Nontypeable Haemophilus influenzae:understanding virulence andcommensal behaviorAlice L. Erwin1,2 and Arnold L. Smith1

1 Microbial Pathogens Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle,

WA 98109-5219, USA2 Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA

Review TRENDS in Microbiology Vol.15 No.8

Haemophilus influenzae is genetically diverse and existsas a near-ubiquitous human commensal or as a path-ogen. Invasive type b disease has been almost elimi-nated in developed countries; however, unencapsulatedstrains – nontypeable H. influenzae (NTHi) – remainimportant as causes of respiratory infections. Respirat-ory tract disease occurs when NTHi adhere to or invaderespiratory epithelial cells, initiating one or more ofseveral proinflammatory pathways. Biofilm formationexplains many of the observations seen in chronic otitismedia and chronic bronchitis. However, NTHi biofilmsseem to lack a biofilm-specific polysaccharide in theextracellular matrix, a source of controversy regardingtheir relevance. Successful commensalism requiresdampening of the inflammatory response and evasionof host defenses, accomplished in part through phasevariation.

Why study Haemophilus influenzae?H. influenzae is a human-restricted gram-negativebacterium that is part of the normal nasopharyngeal floraof most humans. Until twenty years ago, the study of thisspecies focusedonH. influenzae serotypeb,amajorbacterialpathogenof children. Since the introduction in1990of typebpolysaccharide–protein conjugate vaccines, invasive H.influenzae disease has been nearly eliminated in developedcountries. H. influenzae that lack capsular polysaccharidesare referred to as nontypeable (NTHi). Although NTHi aremost commonly associatedwith asymptomatic colonization,they are also pathogenic.H. influenzae is frequently associ-ated with otitis media, chronic bronchitis, and community-acquired pneumonia, and the strains associated with thesemucosal infections are NTHi [1–3]. Colonization by anysingle NTHi is transitory, with new strains acquired everyfew months [4]. A major goal of NTHi research today isidentification of bacterial factors that influence whetheracquisition of anNTHi strain results in disease or in asymp-tomatic colonization. At the time of writing this review,complete or nearly complete genome sequences areavailable for three strains: the otitis media strains86-028NP [5] and R2846 (also known as strain 12) [6] andthe invasive NTHi strain R2866 (also called strain Int1) [7].

Corresponding author: Smith, A.L. (arnold.smith@sbri.org).Available online 27 June 2007.

www.sciencedirect.com 0966-842X/$ – see front matter � 2007 Elsevier Ltd. All rights reserv

At least ten other strains are reportedly being sequenced,most isolated frommiddle ear aspirates [8]. Here, we reviewthe current understanding of the interactions betweenNTHi and host tissue that are thought to lead to disease,and we will summarize the evidence that NTHi strains,including the sequenced strains, differ in these or otheraspects of virulence.

What is H. influenzae?The species H. influenzae is defined by its nutritionalrequirements: both b-nicotinamide adenine dinucleotideand heme must be supplied for growth in ordinary labora-tory conditions. Heme is not required for anaerobic growth,but seems to be important for full virulence. Eachsequenced NTHi strain has two to four hpg genes whichfacilitate heme acquisition from hemoglobin or hemo-globin-haptoglobin complexes. Deletion of the three hpggenes from NTHi 86-028NP resulted in a delayed onset ofinfection in the chinchilla otitis media model, and theinfection was also of shorter duration [9].

Are all H. influenzae equally virulent?Encapsulated H. influenzae are unquestionably morevirulent thanNTHi. Nearly allH. influenzae strains associ-ated with systemic disease possess carbohydrate capsules,and these capsules are essential for virulence in exper-imental infections [10]. For NTHi, several surface struc-tures have been reported to affect virulence, but there is nosingle feature that is characteristic of all disease-associ-ated strains. It is possible that NTHi strains with similarpotential for causing disease might possess different com-binations of virulence-related genes. Further, it is notknown whether NTHi strains differ in virulence.

A recent study provided strong evidence for a group ofHaemophilus isolates with low potential for virulence[11]. In a longitudinal study of 118 patients with chronicobstructive pulmonary disease, Murphy et al. isolatedHaemophilus strains from >600 sputum cultures. Itwas noted that certain isolates initially identified asNTHi (i.e. unencapsulated strains requiring NAD+ andheme) had unusual growth characteristics. Furtherinvestigation identified these isolates as meeting allcharacteristics of H. haemolyticus except that many ofthem lacked hemolytic activity. Correlation of laboratory

ed. doi:10.1016/j.tim.2007.06.004

356 Review TRENDS in Microbiology Vol.15 No.8

findings with clinical data led to the recognition thatacquisitions of ‘non-hemolytic H. haemolyticus’ wererarely, if ever, associated with exacerbations of symptomsof bronchitis, in comparison to acquisitions of ‘true’ NTHi.Similar strains were isolated from pharyngeal cultures ofhealthy children, but not from the middle ear of childrenwith otitis media [11]. Multilocus sequence typing(MLST) and 16S RNA sequencing indicated that the H.haemolyticus strains formed a phylogenetic cluster dis-tinct from NTHi. Investigators should not only evaluatethe possibility that strains isolated from the airway andidentified as NTHi might be H. haemolyticus, but shouldconsider that there might be other distinct subspecies, asyet undiscovered, that differ phenotypically and in viru-lence potential. Most of the studies cited here used well-characterized strains of NTHi that have been typed byMLST and are unlikely to be H. haemolyticus.

H. influenzae virulence is host mediatedThe word virulent is derived from the Latin virulentus(poisonous). For H. influenzae, as for many pathogens, theorganism does not produce a true poison or toxin. Diseaseresults from the response of the host to bacterial factors,particularly endotoxin (lipopolysaccharide). Because oftheir short saccharide chains, the lipopolysaccharides ofH. influenzae are often referred to as lipooligosaccharides(LOS), the term that will be used here. The interactionbetween bacteria and host cells is affected by LOS struc-ture, which varies between strains and also among bac-terial cells within a strain. The initial interaction betweenNTHi and the host is the adherence of bacteria to themucus or cells of the upper airway. The nasopharynxcontains both stratified squamous epithelium and pseu-dostratified ciliated columnar epithelium typical of therespiratory tract; the latter containmucus-secreting gobletcells. Submucosal glands also contribute to surface mucus.Subsequent steps might include invasion of respiratoryepithelial cells, transcytosis to the sub-epithelial compart-ment, or formation of microcolonies and ultimately a bio-film. These processes, and the host response to them, havebeen studied using several different NTHi strains that arenow recognized to differ in genetic content. A summary ofthe reported NTHi–host-cell interaction using differentcells, including transient transfection systems, is depictedin Figure 1.

Primary role of adherenceSeveral H. influenzae adhesins are known (reviewed in[12]). The fimbriae encoded by the hif locus were firstcharacterized in a type b strain, in which they were foundto mediate adherence to all cell types found in the respir-atory tract. Not all NTHi contain the hif locus; when it ispresent it is thought to be most important in the earlysteps of colonization [13]. Many NTHi, including 86-028NP and R2846, contain the hmw1 and hmw2 loci,encoding high molecular weight adhesins. HMW1 andHMW2 have different binding specificities. HMW1 tar-gets sialyl-a2,3 hexose [12], which is not prevalent in theupper respiratory tract [14]. This indicates that for hmw-containing strains, other adhesins are more important inthe initial colonization. Most NTHi strains lacking hmw

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possess genes for a different adhesin, Hia, which issimilar in sequence to the surface fibrils (Hsf) previouslyidentified in H. influenzae type b [12]. Strain R2866includes both the hia and hif loci. Other surface struc-tures shown to bind to eukaryotic cells include the outermembrane proteins Hap [15], outer membrane protein(OMP)-2 [16] and OMP-5 [17]; the lipoprotein PCP [18]; aprotein associated with colony opacity [19]; and a phos-phorylcholine (ChoP) moiety on LOS [20]. Most of thesepotential adhesins have substantial interstrain sequenceheterogeneity. The hif and hmw loci and the licA gene(required for phosphorylcholine synthesis) are subject tophase-variable expression, mediated by changes in lengthof tandem repeat tracts [21–23]. Table 1 summarizes theH. influenzae surface structures and the known cognatehost-cell ligands.

How does respiratory tract colonization occur?After being deposited on respiratory epithelium, NTHimust overcome the innate clearance mechanisms of theupper respiratory tract. Mucociliary clearance and anti-bacterial molecules such as lysozyme, lactoferrin andantimicrobial peptides are all designed to remove infec-tious particles from the airway. Mucus contains the gel-forming mucins, MUC5AC, MUC5B and MUC2; respirat-orymucins bindfimbriatedNTHi [24] and should facilitatebacterial clearance. However, because fimbrial expressionis phase-variable, the emergence of nonfimbriated strainsthat possess other adhesins will facilitate airway coloni-zation.

The secretion of IgA protease by NTHi is inferred tofacilitate colonization in individuals with S-IgA1 thatrecognizes surface molecules of NTHi. The gene (iga)encoding IgA1 protease is present in at least 97% of H.influenzae, but seems to be absent from nonpathogenicHaemophilus species, including H. haemolyticus [25,26].Some NTHi have a second IgA1 protease gene, igaB, theprevalence of which in sputum and middle ear isolates issignificantly greater than in ‘carriage’ isolates (P < 0.001,P = 0.011 and P = 0.0014, respectively) [27]. Neither lyso-zyme nor lactoferrin inhibit the growth of NTHi, but theserine protease activity of lactoferrin cleaves NTHi IgAprotease and the adhesin Hap [28,29]. This activity mightmitigate colonization.

Human b defensin-1 hadminimal activity toward strain12 (R2846), whereas the antibacterial activity of the indu-cible b defensin-2 was found to be comparable to b-lactamantibiotics [30]. The susceptibility of strain 2019 to b

defensin-2 was increased in a mutant (htrB) with reducedacylation of lipid A [31]. These peptides act by damagingthe outer membrane, causing loss of intracellular contentsincluding potassium. The Sap transporter, which is upre-gulated in strain 86-028NP during experimental otitismedia, mediates resistance to b defensins [32] and alsoseems to counteract the cellular potassium loss [33]. AsapA mutant of 86-028NP has markedly attenuated viru-lence in the chinchilla model of otitis media [32]. For theantimicrobial peptide LL-37 (also called hCAP18), thesusceptibility of strain H233 was reduced by expressionof the ChoP moiety on LOS [34].

Figure 1. Intracellular signaling pathways activated by the binding of H. influenzae to the cell surface. The information is extracted from published reports of experiments

using macrophages, respiratory epithelial cells and transient transfection systems. The major intracellular signaling molecules are depicted in color. The interaction

between LBP, CD14 and LOS occurs before they bind to TLR2. Abbreviations and references: CEACAM, carcinoembryonic antigen-related cell-adhesion molecules [37];

Dectin, b-glucan receptor [71]; EGFR, epidermal growth factor receptor [38]; HB ECM, heparin-binding extracellular matrix proteins [70]; HSP70, heat shock protein70

[75,76]; IKKa/b, IkB kinase; LBP, lipopolysaccharide binding protein [73]; LOS, lipooligosaccharide; MEK3/6, MAPK/Erk kinase; MKP-1, MAP Kinase phosphatase-1; MyD88,

myeloid differentiation antigen -88; NF-kB, nuclear factor kB; OMP, outer membrane protein [18,77,79,81]; PI3K, phosphoinositol-3 kinase; PAFR, platelet activating factor

receptor [39]; PKC, protein kinase C; SMAD, receptor serine/threonine kinase for direct transport to nucleus; Src, cytoplasmic tyrosine kinase; TAK1, transforming growth

factor-b activated kinase; TGF-b, transforming growth factor-b [74]; TLR, toll-like receptors [72]; TRAF, tumor necrosis factor receptor-associated receptor factor.

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Dampened inflammation required for commensalism?The nature of the initial binding of bacteria to host cellscould be the crucial event in virulence. Work in severallaboratories, using different strains and cells, indicatesthat binding to epithelial cells by multiple ligands

Table 1. H. influenzae adhesins for eukaryotic cells and their cogn

H. influenzae structure Cognate eukaryotic ligand(s)

Fimbriae (pili) Fibronectin, heparin binding matrix protein

Heat shock protein TLR-2 and TLR-4 sialylated gangliosides (S

Hsf Vitronectin

OMP-2 TLR-2

OMP-5 CECAM

OMP-6 TLR-2

Peptides from OMP-4, OMP-

6 and PCP

TLR-2

LOS ChoP PAFR

Hap Fibronectin, laminin, type IV collagenaThe indication of ‘type b’ or ‘NTHi’ indicates the bacteria in which the interaction was stud

NTHi.

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initiates a proinflammatory response via several pathways(Figure 1). Adherence triggers microvillus formation inrespiratory epithelial cells, leading to engulfment of NTHi[35]. The primary ligands seem to be the Toll-like receptorsTLR2 and TLR4 and the platelet-activating factor receptor

ate ligands

Initiates intracellular signaling Commenta Refs

s Not known Type b [58,69,70]

O4) Inflammation via MyD88/IRAK/NF-kb NTHi [78]

Not known Type b [69]

TNF-a and IL-6 via MyD88 Type b [79,80]

Increased expression of CD105

(less exfoliation)

Strain Rd [37]

Induction of NF-kB NTHi [81]

Synergizes with IFN-b NTHi [18]

PtdIns 3K calcium signaling NTHi 2019 [39]

Not known NTHi N187 [15]

ied; it is not intended to imply that the adhesin or interaction is limited to type b or to

358 Review TRENDS in Microbiology Vol.15 No.8

(PAFR). Signaling by these ligands initiates proinflamma-tory signaling through the mitogen activated kinase cas-cade or by way of p38 activation [36]. Treatment ofepithelial cells with NTHi or bacterial fractions does notlead to appreciable cell death. However, polarized epi-thelial cells infected with H. influenzae strain Rd KW20have been noted to exfoliate. This process seems to beinhibited by binding of the bacterial protein OMP-5 toCECAM, triggering the expression of CD105 [37].

Intracellular residence can shield the NTHi from theaction of antibodies and antibiotics and could provide theorganism a means of avoiding clearance from the respir-atory tract. For intracellular residence to be an effectivepathway in the life cycle of NTHi, there must be a mech-anism for dampening or terminating the inflammatoryprocess. Using a transient transfection system, Mikamiet al. showed that a lysate of NTHi strain 12 (R2846)contained a factor that activated the epidermal growthfactor receptor. Activation of this receptor downregulatesthe p38-mediated proinflammatory response [38]. A sep-arate series of experiments [20,39] studied the interactionof NTHi strain 2019 with PAFR. Binding of ChoP, which isvariably present on NTHi LOS, to the PAFR activates thephosphoinositide 3-kinase (PI3-K); this pathway caninitiate anti-inflammatory pathways by way of negativeregulation of TLR-2, TLR-4 and TLR-9 [40]. Because PI3-Kis activated by NTHi, it is expected that inflammationwould be downregulated early after invasion. ChoPexpressed on the LOS of NTHi engages the PAFR throughmolecular mimicry, and NTHi probably enters the cell in aPAFR-linked pinocytotic vacuole. PAFR is a G-proteincoupled receptor and binds to arrestins which targetvacuolar contents to clathrin-coated pits or to the endocyticpathway, both seem to occur with NTHi. In addition, b-arrestins uncouple G-protein receptors terminating theirsignaling. Limiting the inflammatory response is probablyessential for NTHi to be a successful commensal.

Invasion across respiratory epitheliumAdherence of NTHi to respiratory epithelial cells can alsoresult in transcytosis of organisms into the subepithelialcompartment. NTHi were found in the bronchial submu-cosa of patients with chronic bronchitis >50 years ago,whereas more recent studies identified NTHi betweenepithelial cells and in clusters in the submucosa of similarpatients [41]. Most often the bacteria are extracellular, butthey have been identified in macrophage-like cells [42]. ForNTHi strain 2019 to transcytose murine M cells derivedfrom Peyer’s patches, a wild-type LOS structure wasrequired [43]. Once in the submucosa, NTHi can be pro-cessed for immune presentation.

As noted, NTHi LOS is involved in multiple aspects ofvirulence. LOS structure affects the ability of bacteria toadhere effectively to cells and to invade them. The LOS-dependent intracellular signaling that occurs after NTHienter respiratory epithelial cells seems to be necessary forcolonization of human respiratory epithelium. The coloni-zation of human tissue by NTHi was studied using a modelin which normal human bronchiolar xenografts areimbedded in severe combined immunodeficient (SCID,nu/nu) mice. Inoculation with as few as 10 colony forming

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units (CFU) of NTHi strain R3001 (isolated from apatient with cystic fibrosis) results in colonization of thexenograft. Within 48 h, the xenograft lumen contains107 CFU. By five days, copious mucus is present and thebacterial density is �108 CFU per xenograft. Colonizationin this model was found to be affected by LOS structurebecause mutants of strains R3001 and 2019 in which theacyltransferase gene htrB was inactivated were unable tocolonize the respiratory epithelium in the xenograft model[35].

Does NTHi produce biofilms?Several recent studies have evaluated the concept thatbiofilm production contributes to the pathogenesis of NTHirespiratory infections, particularly those that becomechronic such as otitis media with effusion and chronicbronchitis. Biofilms would be expected to protect bacteriafrom antibiotics and from innate respiratory epithelialclearancemechanisms. Inmost cases, formation of biofilmsis controlled by a regulatory switch [44], and the transitionfrom planktonic to biofilm growth involves production of anextracellular polysaccharide [45]. Although neither ofthese has been well documented for NTHi, there is evi-dence that NTHi can grow in an aggregate form that isconsistent with a biofilm and that this form of growthaffects virulence. NTHi consisting of aggregates of bacteriain a matrix containing LOS, outer membrane proteins andIgA have been observed on immortalized human middleear epithelial cells in vitro [46] and the Calu-3 cell line [47].When grown for extended periods on Calu-3 cells, the cellsbecome less sensitive to gentamicin and continue to elicit aproinflammatory response [47]. Structures consistent witha biofilm have also been observed onmiddle ear tissue fromchinchillas in experimental infections [48,49] and fromchildren with chronic otitis media undergoing tympanost-omy tube placement [50]. Although an extracellular matrixhas been visualized [51], no biofilm-specific exopolysac-charide has been identified. For strain 2019, biofilm-likestructures form on respiratory epithelial cells in vitro and,in the chinchilla model of otitis, media contain an extra-cellular polymer that includes sialic acid [49] in addition toLOS. A recent study of strain 86-028NP from chinchillamiddle ears identified double-stranded DNA and type IVpilin protein in an extracellular matrix [49]. A study ofstrain 9274 found that the extracellular matrix containedthe outer membrane proteins Hap, HMW1 andHMW2, butthat at least four other proteins were detected only inbacterial cells and not in the extracellular matrix [51].Studies using several NTHi strains have shown that pre-vention of sialic acid incorporation bymutation of the sialicacid activator gene siaB or the appropriate sialyltransfer-ase genes reduces virulence in the chinchilla and gerbilmodels of otitis media [52,53]. Both NTHi strains 2019 and86-028NP displayed ChoP expression to a greater extentwhen grown in biofilm conditions, compared with plank-tonic growth [46], and the increase in ChoP expression inbiofilms was associated with decreased endotoxic activity[46]. In strain 86-028NP, mutations in the ChoP transfer-ase gene licD led to a less dense surface growth that did notpersist in the middle ear of chinchillas [54]. It is unlikelythat LOS structure is the only factor affecting NTHi

Review TRENDS in Microbiology Vol.15 No.8 359

biofilm-like structures, as wecA mutants of NTHistrain 2019 do not form the structure and are attenuatedin virulence in the chinchilla model [49]. WecA has beencharacterized in Salmonella enterica as an undecaprenyl-phosphate a-N-acetylglucosaminyltransferase [55]; it hasno known role in LOS biosynthesis in NTHi.

NTHi strains differ in virulence-related phenotypesSome reports support the idea that differences betweenstrains in virulence-related phenotypes correlate withtheir clinical sources. Upon infection of H292 cells (ahuman adenocarcinoma cell line), NTHi strains that per-sist in the lower respiratory tract of patients with chronicbronchitis induced lower levels of IL-6 and IL-8 than non-persisting strains. This was not explained by differences inadherence to theH292 cells, and indicated that the reducedinflammatory potential of certain strains enables them topersist in the lungs [56]. NTHi isolated from the sputum ofpatients with chronic obstructive pulmonary disease whoare undergoing an exacerbation are more adherent torespiratory epithelial cells and induce the secretion ofmoreproinflammatory cytokines than strains causing asympto-matic infection [57]. Although adherence and the ability toinduce inflammation are not tightly linked, the obser-vations support the concept that certain in vitro pheno-types reflect virulence.

For other virulence-related phenotypes, strains varywidely but without apparent correlation with the clinicalsource. Bresser et al. reported that certain NTHi frompatients with chronic bronchitis showed a high level ofadherence to laminin and type I collagen, whereas otherstrains had a substantially lower level of adherence. Therewas no correlation between adherence to these extracellu-lar matrix proteins and whether the strains were persist-ent [58].

Phase variation complicates the study of virulence:serum resistance as an exampleInterpretation of strain comparison data are complicated bythe fact that virulence-related phenotypes are often subjectto phase variation. As each of a dozen or so phase-variablegenes in each strain switches on and off independently, aculture ofH. influenzae consists of a mixture of hundreds ofdifferent variants that differ in LOS structure or in expres-sion of surface antigens such as fimbriae. Such heterogen-eity permits selection of the derivative most fit for theimmediate environment. The practical result is that thephenotype of the laboratory grown culture can differ sub-stantially from that of the same strain when it was in thepatient. In particular, phase-variable aspects of LOS struc-ture have a crucial role in several aspects of virulence,including adherence to cells, induction of inflammationand resistance to complement.

Encapsulated H. influenzae are usually resistant to thecomplement-mediated bactericidal activity of pooled serumfromnormal adults. NTHi are usuallymuchmore sensitive,but display a wide range of susceptibility to human serum[59]. The serum resistance of strain R2866 has been studiedin some detail [60,61]. Strain R2866 (also known as Int1)was of particular interest because it was isolated from thebloodstream of a child with no known abnormality that

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would predispose him to bacteremia [7]. Strain R2866was found to cause bacteremia and meningitis in the infantrat and weanling rat models of invasive disease (unlike anyother NTHi strain tested) [7] and to have unusually highresistance to normal adult serum [62]. For this strain, high-level serum resistance depended on expression of a galacto-syltransferase encoded by the phase-variable gene lgtC [61].Analysis of the tetranucleotide repeat regionwithin the lgtCgene determined that the gene was out of frame (off) in aserum-sensitive variant (R3392) and in frame (on), enablingtranslation in the serum-resistant parent. Chemical anal-ysis of LOS confirmed that the principal glycoform in R2866differed from that in R3392 by a single hexose. lgtC-onvariants of R3392 with increased serum resistance wererecovered after serum selection, confirming the relationshipbetween phase variation and phenotype [60,61].

Several LOS biosynthetic genes in addition to lgtC areknown to affect serum resistance; further, the role ofdifferent genes in serum resistance differs from strain tostrain. lgtC expression (reflected by binding of monoclonalantibody 4C4) had been reported to increase serum resist-ance of other strains, but the effect was much smaller thanthat seen with strain R2866 [22]. It is well established thatsialylation of LOS increases serum resistance [63]. Bycontrast, expression of ChoP increases the sensitivity ofbacteria to human serum, as ChoP binds C-reactiveprotein, activating complement [64]. These and other fea-tures of LOS structure are subject to phase variation, asdescribed for lgtC. The high-level serum resistance ofstrain R2866 was maintained after isolation in the clinicallaboratory, leading to the hypothesis that the association ofthis strain with invasive disease was a result of its abilityto survive in human blood.

In a survey of other NTHi, invasive isolates (from bloodor cerebrospinal fluid of apparently normal children) werenot more resistant to serum than throat or middle earisolates [59]. For some of these cases of invasive disease,unrecognized host factors might have predisposed thesechildren to systemic NTHi infection. For other cases, theNTHi strains might be unusually virulent by mechanismsother than serum resistance. It is likely that for some of theinvasive strains, phase variants with increased sensitivityto serum could have emerged in the laboratory.

Genetic heterogeneity of NTHiIt is well established that NTHi are genetically diverse andit is thought that this observation results in part fromhorizontal genetic exchange. Strains differ in their comp-lement of loci encoding adhesins and IgA proteases, asnoted. In some cases, these differences have been sugg-ested to be associatedwith differences in virulence. Severalloci, including the LOS biosynthetic gene lic2B, the adhe-sin gene hmwA, and the heme acquisition genes hemR andhgpB, have been reported to be more prevalent in otitismedia isolates than in those recovered from the throat ofhealthy children [65,66]. In addition, differential hybrid-ization studies identified several loci that were more com-mon in NTHi strains associated with exacerbations ofsymptoms in patients with chronic obstructive pulmonarydisease than in strains from patients not experiencingexacerbations [67].

Box 1. Outstanding questions

� What is the minimal gene content that permits nontypeable

H. influenzae to initiate an inflammatory response from respira-

tory epithelial cells?

� What bacterial or host factors enable NTHi to persist without

eliciting an inflammatory response?

� What are the changes in respiratory epithelial cells that permit

nontypeable H. influenzae to cause clinical disease?

� Do NTHi form a biofilm containing a biofilm-specific polysacchar-

ide through a process that is regulated?

� Are there groups of nontypeable H. influenzae that are genetically

related and have a propensity to occupy the same niche?

360 Review TRENDS in Microbiology Vol.15 No.8

To consider the possibility that NTHi strains frominvasive infections in children were closely related or con-tained common virulence determinants, PCR was used toevaluate the presence of several genetic loci in a collectionof 52 clinical isolates [59]. The isolates from invasivedisease were found to be as diverse as isolates from otitismedia or from healthy children. The publicly available H.influenzae multilocus sequence typing database (http://haemophilus.mlst.net) currently contains >600 strains,of which >300 are NTHi. There is no obvious phylogeneticclustering of otitis isolates or strains from blood or cere-brospinal fluid. However, few NTHi strains from healthysubjects or from sputum cultures have been typed. It ispossible that such isolates might form clusters distinctfrom the disease-associated strains.

Strain 86-028NP was the first NTHi strain to becompletely sequenced [5]. Annotation of the sequences ofstrains R2846 and R2866 (available at http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) identified a core of �1500open reading frames common to all three NTHi genomes(Figure 2). Nearly all of these are also in the publishedsequence of H. influenzae Rd KW20 [68]. There areno obvious candidates for otitis-specific determinants,supporting the concept that virulence is multifactorialand can be mediated by different mechanisms in different

Figure 2. Comparison of the genomes of the three sequenced nontypeable

H. influenzae. Open reading frames (ORFs) in the nucleotide sequences of str-

ains R2846 and R2866 (available at http://www.ncbi.nlm.nih.gov/sites/entrez?db=

genomeprj&cmd=Retrieve&dopt=Overview&list_uids=9621) were identified by

Glimmer (www.cbcb.umd.edu/software/glimmer). For Rd KW20 and 86-028NP,

the published annotated sequences were used [5,68]. Each pair of nucleotide

sequences was aligned using BLASTN, and the Artemis Comparison Tool (http://

www.sanger.ac.uk/Software/ACT/) was used to visualize the alignments. Gaps in

alignment were noted, and each gene was categorized as being present in one,

two, three or four of the sequenced strains. The results were confirmed by BLASTP

comparisons of each predicted amino acid sequence to the sequences of the other

strains. Most regions of the genomes were shared among all four genomes, but

each strain has several dozen open reading frames that are absent from the other

sequenced strains or shared with only one of the sequenced strain. The Venn

diagram shows the numbers of shared and strain-specific genes for the three NTHi

genomes. Most of the 1522 genes shared by all three NTHi are also in Rd KW20. It

is notable that the two otitis strains (R2846 and 86-028NP) differ from each other in

gene content to as great an extent as they differ from the invasive strain R2866.

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strains. Differences in host risk factors also need to beconsidered.

Concluding remarks and future perspectivesSeveral bacterial-specific and host-specific processes thatare thought to be involved in the pathogenesis of NTHidisease have been described. NTHi strains are geneticallyand phenotypically diverse. Some of the genes encodingproposed virulence determinants are not present in alldisease isolates, indicating that strains can differ in mech-anisms of pathogenesis. It is not yet clear whether NTHistrains differ in their capacity for causing mucosal orsystemic infections, or whether host factors are primarilyresponsible for the outcome of acquisition of a new NTHistrain. The availability of sequenced genomes will facili-tate study of the diversity of NTHi pathogenesis andprovide insights into vaccine candidates for NTHi disease.Other unanswered questions (Box 1) also need to be pur-sued to advance our understanding of NTHi disease andcarriage.

AcknowledgementsThis work was supported in part by grants AI44002, AI46512 and DC005833 from the National Institutes of Health (USA).

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