Molecular and cellular determinants of non-typeable Haemophilus influenzae adherence and invasion

Authors

  • Joseph W. St Geme III

    1. Edward Mallinckrodt Department of Pediatrics and Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8208, St Louis, MO 63117, and Division of Infectious Diseases, St Louis Children’s Hospital, St Louis, MO, USA.
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  • For correspondence at the Department of Pediatrics. E-mail stgeme@borcim.wustl.edu; Tel. (+1) 314 286 2887; Fax (+1) 314 286 2895.

Summary

Non-typeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. Based on information from histopathologic specimens and in vitro studies with human cells and tissues in culture, non-typeable H. influenzae is capable of efficient adherence and appreciable invasion, properties that facilitate the process of colonization. A number of adhesive factors exist, each recognizing a distinct host cell structure and influencing cellular binding specificity. In addition, at least three invasion pathways exist, including one resembling macropinocytosis, a second mediated via the PAF receptor and a third involving β-glucan receptors. Organisms are also capable of disrupting cell–cell junctions and passing between cells to the subepithelial space.

Introduction

Haemophilus influenzae is a Gram-negative bacterium that is a human-specific pathogen. Early studies revealed that not all isolates of H. influenzae are equal in terms of pathogenic potential. In particular, Pittman (1931) reported the observation that most systemic isolates express the type b capsule, whereas most respiratory tract isolates are unencapsulated (referred to as non-typeable).

Since the late 1980s, the incidence of disease caused by H. influenzae type b has dropped precipitously in the United States, reflecting the routine use of H. influenzae conjugate vaccines (Centers for Disease Control and Prevention, 1996). The existing Haemophilus vaccines consist of type b capsular polysaccharide conjugated to one of several non-H. influenzae immunogenic carrier proteins (Ward et al., 1994). Accordingly, these vaccines provide effective protection against disease caused by H. influenzae type b but fail to protect against non-typeable strains.

Non-typeable H. influenzae is a common cause of middle ear infection, sinusitis and conjunctivitis (Turk, 1984; Rao et al., 1999). In addition, this organism is frequently implicated in exacerbations of underlying lung disease, including chronic bronchitis, bronchiectasis and cystic fibrosis, and is an important aetiology of community-acquired pneumonia, especially among children in developing countries and elderly adults (Turk, 1984; Rao et al., 1999). It is sometimes recovered from systemic sites, usually in neonates or in the setting of compromised host immunity (Krasan and St Geme, 1997; Rao et al., 1999).

The pathogenesis of disease caused by non-typeable H. influenzae begins with colonization of the nasopharynx (Murphy et al., 1987), which requires that the organism overcome host clearance mechanisms, including the mucociliary escalator and local immunity. Bacterial adherence to respiratory epithelium represents a mechanism to circumvent mucociliary clearance, whereas entry into host cells (invasion) and penetration between host cells (paracytosis) may facilitate evasion of immune function.

This microreview summarizes our current understanding of the molecular mechanism of non-typeable H. influenzae adherence and invasion.

Haemophilus influenzae adhesins

Upon entry into the respiratory tract, infecting organisms encounter a layer of mucus and the underlying epithelium. Based on experiments with adenoidal tissue and nasal turbinates in organ culture, non-typeable strains of H. influenzae adhere preferentially to mucus, to non-ciliated cells and to areas of damaged epithelium. A number of H. influenzae surface structures influence the process of adherence.

Haemagglutinating pili

Like many Gram-negative bacteria, some isolates of non-typeable H. influenzae are capable of expressing pili. H. influenzae pili are polymeric helical structures ≈ 5 nm in diameter and up to 450 nm long (Stull et al., 1984; St Geme et al., 1996a) (Fig. 1). They are displayed in a peritrichous (circumferential) distribution and mediate agglutination of AnWj-positive human erythrocytes (Guerina et al., 1982; Pichichero et al., 1982). In addition, they promote adherence to human oropharyngeal epithelial cells and enhance adherence to human nasopharyngeal and nasal tissue in organ culture (van Alphen et al., 1986; Loeb et al., 1988; Farley et al., 1990; Read et al., 1991). Studies with glycoconjugate inhibitors indicate that interactions with human erythrocytes and human oropharyngeal epithelial cells occur via recognition of sialylated lactosylceramide derivatives (van Alphen et al., 1991).

Figure 1.

Transmission electron micrographs of H. influenzae pili.

A. Pili on the surface of an organism, visualized after staining with uranyl acetate.

B. The ends of pili, highlighting the tip fibrillum and the closely spaced horizontal striations in the pilus rod. The horizontal striations are characteristic of a tight helix.

C. Alignment of two pilus rods, highlighting the cross-over repeat, which is indicative of a two-stranded helical architecture. The images in (B) and (C) were obtained using freeze–etch electron microscopy, in collaboration with J. Heuser.

Recent work by Kubiet et al. (2000) indicated that pili promote adherence to respiratory mucus as well. These investigators examined binding by isogenic piliated and non-piliated strains to mucin prepared from tracheobronchial secretions from a patient with chronic bronchitis. In assays with H. influenzae derivatives, piliation was associated with a 20-fold increase in binding to mucin. Similarly, in assays with isogenic Escherichia coli strains, expression of H. influenzae pili resulted in a 10- to 20-fold increase in binding to mucin. At this point, the receptor structure in tracheobronchial mucin remains unknown. In addition, it is unclear whether pili recognize mucin from other sites in the respiratory tract.

Studies with H. influenzae type b have established that pili also promote interaction with heparin-binding extracellular matrix proteins. In particular, Virkola et al. (2000) examined isogenic piliated and non-piliated strains and found that pili facilitate bacterial binding to human fibronectin and to mouse heparin-binding growth-associated molecule. Additional experiments with purified pili revealed dose-dependent binding to the N-terminal 30 kDa and the C-terminal 40 kDa fibronectin fragments, which represent the heparin-binding domains of fibronectin. Consistent with these observations, incubation in the presence of 250 μg ml−1 heparin resulted in a 45–75% decrease in binding.

Analysis of a number of type b and non-typeable strains has demonstrated that H. influenzae pili are encoded by a gene cluster consisting of five genes, designated hifA–hifE (van Ham et al., 1994; Gilsdorf et al., 1997). Based on examination by quick freeze–deep etch electron microscopy, H. influenzae pili are composite structures with a thick shaft joined to a short thin tip fibrillum, similar to P pili expressed by uropathogenic E. coli and type 1 pili expressed by Enterobacteriaceae (St Geme et al., 1996a) (Fig. 1B). The H. influenzae pilus shaft consists of repeating HifA subunits and appears to be double-stranded, with a single-start left-handed helix and a double-start right-handed helix, morphologically similar to filamentous actin (St Geme et al., 1996a) (Fig. 1B and C). The tip fibrillum is only 5 nm in length and contains the HifD and HifE subunits (St Geme et al., 1996a; McCrea et al., 1997).

Like P pili, type 1 pili and a number of other Gram-negative bacterial adhesive structures, H. influenzae pili are assembled via the chaperone/usher pathway. HifB is the chaperone and functions in the periplasm, where it stabilizes pilus subunits during their transit from the inner membrane to the outer membrane (St Geme et al., 1996a). Based on molecular modelling and the results of site-directed mutagenesis, HifB appears to facilitate pilus assembly via donor strand exchange, a process first identified in the P pilus and type 1 pilus systems (Krasan et al., 2000). HifC is the usher and is located in the outer membrane, where it presumably facilitates the ordered incorporation of pilus subunits into the growing pilus (Watson et al., 1994).

Analysis of two separate collections of non-typeable H. influenzae isolates indicated that ≈ 15% of strains possess the intact hif gene cluster and are able to express pili (Geluk et al., 1998; Krasan et al., 1999).

Autotransporter proteins

In recent years, proteins belonging to the autotransporter family have been identified with increasing frequency in Gram-negative pathogens (Henderson and Nataro, 2001). Autotransporter proteins are synthesized as preproteins with at least three functional domains, including an N-terminal signal sequence, an internal passenger domain and a C-terminal outer membrane translocator domain (also called a β-domain). The signal sequence directs export of the polypeptide across the inner membrane and is then cleaved by signal peptidase I. Subsequently, the translocator domain inserts into the outer membrane and appears to fold into a β-barrel structure with a central hydrophilic pore, allowing extrusion of the passenger domain across the membrane.

At least three different groups of H. influenzae adhesive structures belong to the autotransporter family of proteins, including Hap, the HMW1/HMW2 proteins and the Hia/Hsf proteins (Fig. 2). Hap is ubiquitous among H. influenzae isolates, including both typeable and non-typeable strains (Rodriguez et al., submitted). HMW1/HMW2-like proteins are expressed by the majority of non-typeable clinical isolates but are generally absent from typeable strains (Barenkamp and Leininger, 1992; Rodriguez et al., submitted). Hia is expressed by nearly all non-typeable strains that lack HMW adhesins (Barenkamp and St Geme, 1996; St Geme et al., 1998). Based on limited studies, a homologue of Hia called Hsf appears to be ubiquitous among typeable H. influenzae, including both type b and non-type b strains (St Geme et al., 1996b).

Figure 2.

The H. influenzae adhesins that belong to the autotransporter family. Hap is a classic autotransporter protein and is synthesized as a precursor protein that undergoes autoproteolysis, with extracellular release of the passenger domain. Autoproteolysis is inhibited by secretory leucocyte protease inhibitor (SLPI), denoted by a star. Hia is synthesized as a precursor protein and remains intact and fully cell associated. HMW1 and HMW2 are examples of unlinked autotransporters. The adhesins are encoded by the hmw1A and hmw2A structural genes, and the outer membrane translocators are encoded by the hmw1B and hmw2B genes. The red stripes represent signal sequences (SS), the blue stripes and symbols represent adhesive passenger domains (HapS, HiaP and HMW1/HMW2), and the green stripes and symbols represent outer membrane translocator domains (Hapβ, Hiaβ and HMW1B/HMW2B). IM, inner membrane; OM, outer membrane.

The H. influenzae Hap protein is an example of a classic autotransporter, analogous to the prototype Neisseria and H. influenzae IgA1 proteases (St Geme et al., 1994; Hendrixson et al., 1997). Once the Hap passenger domain (HapS) is localized on the cell surface, an autoproteolytic cleavage event occurs, resulting in extracellular release of this domain (Fig. 2). Autoproteolysis is mediated by chymotrypsin-like serine protease activity, with the catalytic site consisting of Lys-98, Asp-140 and Ser-243 (Fink et al., 2001). The primary cleavage event occurs at Leu-1036–Asn-1037, and secondary cleavage events occur at Leu-1046–Thr-1047, Phe-1067–Ser-1068 and Phe-1077–Ala-1078, defining a consensus target sequence for cleavage that consists of (Q/R) (A/S) X (L/F) at the P4 to P1 positions (Hendrixson et al., 1997; Fink et al., 2001). Whether HapS cleaves host proteins as well remains unknown.

Hap was first discovered based on its ability to promote adherence and invasion in assays with cultured human epithelial cells (St Geme et al., 1994). More recent evidence indicates that Hap mediates bacterial aggregation and microcolony formation as well (Hendrixson and St Geme, 1998). Hap adhesive activity is localized within the HapS passenger domain (Hendrixson and St Geme, 1998). In considering the paradox that HapS is ultimately released from the surface of the organism, it is notable that Hap autoproteolysis is inhibited by secretory leucocyte protease inhibitor (Hendrixson and St Geme, 1998), a natural component of respiratory secretions that is upregulated in the setting of inflammation and possibly viral infection. One possibility is that the efficiency of Hap autoproteolysis varies according to local conditions, influencing the balance between adhesive activity and extracellular protease activity.

The Hia adhesin was first identified in non-typeable strain 11 (Barenkamp and St Geme, 1996). Based on characterization of this strain, Hia is encoded by a 3.3 kb gene and migrates at a molecular mass of ≈ 115 kDa. Similar to a subset of autotransporter proteins, Hia has an atypical signal peptide with an N-terminal extension that is marked by a series of charged residues (St Geme and Cutter, 2000). Ultimately, cleavage occurs at a predicted signal peptidase I cleavage site between amino acids 49 and 50. The C-terminal 319 amino acids have trans-locator activity and present the passenger domain on the surface of the organism (St Geme and Cutter, 2000). Mutation of the C-terminal phenylalanine results in degradation of the mature protein, suggesting that this residue is essential for targeting Hia to the outer membrane or perhaps for stabilizing Hia in the outer membrane (St Geme and Cutter, 2000). In contrast to Hap and other classic autotransporter proteins, the passenger domain remains uncleaved and completely cell associated in Hia (St Geme and Cutter, 2000) (Fig. 2). Additional studies indicate that the Hia passenger domain harbours high-affinity adhesive activity and mediates interaction with a broad array of respiratory epithelial cell types (St Geme et al., 1996b; Laarmann et al. submitted). Characterization of the receptor structures recognized by Hia is ongoing.

The HMW1 and HMW2 proteins are high-molecular-weight adhesins that were first recognized based on their role as major targets of the antibody response during natural H. influenzae infection (Barenkamp and Bodor, 1990). HMW1 is encoded by the hmw1A gene, which is 4.6 kb and is flanked downstream by the hmw1B and hmw1C genes (Barenkamp and Leininger, 1992; Barenkamp and St Geme, 1994). HMW2 is encoded by the hmw2A gene, which is 4.4 kb and is flanked downstream by the hmw2B and hmw2C genes (Barenkamp and Leininger, 1992; Barenkamp and St Geme, 1994). Overall, the HMW1 and HMW2 adhesins are 71% identical, the hmw1B and hmw2B products are 99% identical, and the hmw1C and hmw2C products are 97% identical (Barenkamp and Leininger, 1992; Barenkamp and St Geme, 1994), suggesting that the hmw1 and hmw2 loci may have evolved by a gene duplication event. Both HMW1 and HMW2 are synthesized as preproteins and are exported across the inner membrane via the Sec system, resulting in cleavage of a 68-amino-acid fragment at a predicted signal peptidase I cleavage site (Grass and St Geme, 2000). Similar to the Hia signal peptide, the 68-amino-acid signal peptide in HMW1 and HMW2 contains an N-terminal extension with a series of charged residues (Grass and St Geme, 2000). Once within the periplasm, HMW1 and HMW2 undergo cleavage between amino acids 441 and 442 and are then translocated across the outer membrane (Barenkamp and Leininger, 1992; St Geme and Grass, 1998). The HMW adhesins are examples of so-called ‘unlinked’ autotransporter proteins and are translocated to the surface of the organism by an HMWB protein (St Geme and Grass, 1998) (Fig. 2). HMW1B and HMW2B are outer membrane proteins that are predicted to form β-barrels and function like the C-terminal β-domain in classic autotransporter proteins. Other examples of unlinked autotransporter proteins include the Serratia marcescens ShlA, the Proteus mirabilis HpmA and the Haemophilus ducreyi HhdA haemolysins, the Bordetella FHA adhesin and the H. influenzae HxuA haeme:haemopexin binding protein (Henderson et al., 2000; Henderson and Nataro, 2001; Jacob-Dubuisson et al., 2001). The cognate outer membrane translocators include ShlB, HpmB, HhdB, FhaC and HxuB respectively (Henderson and Nataro, 2001; Jacob-Dubuisson et al., 2001). In contrast to other unlinked autotransporters, secretion of the HMW proteins requires an additional protein beyond the outer membrane translocator, namely HMW1C or HMW2C (Barenkamp and St Geme, 1994; St Geme and Grass, 1998). Based on recent evidence, the HMWC proteins reside in the cytoplasm and appear to facilitate export of HMW1 and HMW2 across the inner membrane.

The original evidence that HMW1 and HMW2 are adhesins came from studies examining H. influenzae mutants and E. coli transformants in assays with Chang conjunctival epithelial cells (St Geme et al., 1993). These studies confirmed a role for both HMW1 and HMW2 in in vitro adherence. In addition, they indicated that HMW1 is essential whereas HMW2 is dispensable for adherence to Chang cells. Despite the high level of sequence homology between HMW1 and HMW2, experiments with diverse cultured epithelial cell lines indicate that these proteins have distinct cellular-binding specificities (Hultgren et al., 1993; Dawid et al., 2001). With some cell lines, HMW1 mediates higher level adherence than does HMW2, whereas with other cell lines, the reverse is true. Together, these findings suggest that HMW1 and HMW2 interact with specific receptor molecules whose distribution varies from one cell type to another. In addition, these observations imply that the HMW1 and HMW2 proteins interact with distinct eukaryotic receptors and perhaps function at different steps in the process of colonization. Experiments involving modification of the surface of Chang cells indicate that the HMW1 adhesin recognizes a glycoprotein receptor containing N-linked oligosaccharide chains with sialic acid in an α2-3 configuration (St Geme, 1994). Whether HMW1 interacts with the same receptor on other cell types remains unclear. The nature of the HMW2 receptor is still uncharacterized.

Outer membrane proteins

The P2 protein is the most abundant major outer membrane protein in H. influenzae and is a porin that is strongly immunogenic (Hansen et al., 1988). Characterization of multiple non-typeable isolates has demonstrated that P2 is highly variable from one strain to another, in both size and amino acid sequence (Forbes et al., 1992). Further analysis has revealed that variation in P2 sequence also occurs within a clonal population during the course of chronic infection. In individual children with chronic otitis media and in adults with chronic bronchitis resulting from non-typeable H. influenzae, specific regions of P2 vary at a relatively high frequency (Duim et al., 1994; 1996; Smith-Vaughan et al., 1997). P2 antigenic drift allows the organism to evade clearance by potentially protective antibodies and contributes to the development of chronic infection. In overlay assays aimed at defining the determinants of H. influenzae binding to nasopharyngeal mucin, Reddy et al. (1996) discovered that P2 is capable of interacting with mucin, apparently via recognition of sialic acid-containing oligosaccharides. This property may not impart long-term advantage to H. influenzae in a normal host with intact mucociliary function. However, in conditions associated with an abnormality in mucus clearance, such as chronic bronchitis and cystic fibrosis, binding to mucin may facilitate the establishment of infection.

The P5 protein is another major outer membrane protein in H. influenzae and shares homology with E. coli OmpA (Munson et al., 1993). This protein is heat modifiable and is antigenically variable from one strain to another (Munson and Granoff, 1985). Work by Sirakova et al. (1994) indicates that P5 may form a pilus-like surface appendage, prompting the designation ‘P5 fimbrin’ by some investigators. In studies with human oropharyngeal cells, disruption of the P5 gene resulted in reduced adherence, suggesting a role as an adhesin. More recent experiments have provided evidence that P5 interacts with human CEACAM1 (Virji et al., 2000; Hill et al., 2001), a member of the carcinoembryonic antigen (CEA) family of cell adhesion molecules. In particular, based on measurement by fluorescent microscopy, Hill et al. (2001) found that P5-expressing strains were capable of efficient adherence to CHO cells expressing CEACAM1, whereas isogenic P5-deficient variants of selected strains demonstrated minimal adherence. In addition, comparison of isogenic strains revealed that P5 was associated with binding to soluble chimeric CEACAM1 in overlay assays. Analogous to observations with P2, the P5 protein also interacts with nasopharyngeal mucin (Reddy et al., 1996),

OapA is a surface-associated lipoprotein that is responsible for the transparent colony phenotype of H. influenzae and is required for efficient colonization of the nasopharynx in the infant rat model (Weiser et al., 1995). Based on examination of diverse strains, OapA varies in molecular mass between 78 and 84 kDa and has a highly conserved amino acid sequence (Prasadarao et al., 1999). Recent work by Prasadarao et al. (1999) suggests that this protein plays a minor role in H. influenzae adherence to cultured epithelial cells. In studies with Chang conjunctival cells, elimination of expression of OapA in strains Rd and H233 resulted in a three- to ninefold decrease in attachment compared with the isogenic parent strains. In addition, expression of OapA by E. coli DH5α was associated with a threefold increase in adherence over background.

Other proteins

Using thin-layer chromatography, Busse et al. (1997) observed that non-typeable strains of H. influenzae are capable of binding to phosphotidylethanolamine (PE), gangliotriosylceramide (Gg3), gangliotetraosylceramide (Gg4), sulphatoxygalactosylceramide and sulphatoxygalactosylglycerol. In additional studies, these investigators prepared a PE affinity matrix and purified a 46 kDa protein that inhibits binding of whole bacteria to immobilized PE and Gg3. More recently, Hartmann and Lingwood (1997) found that heat shock treatment resulted in a marked increase in non-typeable H. influenzae binding to sulphatoxygalactosylceramide and sulphatoxygalactosylglycerol. Additional analysis suggested that this binding resulted from two Hsp 70-related heat shock proteins that are surface exposed after heat shock (Hartmann et al., 2001).

Lipopolysaccharide

Haemophilus influenzae lipopolysaccharide (also called lipooligosaccharide or LOS) lacks a repeating O antigen and instead contains non-repeating oligosaccharides consisting of glucose, galactose, N-acetylglucosamine, phosphorylcholine and N-acetyl-neuraminic acid in varying combinations (Masoud et al., 1997; Risberg et al., 1999). The oligosaccharides are linked to a triheptose-(2-keto-deoxyoctulosonic acid)-phosphate-lipid A core region and are highly variable from one molecule to another (Preston et al., 1996). In recent work, Swords et al. (2000) found that polystyrene beads coated with LOS purified from H. influenzae strain 2019 were capable of significant adherence to transformed human bronchial epithelial cells. Preincubation of epithelial cells with LOS from wild-type strain 2019 resulted in a dose-dependent inhibition of bead adherence. In contrast, preincubation with LOS from strain 2019 pgB::ermr had no effect on bead adherence. It is noteworthy that strain 2019 pgB::ermr lacks phosphoglu-comutase activity and produces truncated LOS, with no oligosaccharides linked to the core region.

Further analysis revealed that LOS adhesive activity may be dependent upon modification with phosphorylcholine (ChoP) (Swords et al., 2000). Interestingly, ChoP is a host structure as well and is a component of platelet-activating factor (PAF), an ether-linked acetyl-ChoP molecule that mediates inflammatory signals via interaction with the PAF receptor, a G-protein linked receptor. In Streptococcus pneumoniae, adherence and invasion are influenced by interaction between ChoP in the bacterial cell wall and PAF receptor on the host cell surface (Cundell et al., 1995). With this information in mind, Swords et al. (2000) performed confocal microscopy and observed co-localization of adherent bacteria expressing ChoP and host cell PAF receptor. In additional studies, they found that pretreatment with a PAF receptor antagonist had no effect on H. influenzae adherence but resulted in an ≈ 50–70% decrease in invasion (Swords et al., 2000). Together, these findings suggest that LOS ChoP may influence invasion via interaction with PAF receptor and stimulation of a series of signalling events.

Cellular events in invasion

Forsgren et al. (1994) examined adenoids removed from children with chronic secretory otitis media or adenoidal hypertrophy and found viable non-typeable H. influenzae inside reticular crypt epithelial cells and macrophage-like cells. Similarly, Moller et al. (1998) examined lung explants from patients with underlying chronic lung disease and detected H. influenzae in the bronchial epithelium. Consistent with these results, early experiments using cultured human epithelial cells demonstrated that non-typeable H. influenzae is capable of entering and surviving inside cells (St Geme and Falkow, 1990). More recent experiments with human bronchial epithelial cells revealed that H. influenzae invasion begins with extension of host cell microvilli and the formation of lamellipodia (Ketterer et al., 1999) (Fig. 3A). These structures surround adherent bacteria and form a membrane-bound vacuole (Fig. 3B). Based on examination using rhodamine phalloidin and confocal microscopy, cytoskeletal rearrangement is associated with actin polymerization, resulting in an increase in cortical actin and the formation of actin strands both beneath adherent bacteria and around intracellular organisms (Holmes and Bakaletz, 1997; Ketterer et al., 1999). Of note, invasion is blocked by cytochalasin D, an inhibitor of actin polymerization (St Geme and Falkow, 1990; Ketterer et al., 1999).

Figure 3.

A. Adherent bacteria being enveloped by lamellipodia on the surface of adenoidal tissue in culture.

B. A cluster of organisms in a membrane-bound vacuole inside Chang epithelial cells.

In an effort to define the mechanism of H. influenzae invasion more clearly, Ketterer et al. (1999) performed studies with bronchial epithelial cells and impermeant dextran 70 000 labelled with Texas red. Examination by confocal microscopy revealed some vacuoles containing both bacteria and dextran 70 000, suggesting invasion via macropinocytosis. Consistent with this observation, preincubation of cells with PI-3 kinase inhibitors results in a significant reduction in invasion (Swords et al., 2001). At the same time, some vacuoles contain bacteria but no dextran 70 000, and PI-3 kinase inhibitors fail to block invasion completely, suggesting the presence of another invasion pathway (Ketterer et al., 1999; Swords et al., 2001). Indeed, PAF receptor-mediated invasion appears to be independent of macropinocytosis and may be the predominant invasion pathway for most strains.

In recent experiments, Swords et al. (2001) found that pretreatment of bronchial epithelial cells with pertussis toxin resulted in a dose-dependent decrease in H. influenzae invasion. Of note, pertussis toxin ADP ribosylates a subset of Gα proteins, and PAF receptor signalling occurs via coupling to G proteins. Accordingly, one possibility is that PAF receptor-mediated invasion occurs by activation of a pertussis toxin-sensitive heterotrimeric G protein complex. Additional experiments indicate that H. influenzae invasion is associated with increases in cytosolic levels of Ca2+ and is blocked by chelation of cytosolic Ca2+ (via BAPTA-AM) or depletion of intracellular Ca2+ stores (using thapsigargin) (Swords et al., 2001). At this point, the relationship between Ca2+ fluxes and the PAF receptor pathway remains unclear.

Work by Ahren et al. (2001) indicated that entry into monocytes and cultured pneumocytes is partially blocked by laminarin, suggesting the involvement of β-glucan receptors and possibly a third invasion pathway.

Paracytosis

Several studies have suggested that non-typeable H. influenzae is able to pass between cells and invade the subepithelial space. For example, Hers and Mulder (1953) examined tissue sections from patients with acute and chronic muco-purulent bronchitis associated with non-typeable H. influenzae infection and observed bacteria between epithelial cells in the bronchi and bronchioles. Similarly, Farley et al. (1986; 1990) performed experiments with adenoidal tissue in organ culture and noted bacteria between epithelial cells and in the submucosal area. More recently, van Schilfgaarde et al. (1995) used lung epithelial cells and generated polarized cell layers on filter inserts. When non-typeable strains were inoculated onto the apical surface of the cell layers, they were capable of passing through to the basolateral side, without affecting cell viability. Examination by electron microscopy revealed clusters of bacteria between cells, suggesting passage by the process of paracytosis. Based on studies of an H. influenzae genomic library expressed in E. coli, open reading frame HI0638 appears to be sufficient to promote paracytosis (van Schilfgaarde et al., 2000). Mutagenesis suggests that this locus is also essential for paracytosis (van Schilfgaarde et al., 2000). Interestingly, HI0638 encodes a predicted outer membrane lipoprotein, leading to speculation that the HI0638 product may have a direct effect on a signalling pathway involved in the regulation of intercellular junctions.

Summary

Non-typeable H. influenzae initiates infection by colonizing the upper respiratory tract. Based on in vitro studies and histological examination of tissue samples, non-typeable strains are capable of efficient adherence and appreciable invasion, properties of relevance to the process of colonization. Adherence and invasion require the complex interplay of a variety of bacterial and host factors. A number of adhesive factors exist, each recognizing a distinct receptor structure and presumably influencing cellular tropism. In addition, at least three invasion pathways exist, including one resembling macropinocytosis, a second mediated via the PAF receptor and a third involving β-glucan receptors. Organisms are also capable of disrupting cell–cell junctions and passing between cells to the subepithelial space.

The existing literature reflects investigation of diverse non-typeable H. influenzae strains and the use of varied host cell types. In future work, with each H. influenzae adhesin, it will be important to explore the cellular distribution of the relevant receptor, potentially explaining why some strains produce upper respiratory tract infection, others produce lower respiratory tract infection and others produce invasive disease.

Acknowledgements

Much of the work included in this review was supported by Public Health Service Grants DC-02873 and AI-44167 and by awards from the American Heart Association and the March of Dimes.

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