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Pathogenesis Due to Nontypeable Haemophilus influenzae 1
1
The Pathogenesis of Disease Due
to Nontypeable Haemophilus influenzae
Gail G. Hardy, Simone M. Tudor,
and Joseph W. St. Geme, III
1. Introduction
Isolates of Haemophilus influenzae can be separated into encapsulated and
nonencapsulated forms. Encapsulated strains express one of six structurally
and antigenically distinct capsular polysaccharides, designated serotypes a–f
(1). In contrast, nonencapsulated strains are defined by their inability to react
with antisera against the known H. influenzae polysaccharide capsules and are
referred to as nontypeable.
2. Epidemiology
NontypeableH. influenzae is a common commensal organism in the human
nasopharynx and occupies this niche as its natural habitat (2,3). The rate of
nasopharyngeal colonization increases from approx 20% during the first year
of life to over 50% by the age of 5–6 yrs, then remains high through adulthood
(2). Children often harbor multiple strains simultaneously, whereas adults typi-
cally carry only one (3). Based on longitudinal studies, in most cases an indi-
vidual carries a given strain for several weeks to mo, then loses the strain, and
then acquires a new strain (4–6). Transmission generally occurs by airborne
droplets or by direct contact with respiratory secretions.
Isolates of nontypeable H. influenzae account for 20–30% of all episodes of
acute otitis media and possibly a higher percentage of recurrent episodes (7).
In addition, recent studies indicate that this organism is responsible for over
40% of cases of otitis media with effusion (chronic otitis media) (8). Among
patients with acute or chronic sinusitis, nontypeable H. influenzae is the caus-
ative agent in approx one-third (9,10). Similarly, in patients with chronic bron-
From: Methods in Molecular Medicine, vol. 71: Haemophilus influenzae Protocols
Edited by: M. Herbert © Humana Press Inc., Totowa, NJ
1
2 Hardy, Tudor, and St. Geme
chitis and pulmonary exacerbations, several lines of evidence implicate
nontypeableH. influenzae as a common precipitant (11). Nontypeable H. influenzae
is also an important cause of community-acquired pneumonia, especially in
children in developing countries, in patients with underlying chronic lung dis-
ease, and among the elderly (12–14). On occasion, nontypeable H. influenzae
causes systemic disease, with examples including meningitis, septicemia, and
septic arthritis (15).
The pathogenesis of respiratory tract disease due to nontypeable H. influenzae
involves colonization of the nasopharynx followed by contiguous spread to
adjacent sites. In most cases, contiguous spread occurs in the setting of abnor-
malities in nonspecific host defenses. For example, viral upper respiratory
infection disrupts mucociliary activity, mucosal integrity, and neutrophil func-
tion and predisposes to nontypeable H. influenzae otitis media, sinusitis, and
pneumonia. Similarly, underlying lung diseases such as chronic bronchitis,
bronchiectasis, and cystic fibrosis are associated with abnormalities in
mucociliary clearance, thus predisposing to nontypeable H. influenzae bron-
chitis and pneumonia (16,17). Exposure to cigarette smoke results in goblet
cell hyperplasia, mucus hypersecretion, and decreased respiratory epithelial
cell ciliary function, changes that increase the likelihood of localized respira-
tory tract disease due to nontypeable H. influenzae(18).
Most cases of systemic disease due to nontypeable H. influenzae occur in
patients with anatomic abnormalities or compromised immunity (15). For example,
in patients with nontypeable H. influenzae meningitis, typically a communication
exists between the upper respiratory tract and the central nervous system. In indi-
viduals with nontypeable H. influenzae bacteremic disease, a deficiency in humoral
immunity is almost always present, allowing for bacterial invasion of the blood-
stream, then intravascular survival, and then seeding of distant sites (19,20).
Although many cases of nontypeable H. influenzae disease can be treated
successfully with a β-lactam antibiotic such as ampicillin or amoxicillin, resis-
tance is becoming increasingly common, usually reflecting production of a
β-lactamase (21,22). In occasional isolates, resistance results from an altered
penicillin binding protein with diminished affinity for β-lactams. Resistance to
other antibiotics, including trimethoprim-sulfamethoxazole, clarithromycin,
and azithromycin, has also been reported (21).
Even with successful therapy, infection due to nontypeable H. influenzae
can produce long-term complications. As an example, middle ear effusion that
develops during acute otitis media can persist for several weeks or months and
cause deficits in language acquisition, speech development, and cognitive func-
tion (23). The economic impact of nontypeable H. influenzae disease is also
significant, with the cost of physician visits and prescription drugs approach-
ing $1 billion per year in the United States alone (24). H. influenzae otitis
Pathogenesis Due to Nontypeable Haemophilus influenzae 3
media, sinusitis, pneumonia, and bronchitis also incur societal costs related to
time lost from school and work. Beyond morbidity and economic costs,
nontypeable H. influenzae is an important cause of mortality among children
in the developing world who develop pneumonia (25).
3. Bacterial Structure
Nontypeable H. influenzae is typical of Gram-negative bacteria and has a
cell wall that consists of an inner (cytoplasmic) membrane, a periplasm, and an
outer membrane. Analysis of the genome of H. influenzae strain Rd reveals a
system of proteins that comprise the Sec machinery and presumably facilitate
protein export from the cytoplasm (26). The periplasm contains several layers
of peptidoglycan and a protease with homology to the Eschericia coli DegP
(HtrA) protein (27). The outer membrane represents the interface between the
organism and the human host and contains integral proteins, surface-associ-
ated proteins, and lipooligosaccharide.
The major outer membrane proteins include P1, P2, P4, P5, and P6. P1 (also
called protein a) is heat-modifiable and has a molecular mass of 35 kDa at
room temperature and 46–50 kDa after boiling (28–30). P2 (also called protein
b/c) is the most abundant protein in the outer membrane and has a molecular
mass that ranges between 36 and 42 kDa (30). Structural studies indicate that
P2 forms a trimer and has porin activity, allowing molecules up to 1400 Da to
pass through the membrane (31). P4 (or protein e) is a 28–30 kDa lipoprotein
that has been implicated in heme uptake (32,33). P5 (or protein d) is a heat
modifiable protein with homology to E. coli OmpA and has a molecular mass
of approx 27 kDa at room temperature and 35 kDa after boiling (34,35). P6
(also called protein g or PAL) is another outer membrane lipoprotein and has a
molecular mass of 16 kDa (28–30). Comparison of predicted amino acid
sequences from multiple strains reveals that P1, P2, and P5 vary considerably
from one strain to another (36–39). In contrast, P6 is highly conserved, with
little variation in amino acid sequence from strain to strain (40). P4 contains at
least one epitope that is highly conserved among strains (33).
A number of minor outer-membrane proteins exist, including PCP, D15,
OMP 26, protein D, the transferrin-binding proteins (Tbp1 and Tbp2), and sev-
eral heme-binding proteins. PCP is a 16-kDa protein that co-migrates with P6
and makes up approx 0.5% of the outer membrane (41). D15 is a high-mol wt
(103-kDa) protein that is highly conserved among strains and is immunogenic
(27,42). OMP 26 is a 26-kDa protein that elicits clearance of both homologous
and heterologous strains after mucosal immunization in a rat pulmonary chal-
lenge model (43,44). Protein D is a highly conserved, surface-exposed, 42-kDa
lipoprotein that was first identified based on its ability to bind IgD (45). More
recent evidence indicated that protein D has glycerophosphodiester phosphodi-
4 Hardy, Tudor, and St. Geme
esterase activity and facilitates utilization of glycerophosphorylcholine as a
source of choline (45a). The transferrin-binding proteins serve as surface
receptors for human transferrin and facilitate iron acquisition (46).
Lipooligosaccharide is a major component of the H. influenzae cell wall,
accounting for roughly 4% of the dry weight of the organism (47). Similar to
other nonenteric Gram-negative bacteria, H. influenzae lipooligosaccharide con-
tains a lipid A moiety and an oligosaccharide core but lacks O side chains, giving
rise to the term lipooligosaccharide (LOS). A given cell expresses a variety of
LOS structures, with considerable variation in carbohydrate content (48).
4. Population Structure
Over the years, a number of methods have been employed to examine the genetic
and evolutionary relationships between strains of nontypeable H. influenzae.
Examples include biotyping, outer membrane protein typing, lipooligosaccharide
typing, and ribotyping, among others. Perhaps most powerful is multilocus
enzyme electrophoresis, a method that measures electrophoretic mobility of
metabolic enzymes. In particular, chromosomally encoded enzymes are sepa-
rated on a starch gel and then stained histochemically with specific substrates.
Enzyme variants are identified based on differences in mobility (revealing
electromorphs), which reflect changes in net electrostatic charge as a result of
one or more amino acid substitutions (49). Amino acid substitutions in turn reflect
changes in nucleotide sequence. With a given strain, the electrophoretic type is
defined by the combination of electromorphs for the enzymes tested.
Examination of a relatively large number of enzymes within a group of strains
allows accurate estimates of the level of genetic relatedness between strains.
Analysis by multilocus enzyme electrophoresis indicates that encapsulated
H. influenzae strains are clonal and can be segregated into genetically related
clusters, which are grouped into two major phylogenetic divisions (50,51).
Division I contains clusters of types a, b, c, d, and e strains, and division II
contains clusters of types a, b, and f strains. The population structure of
nontypeableH. influenzae is less well defined. Musser et al. examined 65 epi-
demiologically unrelated isolates by multilocus enzyme electrophoresis and
found considerable heterogeneity, with each isolate corresponding to a unique
electrophoretic type (52). Furthermore, comparison with 177 type b isolates
revealed no sharing of electrophoretic types (52). These observations indicate
greater genetic diversity among nontypeable H. influenzae than among
H. influenzae type b and suggest that the nontypeable H. influenzae population
structure lacks clonality. In addition, they suggest that nontypeable strains gen-
erally are not phenotypic variants of type b strains.
More recently, we studied a series of 123 pharyngeal isolates of nontypeable
H. influenzae collected from healthy 3-yr old Finnish children (53). Among
Pathogenesis Due to Nontypeable Haemophilus influenzae 5
these isolates, one was a capsule-deficient type b strain, based on Southern
analysis demonstrating loss of the one functional copy of the nearly duplicated
capb locus. Of the remaining 122 isolates, 31% hybridized with a probe con-
taining the capb locus, suggesting that a subgroup of nontypeable strains might
be more closely related to an encapsulated ancestor. To extend these observa-
tions, we examined the collection of nontypeable strains previously character-
ized in terms of genetic relatedness by Musser et al. (54). Roughly 15% of
these strains hybridized with a probe for the capb locus, in all cases due to the
presence of IS1016, an insertion element associated with cap genes in division
I, encapsulated strains of H. influenzae. Although the strains harboring an
IS1016 element do not define a distinct genetic division, they form small clus-
ters or lineages within the larger population structure. Interestingly, these
strains uniformly lack the family of adhesins most common in nontypeable
strains (see Subheading 5.2.2.). Instead, almost all express a homolog of the
major non-pilus adhesin in encapsulated strains of H. influenzae (seeSubhead-
ing 5.2.3.), again supporting the conclusion that selected nontypeable strains
are more closely related to encapsulated lineages.
Considered together, this information suggests a model in which the prim-
ordialH. influenzae strain was nonencapsulated and spawned two separate but
overlapping lineages. The larger lineage acquired one set of genes involved in
interactions with the host and remained nonencapsulated. The other lineage
acquired a separate set of colonization factors as well as the cap genes and
became encapsulated; subsequent mutations within the cap locus resulted in
evolution of a relatively restricted set of nonencapsulated strains, which repre-
sent a minority of all nontypeable isolates.
5. Establishment on the Mucosal Surface
5.1. Binding to Mucus and Ciliotoxicity
Upon entering the respiratory tract, bacteria interact initially with mucus and
are probably largely eliminated by the mucociliary escalator, which consists of
ciliated respiratory epithelial cells and the associated mucus layer. However, in
at least some circumstances, a small number of organisms may persist. Based
on experiments examining interactions between nonencapsulated H. influenzae
and nasal turbinate tissue in organ culture, residual organisms appear to form
microcolonies within the mucus layer, ultimately elaborating soluble factors
that cause ciliostasis, loss of cilia, and sloughing of ciliated cells (55).
Studies by Kubiet and Ramphal demonstrated that nontypeable H. influenzae
is capable of binding to mucin, a major component of mucus (56). The mecha-
nism of this binding remains poorly understood but probably reflects both spe-
cific and nonspecific interactions. Using a gel overlay assay, Reddy et al. found
that the P2 and P5 outer membrane proteins influence binding to nasopharyn-
6 Hardy, Tudor, and St. Geme
geal mucin (57). Additional experiments using the chinchilla model revealed
that mutants lacking P5 display reduced adherence to Eustacian tube mucus
(58). In the same model, purified P5 is capable of direct binding to mucus and
of blocking adherence by whole organisms (58).
The nontypeable H. influenzae effector responsible for ciliotoxicity was ini-
tially shown to be released from the surface of the organism and to be
nondialyzable, heat stable, and trypsin resistant (59). Subsequent studies by
Johnson and Inzana established that LOS is the relevant substance, as purified
nontypeable H. influenzae LOS reproduces the ciliotoxic effects of infection
with whole organisms (60). In additional work, these investigators demon-
strated that the lipid-free oligosaccharide fraction of LOS has no effect on cili-
ary activity, thus pointing to lipid A as the biologically active moiety (60).
During infection due to nontypeable H. influenzae, LOS may remain associ-
ated with the bacterial surface or may be released (61). These two forms differ
in their biological activities. Secreted LOS is 10-fold more potent in stimulat-
ing the release of monocyte-derived inflammatory mediators such as tumor
necrosis factor alpha (TNF-α), interleukin 1α (IL-1α), and interleukin 6 (IL-6)
(61). In addition, this form is more active in the limulus assay and is more toxic
to mice (61). It remains unknown whether cell-associated LOS and secreted
LOS have differential effects on ciliated respiratory epithelial cells.
Protein D is a second factor that has been implicated in ciliotoxicity (62). This
protein was first identified by Raun et al. and is capable of binding certain human
IgD myeloma proteins (45). Analysis of the predicted amino acid sequence revealed
67% identity with the E. coli periplasmic glycero-phosphodiesterase encoded by
glpQ, and Munson and Sasaki demonstrated that protein D contains glycero-
phosphodiesterase phosphodiesterase activity (63). Recently, Janson and co-work-
ers compared isogenic protein D-expressing and protein D-deficient strains in
assays with human adenoidal tissue in culture and found that protein D was essen-
tial for maximal impairment of ciliary activity and damage to ciliated cells (62). Of
note, protein D did not affect the number of bacteria associated with the mucosal
surface. Consistent with these results, in a rat otitis media model, elimination of
expression of protein D resulted in a 100-fold decrease in virulence (64). At this
point, the mechanism by which protein D influences ciliated epithelium and the
relevance of IgD binding and glycerophosphodiesterase phosphodiesterase activ-
ity remain unclear. However, it is intriguing to consider that the role of protein D in
facilitating acquisition of choline may be important.
5.2. Adherence to Respiratory Epithelium
A fundamental step in the process of colonization involves adherence to the
epithelial surface. Based on experiments with adenoidal tissue and nasal turbinates
in organ culture, nontypeable H. influenzae adheres preferentially to nonciliated
Pathogenesis Due to Nontypeable Haemophilus influenzae 7
cells and to areas of damaged epithelium. In vitro studies using isolated epithelial
cells indicate that a variety of bacterial factors influence adherence.
5.2.1. Pili
Selected isolates of nontypeable H. influenzae express adhesive pili, which
are hairlike surface appendages that extend up to 450 nm in length and are dis-
played circumferentially (65). As viewed by transmission electron microscopy
using the quick-freeze, deep-etch technique, H. influenzae pili are composite
structures consisting of a relatively rigid helical rod joined to a very thin tip (66).
The helical rod is approx 6–7 nm in diameter and appears to be two-stranded,
similar to filamentous actin and different from other well-characterized pili (66).
TheH. influenzae gene cluster contains a total of five genes, designated hifA–E
(67–71). hifA encodes the HifA major structural subunit and is transcribed
divergently from the rest of the gene cluster (67).hifB encodes the HifB chaper-
one, which resides in the periplasm and serves to stabilize subunits against pre-
mature degradation and to target subunits to the outer membrane (66). hifC
encodes the HifC protein, which shares homology with outer-membrane ushers
and presumably sits in the outer membrane and facilitates the translocation of
subunits across the outer membrane, allowing their incorporation into growing
pili(69).hifD encodes the HifD minor subunit, and hifE encodes the HifE minor
subunit (67,68). Based on localization by immunoelectron microscopy, both
HifD and HifE appear to make up the tip fibrillum (66,72). An in-frame dele-
tion in hifD results in a decreased number of pili, suggesting that HifD serves to
nucleate pilus assembly (68). Antiserum against HifE eliminates pilus-medi-
ated adherence, arguing that this protein represents the pilus adhesin (72). Con-
sistent with this possibility, HifE is larger than most structural subunits and
shares significant homology with other pilus adhesins.
The adhesive activity of H. influenzae pili was first appreciated in the early
1980s, when two groups independently noted a correlation between piliation
and agglutination of human erythrocytes and adherence to human oropharyn-
geal epithelial cells (73,74). Subsequently, van Alphen demonstrated that pilus-
mediated hemagglutination correlates with expression of the AnWj antigen
(formerly called the Anton antigen) and is inhibited by anti-AnWj antiserum
(75). Interestingly, human oropharyngeal epithelial cells lack this antigen, and
pilus-dependent attachment to these cells is unaffected by anti-AnWj antise-
rum (75). Nevertheless, the same anti-pilus monoclonal antibody blocks
agglutination of erythrocytes and attachment to oropharyngeal epithelial cells
(76), suggesting that pili may recognize the same structure on both erythro-
cytes and epithelial cells, apparently in the context of either the AnWj antigen
or some other cell membrane receptor. Consistent with this consideration, com-
pounds containing sialyllactosylceramide, including the gangliosides GM1,
8 Hardy, Tudor, and St. Geme
GM2, GM3, and GD1a, are able to inhibit hemagglutination as well as adher-
ence to epithelial cells (77).
5.2.2. HMW1 and HMW2
Following the identification of H. influenzae pili, a series of observations
suggested that nontypeable strains express nonpilus adhesins as well. In par-
ticular, several studies demonstrated that nonpiliated strains are capable of effi-
cient adherence to cultured human epithelial cells (78). Additional experiments
revealed that nonpiliated strains are capable of appreciable association with the
mucosal surface of nasal turbinates in culture (55). A major clue regarding the
nature of these adhesins came from work by Barenkamp and co-workers, who
identified a family of surface-exposed high-molecular-weight proteins that are
major targets of the serum antibody response to infection (79,80). The proto-
type members of this family are proteins called HMW1 and HMW2, which are
related proteins expressed by the same strain (79). Overall, HMW1 and HMW2
are 80% similar and 71% identical. Interestingly, they share significant sequence
similarity with the filamentous hemagglutinin agglutination (FHA) protein, an
adhesin and colonization factor expressed by Bordetella pertussis(81,82).
The HMW1 and HMW2 adhesins are encoded by separate gene clusters, each
containing three genes called hmwA, hmwB,andhmwC. The hmw1A and hmw2A
genes encode the adhesins (HMW1 and HMW2), the hmw1B and hmw2B genes
encode 60-kDa outer membrane proteins (HMW1B and HMW2B) that translo-
cate the adhesins to the cell surface, and the hmw1C and hmw2C genes encode
73-kDa cytoplasmic proteins (HMW1C and HMW2C) that appear to stabilize
the adhesins prior to export from the cytoplasm (83,84). Of note, the predicted
amino acid sequences of HMW1B and HMW2B are 99% identical, and the
predicted sequences of HMW1C and HMW2C are 97% identical. Consistent with
this homology, in assays examining secretion of HMW1 and HMW2, HMW1B/
HMW1C are able to substitute for HMW2B/HMW2C and vice versa (84).
The HMW1 and HMW2 adhesins are synthesized as 160-kDa and 155-kDa
preproteins, respectively. In each case, a 68-amino acid N-terminal fragment
directs export from the cytoplasm via the Sec machinery (85). Following cleav-
age between amino acids 68 and 69 by leader peptidase, the proteins are
released into the periplasm (85). With both proteins, the segment between
amino acids 69 and 441 serves as an intramolecular chaperone and mediates
interaction with the HMW1B or HMW2B outer-membrane translocator (85).
While associated with the periplasmic face of the outer membrane, HMW1 and
HMW2 are cleaved between amino acids 441 and 442, resulting in mature 125-kDa
and 120-kDa proteins, respectively (79,85). Subsequently, they are transported
to the surface of the organism via the HMWB proteins (84).
In studies using diverse cultured human epithelial cell lines, HMW1 and
HMW2 exhibit differing cellular binding specificities (86), suggesting that they
Pathogenesis Due to Nontypeable Haemophilus influenzae 9
recognize distinct host cell receptor structures. More detailed experiments with
Chang conjunctival cells have demonstrated that HMW1 interacts with a gly-
coprotein that contains N-linked oligosaccharide chains with sialic acid in an
α-2,3 configuration (87). At this point, the nature of the HMW2 receptor
remains unknown.
5.2.3. Hia
Approximately 25% of nontypeable strains lack proteins that belong to the
HMW1/HMW2 family of proteins. Nevertheless, nearly all of these strains
remain capable of efficient adherence to cultured human epithelial cells (54).
Furthermore, they express high-molecular-weight proteins that are major tar-
gets of the antibody response to infection (80). With this information in mind,
recent work has demonstrated that these strains express an adhesin called Hia
(88). The prototype Hia protein is 115 kDa in size and is encoded by a 3.3-kb
gene (88). Interestingly, Hia shares significant homology with the major
nonpilus adhesin present in H. influenzae type b, a protein called Hsf (89).
Overall, Hia and Hsf share 72% identity and 80% similarity. They are most
similar at their N-terminal and C-terminal ends and also contain a conserved
internal domain that is repeated three times in Hsf, thus accounting for the
larger size of Hsf (approx 240 kDa).
Adherence assays with E. coli transformants harboring the hia gene
demonstrate that Hia is capable of reaching the bacterial surface and mediating
in vitro adherence independent of other H. influenzae gene products, with the
probable exception of the Sec proteins, which are present in both H. influenzae
and E. coli (88). Recent evidence indicates that Hia belongs to the growing
family of autotransporters, IgA1 protease-like proteins present in Gram-nega-
tive bacteria (89a). However, in contrast to other well-characterized auto-
transporter proteins, Hia undergoes no processing event on the bacterial surface
and remains cell-associated in the full-length form.
5.2.4. Hap
Mutants deficient in expression of HMW1 and HMW2 or Hia remain capable
of low-level adherence to cultured human epithelial cells, suggesting that an
additional adhesin exists. To address this possibility, we prepared a genomic
library from a virulent clinical isolate and identified a gene called hap, which
confers a capacity for intimate interaction with cultured epithelial cells (90).
Analysis of the predicted amino acid sequence of the Hap protein reveals sig-
nificant sequence homology with the H. influenzae and Neisseria IgA proteases.
This homology includes the region identified as the serine protease catalytic
site of the IgA1 proteases. Overall, there is 30–35% identity and 51–55% simi-
larity between Hap and the H. influenzae and N. gonorrhoeae IgA1 proteases.
