P5 1.65 7.39E 03 0.043

Gene designations from analysis with our SMRT produced genomes for each strain; differences are represented as ON:OFF; statistical signicance is measured using Stouffers P value and Stouffers adjusted (Adj) value, as the results are the interpretation and comparison of two independently prepared sets of OMPs from each ON/OFF pair. Schematics of differentially regulated genes with locations of ModA methylation motifs are depicted in Supplementary Fig. 3.*Only samples with an ON:OFF ratio 41.5 or o0.7 were included. Complete iTRAQ data for all the ve strain pairs are presented as Supplementary Data 2.

wIndicates identication by microarray and iTRAQ.

zIdentied by western blotting and iTRAQ.yHia was subsequently shown to be regulated in a modA10-independent manner.

0

surface40. The increase in an accessory protein in modA2ON with specicity for both HMW1/2A proteins would explain the increase in both HMW-A proteins in modA2ON seen in our western blotting with the modA2 strain pair (Fig. 3b). NanM, a sialic acid mutarose, was also present in greater amounts in the outer membrane of modA2ON cells (Table 2).

Phase variation of modA inuences susceptibility to antibiotics. Phasevarion-mediated differential susceptibility to antibiotics has been reported in other human-adapted pathogens41, so we sought to investigate whether this was also the case for our ve modA ON/OFF pairs in NTHi. Minimum inhibitory concentration (MIC) analysis showed that phase-variable expression of modA2, modA5 and modA10 led to 2-fold changes in susceptibility to ampicillin, erythromycin and gentamicin, respectively (Supplementary Table 2). These ndings suggest that gene regulation through DNA methylation is an additional element that may contribute to antibiotic susceptibility.

ModA4 mediates evasion of opsonophagocytic killing. Differential expression of HMW protein had been observed in strain C486, containing the modA4 phasevarion. Antiserum was available that would recognize the HMW protein in this strain. We tested our C486 modA4 ON/OFF strain pair in an opsonophagocytic killing assay42. The modA4ON strain was signicantly (Po0.05 at all the data points; calculated using Students t-test)

more susceptible to killing at every dilution of antibody tested compared with modA4OFF (Fig. 4). No killing was observed with this strain pair using pre-immune sera from animals used to raise anti-HMW sera, nor was killing observed with antisera raised against the protein Hia (data not shown).

Differentially expressed genes in the modA2 phasevarion. Our analysis of samples from a wide variety of sources showed that

modA2 is the most prevalent modA allele present in NTHi isolates (Fig. 1), suggesting that modA2 may have an important role in both asymptomatic colonization and disease. iTRAQ proteomic analysis also showed more differences in expression prole analysis between modA2ON and modA2OFF than the other four modA strain pairs. These observations could lead to a greater potential for phenotypic and virulence differences in vivo (Table 2). We therefore selected the modA2 phasevarion for microarray analysis. RNA was isolated and compared from the modA2ON strain and the modA2::kan mutant. We found 36

100

modA4ON

modA4OFF

80

Percent killing

60

40

20

10 20 40 80 160

Serum dilution

Figure 4 | Opsonophagocytic killing curves using strain C486 modA4 ON/OFF strain pair. Assays were carried out using guinea pig antiserum raised against puried HMW proteins from NTHi strain 12, with NTHi strain C486 harbouring the modA4 ON/OFF pair. Three independent assays were carried out per strain, with error bars representing one s.d. Statistical signicance was calculated using Students t-test, and was o0.05 at all dilutions of antiserum. Individual P values1:10 0.034; 1:20 0.0185;

1:40 0.0012; 1:80 0.009; 1:160 0.0065.

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modA2 natural ON and OFF states impacted virulence, a variant of NTHi strain 723 selected for its modA2ON status (B90% ON,22 AGCC repeats; modA2(22)ON) and a variant selected for its modA2OFF status (B90% OFF. 24 AGCC repeats; modA2(24)-

OFF) were used as challenge strains in the well-established chinchilla model of OM49. These variants were free to phase-vary during the 22 days of the experiment. In two separate studies, cohorts of ve chinchillas each (n 10 total) were challenged

intranasally and transbullarly with NTHi strain 723 modA2(22)ON or strain 723 modA2(24)OFF. Middle ear uids were collected at regular intervals to assess any differences between these strains in their ability to infect the middle ear.Samples taken directly from the left and right middle ear were immediately snap frozen in liquid nitrogen, and were not subcultured, thereby reecting the exact ON/OFF status of the bacterial population from the site and time of sampling.

The ON/OFF status of the repeat tracts in both the modA2(22)ON and modA2(24)OFF infected cohorts from the starting inoculum (day 0) and subsequent days 4, 7, 10, 14, 18 and 22 after challenge were veried by fragment analysis (Fig. 5a; full data set presented in Supplementary Table 4). No gross difference in colony-forming units (c.f.u.) number or mortality was observed (Fig. 5b,c). Figure 5a shows a heat map of the modA2ON status of the inoculum, and the bacteria isolated from both middle ears.Animals challenged with the predominantly modA2ON variant (green) remained ON, whereas those animals challenged with the predominantly modA2OFF variant (red) showed a consistent switch from the OFF to the ON state during the course of the experimental OM study (Fig. 5a). This observation suggests that

Table 3 | Differentially expressed genes in the NTHi strain 723 modA2ON microarray.

Gene Microarray ID Description Ratio qRT-PCR B-Stat Reduced expression in the H. inuenzae strain 723 modA2 mutant723_01712 NTHI0007 Formate dehydrogenase major subunit fdxG 1.50 2.77

723_01520 Hu203001461 Alcohol dehydrogenase, class III 1.53 2.42

723_01710 NTHI0010 Formate dehydrogenase, iron-sulfur subunit fdxH 1.53 1.66

723_00429 Hu103001790 L-lactate permease lctP 1.79 3.08

723_00946 Hu203000185 DL-methionine transporter ATP-binding subunit metN 1.96 3.560.741* 4.80

Increased expression in the H. inuenzae strain 723 modA2 mutant723_01433 ORF02024 Haem/haemopexin-binding precursor hxuA 1.45 0.86 723_00220 NTHI1806 Glycogen synthase glgA 1.49 1.84 723_00222 NTHI1808 Glycogen debranching enzyme glgX 1.52 1.27 723_01160 HI0534 Aspartate ammonia-lyase aspA 1.52 2.66 723_00775 ORF01899 Fumarate reductase subunit D frdD 1.56 0.89 723_01524 HI0181 Formate transporter focA 1.57 1.14 723_01327 Hu203001649 Iron (chelated) ABC transporter yfeB 1.61 1.32 723_00221 NTHI1807 Glucose-1-phosphate adenylyltransferase glgC 1.61 3.76 723_00223 NTHI1809 Glycogen branching enzyme glgB 1.66 2.21 723_00419 HI0809 Phosphoenolpyruvate carboxykinase pckA 1.69 1.00 723_01614 ORF00565 Iron (III) ABC transporter, permease protein hitB 1.75 4.39 723_01328 Hu203001648 Iron chelated ABC transporter permease yfeC 1.90 1.69 723_01329 Hu203001647 Iron chelated ABC transporter permease yfeD 2.08 3.98 723_00591 Hu203000516 Anaerobic DMSO chain C dmsC 2.09 1.26 723_01434 Hu103000469 Haem/haemopexin-binding hxuBw 2.14 1.15 723_01326 Hu203001650 Iron chelated ABC transporter yfeA 2.17 2.1660.667* 5.83 723_00589 Hu203000518 Anaerobic DMSO chain A dmsA 2.22 2.140.839* 2.38 723_00437 HI1210 Malate dehydrogenase mdh 2.28 5.50 723_00590 Hu203000517 Anaerobic DMSO chain B dmsB 2.65 2.67 723_01615 Hu203001388 Iron-utilization hitAw 4.04 2.080.397* 4.72

The genes listed are either down- or upregulated in the H. inuenzae 723 modA2::kan mutant strain compared with the modA2ON strain. The identity of the gene is indicated with our SMRT-derived genomic annotation (accession number CP007472), the original identier from the microarray, and a description of each gene. The average ratio presented is the mean of NTHi strain 723 modA2::kan mutant:723 modA2ON from six replicate spots on three independent microarrays, incorporating a dye swap. Only those genes with an expression value 41.4-fold were included in this table.

*Gene expression was conrmed by quantitative RTPCR in NTHi strain 723 modA2ON and 723 modA2OFF strain variants.wIdentied by both microarray and iTRAQ. B-stat (B-statistic) represents the log-odds that the gene is differentially expressed. A threshold in the B statistic of 0.0 was adopted as genes with a B score 40 have a 450% probability of being truly differentially expressed. All the microarray data are presented in Supplementary Table 3. Schematics of differentially regulated genes with locations of ModA methylation motifs are depicted in Supplementary Fig 3.

genes with an expression ratio of 1.4-fold or greater, with 27 genes upregulated in modA2::kan relative to modA2ON and nine genes downregulated, thereby demonstrating modA2 phase variation has a global impact on gene expression (Table 3; full results in Supplementary Table 3). Quantitative real-time PCR specic for several of the differentially expressed genes conrmed the array data (Table 3). This analysis adds further evidence to our observations using iTRAQ quantitative mass spectrometry that there is differential regulation of genes involved in iron acquisition in the modA2 strain pair (Table 2; Table 3). For example both hxuB 723_01434 and hitA 723_1615 are identied as differentially regulated by both iTRAQ and microarray analysis. Microarray analysis revealed several additional iron-regulated genes that were not identied by iTRAQ as they are not OMPs. These include hitB, which encodes a cytoplasmic permease, with both HitA and HitB being essential for the utilization of iron by NTHi43; and hxuA, a secreted protein involved in the binding of haemhaemopexin. Functional HxuA and HxuB proteins are required for virulence in H. inuenzae44. Also upregulated in 723 modA2::kan was the yfeABCD operon, which encodes an iron transport system45. This locus has homology to the yfeABCD locus of Yersinia pestis, where it is associated with virulence46. Several genes involved in anaerobic metabolism also showed increased expression in modA2::kan (Table 3). Many enzymes involved in anaerobic respiration appear to play an important role in colonization and virulence in bacterial pathogens47,48.

Strain 723 modA2ON is preferentially selected in vivo. To determine whether the gene expression differences between the

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Day 0 4 7 10 14 18 22

Death ON OFF

No data

22ON 24OFF

Figure 6 | Biolm formation using modA2ON and OFF strains. (a) Shows biolm formation using modA2(22)ON. (b) Shows biolm formation using modA2(24)OFF after 24 h growth on chambered coverglass and visualized using confocal microscopy following LIVE/DEAD BacLight staining. Scale bar shown represents 50 mm.

1.E+10

Avgerage c.f.u. ml1 MEE exudate

1.E+09

1.E+08

1.E+07

1.E+06

1.E+05

1.E+04

1.E+03

1.E+02

22ON

24OFF

1.E+01

1.E+00

0 2 4 6 8 10 12 14 16 18 20 22

Days after challenge

0 0 2 4 6 8 10 12 14 16 18 20 22Days after challenge

100

90

80

70

Percent survival

60

50

40

30

20

22ON

24OFF

10

Figure 5 | Results from chinchilla middle ear samples. (a) Heat map showing fragment analysis results from animals challenged with either NTHi 723 modA2(22)ON or modA2(24)OFF. The time of sampling is indicated at the top of the gure by days after initial infection, for example, day 0 initial inoculum, day 4 4 days after initial infection and so on. All

fragment analysis data are presented in Supplementary Table 4; (b) middle ear exudate (MEE) bacterial CFU mean counts from animals infected with modA2(22)ON and modA2(24)OFF. Error bars represent s.d.; and (c) survival of animals infected with modA2(22)ON and modA2(24)OFF.

selection for modA2ON over modA2OFF occurred during overt infection of the middle ear.

modA2ON forms more robust biolms in vitro than modA2OFF. Given the selection for modA2ON in the chinchilla middle ear and the importance of biolm formation in chronicity and recurrence of OM, we assayed these ON and OFF variants for relative ability to form a biolm in vitro. Chamberslides were inoculated with either NTHI strain 723 modA2(22)ON or modA2(24)OFF and allowed to

form biolms for 24 h before being stained with a viable bacterial stain and subjected to confocal imaging. As shown in Fig. 6, biolms formed by the modA2ON variant were notably more robust than those formed by modA2OFF.

DiscussionPhenotypic analysis of all modA ON/OFF pairs showed that all phasevarions inuence the protein prole of the outer membrane, with gross phenotypic changes evident with the ve strain pairs on silver-stained gels (Fig. 3). Our in-depth analysis of multiple current vaccine candidates50 through western blotting, and iTRAQ quantitative mass-spectrometry of OMPs, showed that modA phase variation inuenced the expression of several of these proteins, with some current vaccine candidates50 differentially expressed in multiple phasevarions (HMW1/2A, OMP P6). Most compelling in this analysis was the expression and phenotypic differences related to HMW1/2A. The HMW protein is a well-characterized adhesin expressed by NTHi, and has been investigated as a potential vaccine candidate33,34. However, our ndings suggest that differential expression is occurring in three separate modA phasevarions. This inuence appears to be direct (modA4 and modA5 directly affect HMW-A expression) or indirect (modA2 ON cells show higher expression of the accessory protein HMW2B). Although HMW1/2A is known to be phase variable itself, through heptanucleotide repeats in its promoter region37, sequencing of HMW in all the three strain pairs show that phase-variable expression of HMW1/2A itself is likely not responsible for the large differences in expression demonstrated by western blot and ELISA in the modA2, modA4 and modA5 ON/OFF strain pairs. The difference in HMW expression mediated by the modA4 phasevarion is sufcient to result in marked differences in the rate of HMW-specic opsonophagocytic killing (Fig. 4). This would likely result in selection for a modA4OFF subpopulation that express lower amounts of HMW. HMW has adhesion functions51 after selection has been relaxed; therefore, selection for adherent cells may counter select for the subpopulation of modA4OFF.

In this study, iTRAQ proteomic analysis of the OMP prole, and detailed gene expression studies of NTHi strain 723, containing modA2, the most prevalent modA allele found in all the clinical isolates (Fig. 1), showed that ModA2 has a major effect on gene expression and phenotype. Together with animal studies showing consistent selection for cells that have switched from modA2OFF to ON in our chinchilla model system of OM, this indicates that switching of the modA2 phasevarion plays an

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important role in niche adaptation to the middle ear. This could be due, in part, to increased HMW expression, despite this making the cells more susceptible to an adaptive immune response. Here, the balance between adherence to host cells, a key step in colonization, and expression of this immunogenic surface protein appears to have been achieved: a modA2ON strain (increased in HMW1/2A expression) may be more adherent when compared with modA2OFF (decreased HMW1/2A). Perhaps the advantage of increased adhesion early in infection through increased HMW1/2A expression gives a selective advantage, with a gradual decrease in expression of HMW1/2A occurring over prolonged periods of host colonization, thus reducing the potential for an adaptive immune response to HMW1/2A52.

Expression of iron and haem-acquisition-associated genes have previously been shown to increase in NTHi recovered from middle ear effusions in patients with OM53. Thus, strains lacking a functional ModA2 protein (modA2OFF and modA2::kan) appear to be primed to cope well in the iron-restricted host environment through increased production of a number of iron-acquisition factors. However, this may, in fact, be counterproductive: high iron concentrations are known to increase oxidative stress through the Fenton reaction54, and may, in fact, decrease the tness of modA2OFF strains in some host environments. Several genes involved in anaerobic metabolism are upregulated in modA2::kan (Table 3). Dimethylsulfoxide reductase is a virulence factor in the Pasteurellaceae55; and phosphoenolpyruvate carboxykinase, which feeds phosphoenolpyruvate into gluconeogenesis to generate a pool of glucose phosphate, is key to the pathogenesis of Staphylococcus aureus56. Several gene-encoding enzymes involved in glycogen synthesis are also upregulatedglycogen is important for biolm formation in Salmonella enteritidis57 and E. coli58. However, many of these genes were shown to be key when studying mutants in these systems: an all or nothing approach. The subtle changes resulting from modA2 phase variation (for example, phosphoenolpyruvate carboxykinase is only B1.7-fold upregulated in modA2::kan; enzymes involved in glycogen synthesis are all B1.5-fold upregulated in modA2::kan) appear to give no competitive advantage to modA2OFF compared with modA2ON when infecting the middle ear. Indeed, the changes in modA2ON are, in fact, preferentially selected. Whether this is due to the increased adhesion afforded by increased HMW-A proteins in modA2ON, the potential for increased oxidative stress through increased iron accumulation in modA2OFF leading to decreased tness, some result of metabolic ux due to differences in the levels of respiratory and associated enzymes, or a combination of all these factors, remains to be elucidated. Moreover, as the NTHi 723 modA2ON strain was able to form more robust biolms in vitro, this may also contribute to selection for the ON variant in the middle ear niche in vivo. Thus, selection against modA2OFF, and selection for modA2ON, may combine in vivo.

Recent work17,59,60 supports a key role for phase variation of individual factors in niche adaptation, independent of the established role of phase variation in immunoevasion. In this study, we have focused on the biphasic epigenetic switch that results in pleiotropic differential regulation of a phase-variable regulon of genesthe phasevarion. Previous studies have suggested a role for phasevarions in virulence6,7,9,10,16,41, but the impact of this novel biphasic epigenetic switch was untested in vivo. In this study, we observed for the rst time a consistent selection for individuals that switched from modA2OFF to ON within the middle ear niche in the chinchilla model of OM. This in vivo data reveals a clear tness advantage for modA2ON in this niche. Furthermore, we have shown that ve phase-variable modA alleles predominate in clinical OM isolates and healthy

carriers, suggesting a link between NTHi phasevarions and the potential to transmit and cause disease. All candidate phasevarions examined were shown to regulate multiple genes, including potential vaccine candidates. Dening the stable immunological target that NTHi represents requires a full analysis of the impact of phasevarions on NTHi gene expression, and future vaccine candidates will need to be assessed to conrm that their expression is not inuenced by the epigenetic changes that result from phasevarion ON/OFF switching. Our recent studies describing a distinct, six-phase epigenetic switch in the major Gram-positive pathogen Streptococcus pneumoniae61 indicates that bacterial epigenetics is a key emerging eld in bacterial pathogenesis and a new challenge to vaccine development for these important human pathogens.

Methods

Bacterial strains and cultures. NTHi strains 723, 477 and 1209 were received from the Finnish Otitis Media study group62. NTHi strain C486 was isolated from a child with otitis media63. Strain R2866 was isolated from a child with sepsis64. NTHi were routinely cultured in BHI broth (Oxoid) supplemented (sBHI) with hemin (1% v/v) and NAD (2 mg ml 1) or sBHI agar (as broth but with 1% w/v bacteriological agar; Oxoid). Liquid cultures were grown aerobically at 37 C with shaking at 90 r.p.m. Plates were grown at 37 C supplemented with 5% (v/v) CO2.

E. coli DH5a (Coli Genetic Stock Centre, Yale University, USA) and BL21(DE3) (Merck Millipore) strains were grown at 37 C in Luria-Bertani (LB) broth supplemented with ampicillin (100 mg ml 1) or kanamycin (50 mg ml 1) as required.

Molecular biology. All restriction endonucleases were purchased from New England Biolabs. Primers were purchased from Sigma-Aldrich and are detailed in Supplementary Table 5. PCR was carried out as recommended by the manufacturers instructions (Promega; EMD Millipore, USA). Sequencing was carried out using Big Dye 3.1 (Perkin Elmer) and PCR products puried using the Qiagen PCR purication kit according to the manufacturers instructions. Samples were sequenced by the Grifth University DNA sequencing facility (GUDSF), Brisbane, Australia, or the Australian Equine Genetics Research Facility, University of Queensland, Brisbane. Primers used for sequencing the HMW promoter region (HMW1-F; HMW2-F; HMW3-F and HMW-R; Supplementary Table 5) were based on those used previously37. The modA DRD region was analysed as previously described6. Briey, PCR products encompassing the DRD were amplied using primers Him6 and Him11 (Supplementary Table 5), sequenced and compared with modA allele reference sequences described6,15. Natural ON and OFF strains were isolated by fragment length analysis of the modA repeat tract of multiple single colonies using the uorescently labelled forward primer Him1F and the reverse primer Him3 (Supplementary Table 5)6, and fragments were analysed by AEGRC or GUDSF. Strains containing 490% ON or OFF were considered to be natural ON or OFF, respectively, and were used in subsequent studies.

PCR products generated for cloning into the pET51 Ek/LIC cloning vector (EMD Millipore) were prepared using KOD Hot-start DNA polymerase (EMD Millipore) using gene-specic primers for modA2 or siaB, (Supplementary Table 5) according to the manufacturers instructions. Overexpression of ModA2 and SiaB was carried out using E. coli BL21 cells, which were induced by the addition of IPTG to a nal concentration of 0.5 mM for 2 h at 37 C with shaking at 120 r.p.m.

RNA extraction. Triplicate cultures of NTHi strains 723 modA2ON and 723 modA2::kan were grown to exponential phase (optical density at 600 nm 0.3 to

0.4) in sBHI broth before RNA extraction. Growth rates of strain pairs used to make RNA for microarray comparison were equivalent, ensuring that the samples taken were in the same growth phase. Culture media for RNA preps were free of antibiotics. Approximately 100 mg of total RNA was prepared from each sample using the RNeasy Midi Kit according to the manufacturers instructions (Qiagen). The triplicate samples were pooled and the integrity and concentration of RNA was determined via micro-uidic analysis on a bio-analyser (Agilent Technologies).

Microarray analysis. All the microarray analyses were performed onH. inuenzae genome arrays (TIGR; http://pfgrc.tigr.org/

Web End =http://pfgrc.tigr.org/ ). Each microarray consists of 4,454 70-mer oligonucleotides representing ORFs from H. inuenzae strains Rd KW20, 86-028NP, R2846 and R2866. Methods and analysis were as previously described65. Briey, 5 mg of each total RNA sample was labelled using random hexamers and direct incorporation of uorescently Cy3- or Cy5-labelled nucleotides as described66. Hybridizations were performed in triplicate and incorporated a dye swap to account for dye bias. After hybridization, arrays were washed and scanned on an Agilent G2565BA microaray scanner. Images of the hybridizations were analysed using Imagene 5.5 (BioDiscovery) and the mean

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foreground, mean background and spot/signal quality determined. Primary data were imported into an in-house installation of BASE (http://kidney.scgap.org/base

Web End =http://kidney.scgap.org/ http://kidney.scgap.org/base

Web End =base ). After print-tip intensity-independent Lowess normalization, differential expression was dened using a robust statistical method rather than simple fold change. All the genes were ranked using the B-statistic method where both fold change and variance of signals in replicates are used to determine the likelihood that genes are truly differentially expressed.

Quantitative real-time PCR. Oligonucleotides (Supplementary Table 5) were designed using Primer Express 1.0 software (ABI Prism; PE Biosystems). Real-time PCR reactions were performed in triplicate using RNA isolated from 723 mod-A2ON and 723 modA2OFF. cDNA was synthesized using NEB Protoscript II and random hexamers (Invitrogen; 50 ng ml 1) according to the manufacturers instructions. Reverse transcriptase reactions lacking Protoscript II were performed as a negative control. All the real-time PCR reactions were performed in a 25-ml mixture containing a 1 in 5 dilution of the cDNA preparation (5 ml), 10xSYBR Green buffer (PE Applied Biosystems) and 2 mM of each primer. 16S RNA was used as the standard control in each quantitative PCR. Amplication and detection of specic products were performed with the ABI Prism 7700 sequence-detection system (PE Applied Biosystems) with the following cycle prole: 95 C for 10 min, followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. The data were analysed with ABI prism 7700 (version 1.7) analysis software. Relative gene expression between 723 modA2OFF and 723 modA2ON was determined using the 2 DDCT relative quantication method.

SMRT sequencing and methylome analysis. Genomic DNA from each natural modAON and kanamycin knockout pair was prepared using the Qiagen genomic DNA midi kit according to the manufacturers instructions. SMRT and methylome analysis was carried out as done previously21,22. Briey, genomic DNA was sheared to an average length of B10 kb using g-TUBEs (Covaris, Woburn, MA, USA) and

SMRTbell template-sequencing libraries were prepared using sheared DNA. DNA was end repaired, then ligated to hairpin adaptors. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA). Primer was annealed and samples were sequenced on the PacBio RS II (Menlo Park, CA, USA) using standard protocols for long insert libraries. Plasmid midipreps from E. coli cells expressing NTHi 723 ModA2 and a negative control expressing a non-methylase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturers instructions, and analysed as above.

Preparation of OMPs from NTHi. NTHi modA ON/OFF pairs were grown in sBHI broth (50 ml) at 37 C overnight with shaking at 100 r.p.m. The cells were pelleted at 4,500 r.p.m. for 15 min at 4 C, resuspended in 4 ml 10 mM HEPESNaOH pH7.5 and OMPs were prepared as detailed previously67. Briey, cells were lysed by sonication, and debris pelleted as above. Sarcosyl was added to the claried supernatant to a nal concentration of 1% and incubated at 25 C for 30 min. Supernatants were then centrifuged at 110,000g for 90 min. Pellets were resuspended in 10 mM HEPES-NaOH pH7.5, sarcosyl added to a nal concentration of 1%, and incubation and centrifugation steps repeated twice more. Final pellets containing the OMP-enriched fraction were resuspended in 100 ml of 10 mM HEPES-NaOH pH7.5 and the protein concentration quantied using the BCA protein assay kit according to the manufacturers instructions (Thermo Scientic).

Western blot analysis. Each of the OMP preparations (5 mg) were run on the Novex Bis-Tris pre-cast gel system with MOPS running buffer according to the manufacturers instructions (Life Technologies). Ammoniacal silver staining was carried out to visualize proteins. Western blotting was carried out using nitro-cellulose membranes (Bio-Rad) and standard protocols68. All mouse (AD6 anti-HMW1/2A34; 1F4 anti-Hia35) and rabbit (LB1 anti-OMP P5 (ref. 28); chimV4 anti-OMP P5; anti-OMP P5) primary antibodies were used at a dilution of 1:2,500; all chinchilla (anti-OMP P2; anti-LPD27; anti-PDM27; anti-OMP P5/P6) primary antibodies at a dilution of 1:250. Anti-mouse-AP and anti-rabbit-AP secondary antibodies were used at a dilution of 1:5,000 (Sigma-Aldrich); protein A-AP secondary antibody was used at a dilution of 1:500 (Sigma-Aldrich) in blots where chinchilla primary antibodies were used. Blots were developed using SigmaFAST NBT/BCIP tablets according to the manufacturers instructions (Sigma-Aldrich). All the primary antibodies were raised by the authors laboratories, with specic references for those described previously.

ELISA assay. ELISA assays were carried out using standard protocols68 in 96-well Maxisorb plates (NUNC; Thermo Scientic). Cells were diluted to a 0D600 of 0.2

(2 108 c.f.u. ml 1), with 50 ml added per well. All the strains were assayed in

triplicate. Primary antibody AD6 against HMW34 was used at a starting concentration of 1:200 (NTHi strains C486 and 477) or 1:10,000 (NTHi strain 723) and serially diluted two-fold in 1 PBS pH 7.9. Secondary antibody (goat anti-

mouse HRP conjugate; Sigma-Aldrich) was used at a concentration of 1:10,000. Antibody was detected using TMB single-substrate solution as recommended by

the manufacturer (Sigma-Aldrich). The data were plotted as antibody dilution(x axis) versus absorbance at 450 nm (y axis), and data from specic titres in the linear range of the response curve picked for statistical analysis.

iTRAQ analysis. iTRAQ 1D nanoLC ESI MS/MS was carried out by the Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia. Approximately 10 mg of OMP preparation from duplicate samples of each modA ON/OFF pair was supplied for the analysis. Samples were buffer exchanged into 0.25 M TEAB and 0.05% SDS and quantied samples were reduced with TCEP, alkylated with MMTS and digested with trypsin. Digested samples were labelled, passed through an SDS removal column (Thermo Fisher) and dried. Labelled samples were resuspended in 50 ml of loading/desalting solution (0.1%

formic acid and 2% acetonitrile 97.9% water). Sample (20 ml) was injected onto a peptide trap (Michrome peptide Captrap) for pre-concentration and desalted with0.1% formic acid, 2% acetonitrile. Peptides were eluted from the column using a linear solvent gradient, with steps, from mobile phase A: mobile phase B (98:2) to mobile phase A: mobile phase B (65:35) where mobile phase A is 0.1% formic acid and mobile phase B is 90% ACN/0.1% formic acid at 600 nl min 1 over a 100-min period. After peptide elution, the column was cleaned with 95% buffer B for 15 min and then equilibrated with buffer A for 25 min before next sample injection. The reverse phase nanoLC eluent was subject to positive ion nanoow electrospray analysis in an information dependent acquisition mode (IDA). In IDA mode, a TOFMS survey scan was acquired (m/z 4001,500, 0.25 s), with the 10 most intense multiply charged ions (counts 4150) in the survey scan sequentially subjected to

MS/MS analysis. MS/MS spectra were accumulated for 200 ms in the mass range m/z 1001,500 with the total cycle time of 2.3 s. The experimental nanoLC ESI ms/ms data were submitted to ProteinPilot V4.2b (AB Sciex) for data processing.

MIC assay. The MIC was measured by broth microdilution in triplicate experiments as described previously69 using mid-log phase NTHi cells grown aerobically in sBHI. Briey, 50 ml of each culture was added to 96-well plates in which antibiotics had been serially diluted, and plates grown at 37 C with 5% CO2 for 24 h. The MIC (mg l 1) was determined as the last dilution at which turbidity was observed following overnight growth. All the assays were performed in triplicate.

Opsonophagocytic killing assays. The growth conditions of the bacteria, the growth and differentiation of the HL-60 cells, (ATCC CCL-240) and the opsonophagocytic assay itself were performed as described previously33,42. The opsonophagocytic assay was performed in 5-ml capped polystyrene tubes (Sarstedt, Newton, NC). The complement source was human serum collected from a single healthy adult that was adsorbed to remove serum IgG by passing aliquots repeatedly over a protein G afnity column at 4 C. Antibody (guinea pig anti-strain 12 HMW1/HMW2 antiserum42) was serially diluted, and B5 103 c.f.u.

mid-log phase bacterial cells were added to each dilution, and tubes incubated at 37 C with 5% CO2 for 15 min. Following this, complement was added, followed by immediate addition of differentiated HL-60 cells. Tubes were incubated at 37 C with 200 r.p.m. horizontal shaking. At the end of the 90-min incubation period, c.f.u. were calculated by plating 10 ml of each culture and incubating overnight at 37 C with 5% CO2. The per cent killing at each serum dilution was calculated by determining the ratio of the bacterial colony count at each dilution to thatof the complement control using the modA4 ON/OFF pair (NTHi strain C486). Pre-immune sera and anti-Hia sera were used as negative controls.

Biolm formation on chambered coverglass slides. Formation of NTHI biolms was performed in eight-well-chambered coverglass slides (Thermo Scientic, Waltham, MA) as described previously70. Briey, mid-log phase cultures of NTHi strain 723 modA2ON and modA2OFF grown in sBHI were diluted with fresh pre-warmed media and used to inoculate 4 104 c.f.u. in 200 ml total volume per well.

Slides were incubated at 37 C with 5% atmospheric CO2 and the growth medium was replaced with fresh medium after 16 h. Twenty-four hours after seeding, biolms were stained with LIVE/DEAD BacLight stain (Life Technologies) and xed overnight in xative (1.6% paraformaldehyde, 2.5% glutaraldehyde, 4% acetic acid in 0.1 M phosphate buffer, pH 7.4). Fixative was replaced with saline before imaging on a Zeiss 510 Meta-laser scanning confocal microscope; images were rendered with Zeiss Zen software.

Chinchilla model of NTHi-induced OM. Adult chinchillas (Chinchilla lanigera) were supplied by Rauschers Chinchilla Ranch, LaRue, OH. Chinchillas were not sex-differentiated, and were classed as adult if weighing between 500700 grams. Chinchillas were allowed to acclimate in the vivarium for 710 days before beginning the study. In two separate studies, cohorts of ve chinchillas each were established, then challenged intranasally and transbullarly with either: (1) NTHi strain 723 modA2(22)ON or (2) NTHi strain 723 modA2(24)OFF at a challenge dose of B1 108 c.f.u. intranasally and 750 c.f.u. transbullarly. A total of 10

animals (20 ears) were challenged with each of these variants of NTHI strain 723. The animals were then monitored daily via video otoscopy for 22 days. Delivered doses were conrmed by dilution plate counts of inocula on chocolate agar. On days 2, 4, 7, 10, 14, 18 and 22 after challenge epitympanic taps (removal of a small

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aliquot of uid from the middle ear space) were performed as described previously. On collection of samples, 10 ml were placed in a sterile Eppendorf tube on ice. This aliquot was used for evaluation of the colony-forming unit of NTHi per ml sample. The remainder of the collected samples was immediately centrifuged at 4 C for3 min, the supernatant was carefully removed and the pellet was snap frozen in liquid nitrogen and stored at 80 C. On day 22 (unless removed from the study

early owing to morbidity), anaesthetized chinchillas were euthanized and the bullae were dissected from the skull. The left bulla from each chinchilla was aseptically opened and imaged. The middle ear mucosa, including any adherent mucosal biolm was removed, homogenized in sterile PBS and snap frozen.

With regard to animal treatment and handling, all protocols were approved by the Nationwide Childrens Hospital Animal Care and Use Committee, in accordance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals.

Fragment analysis to determine modA2 ON/OFF status in chinchilla samples.

Fluid samples were taken from the left and right middle ears on days 0, 2, 4, 7, 10, 14, 18 and 22 from each cohort of 10 chinchillas that had been challenged with either strain 723 modA2(22)ON or strain 723 modA2(24)OFF. The ratio of mod-A2ON and modA2OFF from the starting inoculum (day 0) compared with days 4, 7, 10, 14, 18 and 22 was veried via fragment analysis for each of the chinchillas. Samples were thawed briey on ice, with 1 ml serving as the template in a 25 ml

GoTaq PCR reaction (Promega) using primers Him1F and Him3 (Supplementary Table 5) as described previously6. Samples were run using GeneScan uorescent-PCR fragment-length analysis by GUDSF, and analysed using Peak Scanner software (Applied Biosystems).

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Acknowledgements

Work was supported by NHMRC (Australia) Project Grant 1034401 to M.P.J. andL.O.B., NHMRC Program Grant 565526 to A.G.M. and M.P.J., NHMRC Program Grant to M.P.J. 1071659, Grant NIH/NIDCD (USA) R01DC003915 to L.O.B., and grant NIH/ NIAID (USA) R01AI 81887 to S.J.B. iTRAQ analysis was undertaken at APAF, the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS).

Author contributions

L.O.B. and M.P.J. conceived the project; J.M.A., L.O.B. and M.P.J. designed the experiments; J.M.A., Y.N.S., K.L.F., M.B., T.A.C., J.A.J., K.L.B., F.E.-C.J., P.M.P. and S.J.B. performed the experiments; J.M.A. and Y.N.S. analysed the data; A.G.M., S.J.B. S.M.G., J.K. and A.L.S. provided advice; J.M.A., L.O.B. and M.P.J. wrote the paper.

Additional information

Accession codes. The SMRT methylome data for strains 723, C486, 477, 1209 and R2866 have been deposited in REBASE. Annotated genomic sequences for strains 723, C486, 477 and 1209 have been deposited in NCBI GenBank with accession codes CP007472 (strain 723), CP007471 (strain C486), CP007470 (strain 477) and JMQP01000000 (strain 1209). iTRAQ proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identier PXD002210. The microarray data have been deposited in the NCBI Gene Expression Omnibus database with accession codes GSE69831.

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Competing nancial interests: T.A.C., M.B. and J.K. are full-time employees of Pacic Biosciences, a company commercializing the use of single molecule, real-time (SMRT) sequencing. The remaining authors declare no competing nancial interests.

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How to cite this article: Atack, J. M. et al. A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus inuenzae. Nat. Commun. 6:7828 doi: 10.1038/ncomms8828 (2015).

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