Revisión sobre virulencia del Haemophilus no tipificable

1. Nontypeable Haemophilus influenzae:
understanding virulence and
commensal behavior
Alice 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, USA
2
Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA
Haemophilus influenzae is genetically diverse and exists
as a near-ubiquitous human commensal or as a path-
ogen. Invasive type b disease has been almost elimi-
nated in developed countries; however, unencapsulated
strains – nontypeable H. influenzae (NTHi) – remain
important as causes of respiratory infections. Respirat-
ory tract disease occurs when NTHi adhere to or invade
respiratory epithelial cells, initiating one or more of
several proinflammatory pathways. Biofilm formation
explains many of the observations seen in chronic otitis
media and chronic bronchitis. However, NTHi biofilms
seem to lack a biofilm-specific polysaccharide in the
extracellular matrix, a source of controversy regarding
their relevance. Successful commensalism requires
dampening of the inflammatory response and evasion
of host defenses, accomplished in part through phase
variation.
Why study Haemophilus influenzae?
H. influenzae is a human-restricted gram-negative
bacterium that is part of the normal nasopharyngeal flora
of most humans. Until twenty years ago, the study of this
speciesfocusedonH.influenzaeserotype b,a majorbacterial
pathogen ofchildren. Sincethe introduction in 1990oftypeb
polysaccharide–protein conjugate vaccines, invasive H.
influenzae disease has been nearly eliminated in developed
countries. H. influenzae that lack capsular polysaccharides
are referred to as nontypeable (NTHi). Although NTHi are
most commonly associated with 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 these
mucosal infections are NTHi [1–3]. Colonization by any
single NTHi is transitory, with new strains acquired every
few months [4]. A major goal of NTHi research today is
identification of bacterial factors that influence whether
acquisition of an NTHi strain results in disease or in asymp-
tomatic colonization. At the time of writing this review,
complete or nearly complete genome sequences are
available for three strains: the otitis media strains
86-028NP [5] and R2846 (also known as strain 12) [6] and
the invasive NTHi strain R2866 (also called strain Int1) [7].
At least ten other strains are reportedly being sequenced,
most isolated from middle ear aspirates [8]. Here, we review
the current understanding of the interactions between
NTHi 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 other
aspects of virulence.
What is H. influenzae?
The species H. influenzae is defined by its nutritional
requirements: both b-nicotinamide adenine dinucleotide
and 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. Each
sequenced NTHi strain has two to four hpg genes which
facilitate heme acquisition from hemoglobin or hemo-
globin-haptoglobin complexes. Deletion of the three hpg
genes from NTHi 86-028NP resulted in a delayed onset of
infection in the chinchilla otitis media model, and the
infection was also of shorter duration [9].
Are all H. influenzae equally virulent?
Encapsulated H. influenzae are unquestionably more
virulent than NTHi. Nearly all H. 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 no
single feature that is characteristic of all disease-associ-
ated strains. It is possible that NTHi strains with similar
potential for causing disease might possess different com-
binations of virulence-related genes. Further, it is not
known whether NTHi strains differ in virulence.
A recent study provided strong evidence for a group of
Haemophilus isolates with low potential for virulence
[11]. In a longitudinal study of 118 patients with chronic
obstructive pulmonary disease, Murphy et al. isolated
Haemophilus strains from >600 sputum cultures. It
was noted that certain isolates initially identified as
NTHi (i.e. unencapsulated strains requiring NAD+ and
heme) had unusual growth characteristics. Further
investigation identified these isolates as meeting all
characteristics of H. haemolyticus except that many of
them lacked hemolytic activity. Correlation of laboratory
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Corresponding author: Smith, A.L. (arnold.smith@sbri.org).
Available online 27 June 2007.
www.sciencedirect.com 0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.06.004

2. findings with clinical data led to the recognition that
acquisitions of ‘non-hemolytic H. haemolyticus’ were
rarely, if ever, associated with exacerbations of symptoms
of bronchitis, in comparison to acquisitions of ‘true’ NTHi.
Similar strains were isolated from pharyngeal cultures of
healthy children, but not from the middle ear of children
with 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 evaluate
the possibility that strains isolated from the airway and
identified as NTHi might be H. haemolyticus, but should
consider that there might be other distinct subspecies, as
yet 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 by
MLST and are unlikely to be H. haemolyticus.
H. influenzae virulence is host mediated
The word virulent is derived from the Latin virulentus
(poisonous). For H. influenzae, as for many pathogens, the
organism does not produce a true poison or toxin. Disease
results from the response of the host to bacterial factors,
particularly endotoxin (lipopolysaccharide). Because of
their short saccharide chains, the lipopolysaccharides of
H. influenzae are often referred to as lipooligosaccharides
(LOS), the term that will be used here. The interaction
between 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 between
NTHi and the host is the adherence of bacteria to the
mucus or cells of the upper airway. The nasopharynx
contains both stratified squamous epithelium and pseu-
dostratified ciliated columnar epithelium typical of the
respiratory tract; the latter contain mucus-secreting goblet
cells. Submucosal glands also contribute to surface mucus.
Subsequent steps might include invasion of respiratory
epithelial 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, have
been studied using several different NTHi strains that are
now recognized to differ in genetic content. A summary of
the reported NTHi–host-cell interaction using different
cells, including transient transfection systems, is depicted
in Figure 1.
Primary role of adherence
Several H. influenzae adhesins are known (reviewed in
[12]). The fimbriae encoded by the hif locus were first
characterized in a type b strain, in which they were found
to mediate adherence to all cell types found in the respir-
atory tract. Not all NTHi contain the hif locus; when it is
present it is thought to be most important in the early
steps of colonization [13]. Many NTHi, including 86-
028NP and R2846, contain the hmw1 and hmw2 loci,
encoding high molecular weight adhesins. HMW1 and
HMW2 have different binding specificities. HMW1 tar-
gets sialyl-a2,3 hexose [12], which is not prevalent in the
upper respiratory tract [14]. This indicates that for hmw-
containing strains, other adhesins are more important in
the initial colonization. Most NTHi strains lacking hmw
possess genes for a different adhesin, Hia, which is
similar in sequence to the surface fibrils (Hsf) previously
identified in H. influenzae type b [12]. Strain R2866
includes both the hia and hif loci. Other surface struc-
tures shown to bind to eukaryotic cells include the outer
membrane proteins Hap [15], outer membrane protein
(OMP)-2 [16] and OMP-5 [17]; the lipoprotein PCP [18]; a
protein associated with colony opacity [19]; and a phos-
phorylcholine (ChoP) moiety on LOS [20]. Most of these
potential adhesins have substantial interstrain sequence
heterogeneity. The hif and hmw loci and the licA gene
(required for phosphorylcholine synthesis) are subject to
phase-variable expression, mediated by changes in length
of tandem repeat tracts [21–23]. Table 1 summarizes the
H. influenzae surface structures and the known cognate
host-cell ligands.
How does respiratory tract colonization occur?
After being deposited on respiratory epithelium, NTHi
must overcome the innate clearance mechanisms of the
upper respiratory tract. Mucociliary clearance and anti-
bacterial molecules such as lysozyme, lactoferrin and
antimicrobial peptides are all designed to remove infec-
tious particles from the airway. Mucus contains the gel-
forming mucins, MUC5AC, MUC5B and MUC2; respirat-
ory mucins bind fimbriated NTHi [24] and should facilitate
bacterial clearance. However, because fimbrial expression
is phase-variable, the emergence of nonfimbriated strains
that possess other adhesins will facilitate airway coloni-
zation.
The secretion of IgA protease by NTHi is inferred to
facilitate colonization in individuals with S-IgA1 that
recognizes 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 nonpathogenic
Haemophilus species, including H. haemolyticus [25,26].
Some NTHi have a second IgA1 protease gene, igaB, the
prevalence of which in sputum and middle ear isolates is
significantly 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 the
serine protease activity of lactoferrin cleaves NTHi IgA
protease and the adhesin Hap [28,29]. This activity might
mitigate colonization.
Human b defensin-1 had minimal activity toward strain
12 (R2846), whereas the antibacterial activity of the indu-
cible b defensin-2 was found to be comparable to b-lactam
antibiotics [30]. The susceptibility of strain 2019 to b
defensin-2 was increased in a mutant (htrB) with reduced
acylation of lipid A [31]. These peptides act by damaging
the outer membrane, causing loss of intracellular contents
including potassium. The Sap transporter, which is upre-
gulated in strain 86-028NP during experimental otitis
media, mediates resistance to b defensins [32] and also
seems to counteract the cellular potassium loss [33]. A
sapA mutant of 86-028NP has markedly attenuated viru-
lence in the chinchilla model of otitis media [32]. For the
antimicrobial peptide LL-37 (also called hCAP18), the
susceptibility of strain H233 was reduced by expression
of the ChoP moiety on LOS [34].
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3. Dampened inflammation required for commensalism?
The nature of the initial binding of bacteria to host cells
could be the crucial event in virulence. Work in several
laboratories, using different strains and cells, indicates
that binding to epithelial cells by multiple ligands
initiates a proinflammatory response via several pathways
(Figure 1). Adherence triggers microvillus formation in
respiratory epithelial cells, leading to engulfment of NTHi
[35]. The primary ligands seem to be the Toll-like receptors
TLR2 and TLR4 and the platelet-activating factor receptor
Table 1. H. influenzae adhesins for eukaryotic cells and their cognate ligands
H. influenzae structure Cognate eukaryotic ligand(s) Initiates intracellular signaling Commenta
Refs
Fimbriae (pili) Fibronectin, heparin binding matrix proteins Not known Type b [58,69,70]
Heat shock protein TLR-2 and TLR-4 sialylated gangliosides (SO4) Inflammation via MyD88/IRAK/NF-kb NTHi [78]
Hsf Vitronectin Not known Type b [69]
OMP-2 TLR-2 TNF-a and IL-6 via MyD88 Type b [79,80]
OMP-5 CECAM Increased expression of CD105
(less exfoliation)
Strain Rd [37]
OMP-6 TLR-2 Induction of NF-kB NTHi [81]
Peptides from OMP-4, OMP-
6 and PCP
TLR-2 Synergizes with IFN-b NTHi [18]
LOS ChoP PAFR PtdIns 3K calcium signaling NTHi 2019 [39]
Hap Fibronectin, laminin, type IV collagen Not known NTHi N187 [15]
a
The indication of ‘type b’ or ‘NTHi’ indicates the bacteria in which the interaction was studied; it is not intended to imply that the adhesin or interaction is limited to type b or to
NTHi.
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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4. (PAFR). Signaling by these ligands initiates proinflamma-
tory signaling through the mitogen activated kinase cas-
cade or by way of p38 activation [36]. Treatment of
epithelial cells with NTHi or bacterial fractions does not
lead to appreciable cell death. However, polarized epi-
thelial cells infected with H. influenzae strain Rd KW20
have been noted to exfoliate. This process seems to be
inhibited by binding of the bacterial protein OMP-5 to
CECAM, triggering the expression of CD105 [37].
Intracellular residence can shield the NTHi from the
action of antibodies and antibiotics and could provide the
organism a means of avoiding clearance from the respir-
atory tract. For intracellular residence to be an effective
pathway in the life cycle of NTHi, there must be a mech-
anism for dampening or terminating the inflammatory
process. Using a transient transfection system, Mikami
et al. showed that a lysate of NTHi strain 12 (R2846)
contained a factor that activated the epidermal growth
factor receptor. Activation of this receptor downregulates
the p38-mediated proinflammatory response [38]. A sep-
arate series of experiments [20,39] studied the interaction
of NTHi strain 2019 with PAFR. Binding of ChoP, which is
variably present on NTHi LOS, to the PAFR activates the
phosphoinositide 3-kinase (PI3-K); this pathway can
initiate anti-inflammatory pathways by way of negative
regulation of TLR-2, TLR-4 and TLR-9 [40]. Because PI3-K
is activated by NTHi, it is expected that inflammation
would be downregulated early after invasion. ChoP
expressed on the LOS of NTHi engages the PAFR through
molecular mimicry, and NTHi probably enters the cell in a
PAFR-linked pinocytotic vacuole. PAFR is a G-protein
coupled receptor and binds to arrestins which target
vacuolar contents to clathrin-coated pits or to the endocytic
pathway, both seem to occur with NTHi. In addition, b-
arrestins uncouple G-protein receptors terminating their
signaling. Limiting the inflammatory response is probably
essential for NTHi to be a successful commensal.
Invasion across respiratory epithelium
Adherence of NTHi to respiratory epithelial cells can also
result in transcytosis of organisms into the subepithelial
compartment. NTHi were found in the bronchial submu-
cosa of patients with chronic bronchitis >50 years ago,
whereas more recent studies identified NTHi between
epithelial cells and in clusters in the submucosa of similar
patients [41]. Most often the bacteria are extracellular, but
they have been identified in macrophage-like cells [42]. For
NTHi strain 2019 to transcytose murine M cells derived
from Peyer’s patches, a wild-type LOS structure was
required [43]. Once in the submucosa, NTHi can be pro-
cessed for immune presentation.
As noted, NTHi LOS is involved in multiple aspects of
virulence. LOS structure affects the ability of bacteria to
adhere effectively to cells and to invade them. The LOS-
dependent intracellular signaling that occurs after NTHi
enter respiratory epithelial cells seems to be necessary for
colonization of human respiratory epithelium. The coloni-
zation of human tissue by NTHi was studied using a model
in which normal human bronchiolar xenografts are
imbedded in severe combined immunodeficient (SCID,
nu/nu) mice. Inoculation with as few as 10 colony forming
units (CFU) of NTHi strain R3001 (isolated from a
patient with cystic fibrosis) results in colonization of the
xenograft. Within 48 h, the xenograft lumen contains
107
CFU. By five days, copious mucus is present and the
bacterial density is 108
CFU per xenograft. Colonization
in this model was found to be affected by LOS structure
because mutants of strains R3001 and 2019 in which the
acyltransferase gene htrB was inactivated were unable to
colonize the respiratory epithelium in the xenograft model
[35].
Does NTHi produce biofilms?
Several recent studies have evaluated the concept that
biofilm production contributes to the pathogenesis of NTHi
respiratory infections, particularly those that become
chronic such as otitis media with effusion and chronic
bronchitis. Biofilms would be expected to protect bacteria
from antibiotics and from innate respiratory epithelial
clearance mechanisms. In most cases, formation of biofilms
is controlled by a regulatory switch [44], and the transition
from planktonic to biofilm growth involves production of an
extracellular polysaccharide [45]. Although neither of
these has been well documented for NTHi, there is evi-
dence that NTHi can grow in an aggregate form that is
consistent with a biofilm and that this form of growth
affects virulence. NTHi consisting of aggregates of bacteria
in a matrix containing LOS, outer membrane proteins and
IgA have been observed on immortalized human middle
ear epithelial cells in vitro [46] and the Calu-3 cell line [47].
When grown for extended periods on Calu-3 cells, the cells
become less sensitive to gentamicin and continue to elicit a
proinflammatory response [47]. Structures consistent with
a biofilm have also been observed on middle ear tissue from
chinchillas in experimental infections [48,49] and from
children with chronic otitis media undergoing tympanost-
omy tube placement [50]. Although an extracellular matrix
has been visualized [51], no biofilm-specific exopolysac-
charide has been identified. For strain 2019, biofilm-like
structures 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 to
LOS. A recent study of strain 86-028NP from chinchilla
middle ears identified double-stranded DNA and type IV
pilin protein in an extracellular matrix [49]. A study of
strain 9274 found that the extracellular matrix contained
the outer membrane proteins Hap, HMW1 and HMW2, but
that at least four other proteins were detected only in
bacterial cells and not in the extracellular matrix [51].
Studies using several NTHi strains have shown that pre-
vention of sialic acid incorporation by mutation of the sialic
acid activator gene siaB or the appropriate sialyltransfer-
ase genes reduces virulence in the chinchilla and gerbil
models of otitis media [52,53]. Both NTHi strains 2019 and
86-028NP displayed ChoP expression to a greater extent
when grown in biofilm conditions, compared with plank-
tonic growth [46], and the increase in ChoP expression in
biofilms 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 not
persist in the middle ear of chinchillas [54]. It is unlikely
that LOS structure is the only factor affecting NTHi
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5. biofilm-like structures, as wecA mutants of NTHi
strain 2019 do not form the structure and are attenuated
in virulence in the chinchilla model [49]. WecA has been
characterized in Salmonella enterica as an undecaprenyl-
phosphate a-N-acetylglucosaminyltransferase [55]; it has
no known role in LOS biosynthesis in NTHi.
NTHi strains differ in virulence-related phenotypes
Some reports support the idea that differences between
strains in virulence-related phenotypes correlate with
their clinical sources. Upon infection of H292 cells (a
human adenocarcinoma cell line), NTHi strains that per-
sist in the lower respiratory tract of patients with chronic
bronchitis induced lower levels of IL-6 and IL-8 than non-
persisting strains. This was not explained by differences in
adherence to the H292 cells, and indicated that the reduced
inflammatory potential of certain strains enables them to
persist in the lungs [56]. NTHi isolated from the sputum of
patients with chronic obstructive pulmonary disease who
are undergoing an exacerbation are more adherent to
respiratory epithelial cells and induce the secretion of more
proinflammatory cytokines than strains causing asympto-
matic infection [57]. Although adherence and the ability to
induce 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 vary
widely but without apparent correlation with the clinical
source. Bresser et al. reported that certain NTHi from
patients with chronic bronchitis showed a high level of
adherence to laminin and type I collagen, whereas other
strains had a substantially lower level of adherence. There
was 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 example
Interpretation of strain comparison data are complicated by
the fact that virulence-related phenotypes are often subject
to phase variation. As each of a dozen or so phase-variable
genes in each strain switches on and off independently, a
culture of H. influenzae consists of a mixture of hundreds of
different 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 the
immediate environment. The practical result is that the
phenotype of the laboratory grown culture can differ sub-
stantially from that of the same strain when it was in the
patient. In particular, phase-variable aspects of LOS struc-
ture have a crucial role in several aspects of virulence,
including adherence to cells, induction of inflammation
and resistance to complement.
Encapsulated H. influenzae are usually resistant to the
complement-mediated bactericidal activity of pooled serum
from normal adults. NTHi are usually much more sensitive,
but display a wide range of susceptibility to human serum
[59]. The serum resistance of strain R2866 has been studied
in some detail [60,61]. Strain R2866 (also known as Int1)
was of particular interest because it was isolated from the
bloodstream of a child with no known abnormality that
would predispose him to bacteremia [7]. Strain R2866
was found to cause bacteremia and meningitis in the infant
rat and weanling rat models of invasive disease (unlike any
other NTHi strain tested) [7] and to have unusually high
resistance 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 region within the lgtC
gene determined that the gene was out of frame (off) in a
serum-sensitive variant (R3392) and in frame (on), enabling
translation in the serum-resistant parent. Chemical anal-
ysis of LOS confirmed that the principal glycoform in R2866
differed from that in R3392 by a single hexose. lgtC-on
variants of R3392 with increased serum resistance were
recovered after serum selection, confirming the relationship
between phase variation and phenotype [60,61].
Several LOS biosynthetic genes in addition to lgtC are
known to affect serum resistance; further, the role of
different genes in serum resistance differs from strain to
strain. lgtC expression (reflected by binding of monoclonal
antibody 4C4) had been reported to increase serum resist-
ance of other strains, but the effect was much smaller than
that seen with strain R2866 [22]. It is well established that
sialylation of LOS increases serum resistance [63]. By
contrast, expression of ChoP increases the sensitivity of
bacteria to human serum, as ChoP binds C-reactive
protein, activating complement [64]. These and other fea-
tures of LOS structure are subject to phase variation, as
described for lgtC. The high-level serum resistance of
strain R2866 was maintained after isolation in the clinical
laboratory, leading to the hypothesis that the association of
this strain with invasive disease was a result of its ability
to survive in human blood.
In a survey of other NTHi, invasive isolates (from blood
or cerebrospinal fluid of apparently normal children) were
not more resistant to serum than throat or middle ear
isolates [59]. For some of these cases of invasive disease,
unrecognized host factors might have predisposed these
children to systemic NTHi infection. For other cases, the
NTHi strains might be unusually virulent by mechanisms
other than serum resistance. It is likely that for some of the
invasive strains, phase variants with increased sensitivity
to serum could have emerged in the laboratory.
Genetic heterogeneity of NTHi
It is well established that NTHi are genetically diverse and
it is thought that this observation results in part from
horizontal genetic exchange. Strains differ in their comp-
lement of loci encoding adhesins and IgA proteases, as
noted. In some cases, these differences have been sugg-
ested to be associated with differences in virulence. Several
loci, including the LOS biosynthetic gene lic2B, the adhe-
sin gene hmwA, and the heme acquisition genes hemR and
hgpB, have been reported to be more prevalent in otitis
media isolates than in those recovered from the throat of
healthy children [65,66]. In addition, differential hybrid-
ization studies identified several loci that were more com-
mon in NTHi strains associated with exacerbations of
symptoms in patients with chronic obstructive pulmonary
disease than in strains from patients not experiencing
exacerbations [67].
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6. To consider the possibility that NTHi strains from
invasive infections in children were closely related or con-
tained common virulence determinants, PCR was used to
evaluate the presence of several genetic loci in a collection
of 52 clinical isolates [59]. The isolates from invasive
disease were found to be as diverse as isolates from otitis
media 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 phylogenetic
clustering of otitis isolates or strains from blood or cere-
brospinal fluid. However, few NTHi strains from healthy
subjects or from sputum cultures have been typed. It is
possible that such isolates might form clusters distinct
from the disease-associated strains.
Strain 86-028NP was the first NTHi strain to be
completely sequenced [5]. Annotation of the sequences of
strains R2846 and R2866 (available at http://www.ncbi.
nlm.nih.gov/genomes/lproks.cgi) identified a core of 1500
open reading frames common to all three NTHi genomes
(Figure 2). Nearly all of these are also in the published
sequence of H. influenzae Rd KW20 [68]. There are
no obvious candidates for otitis-specific determinants,
supporting the concept that virulence is multifactorial
and can be mediated by different mechanisms in different
strains. Differences in host risk factors also need to be
considered.
Concluding remarks and future perspectives
Several bacterial-specific and host-specific processes that
are thought to be involved in the pathogenesis of NTHi
disease have been described. NTHi strains are genetically
and phenotypically diverse. Some of the genes encoding
proposed virulence determinants are not present in all
disease isolates, indicating that strains can differ in mech-
anisms of pathogenesis. It is not yet clear whether NTHi
strains differ in their capacity for causing mucosal or
systemic infections, or whether host factors are primarily
responsible for the outcome of acquisition of a new NTHi
strain. The availability of sequenced genomes will facili-
tate study of the diversity of NTHi pathogenesis and
provide 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 and
carriage.
Acknowledgements
This work was supported in part by grants AI44002, AI46512 and DC
005833 from the National Institutes of Health (USA).
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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=
genomeprjcmd=Retrievedopt=Overviewlist_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.
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?
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