2.2. Chlamydia pneumoniae

2.2.1. Taxonomy

Although they were first considered as protozoa and later as viruses, chlamydiae are gram-negative obligate intracellular eubacteria. Originally, they were taxonomically categorised into their own order Chlamydiales, with one family, Chlamydiaceae, and a single genus, Chlamydia (Moulder et al. 1984). The genus included four species: C. trachomatis, C. psittaci (Moulder et al. 1984), C. pneumoniae (Grayston et al. 1989) and C. pecorum (Fukushi & Hirai 1992).

In 1999, it was recommended by Everett et al. (1999) that the genus Chlamydia should be divided in two genera, Chlamydia and Chlamydophila, containing altogether nine species (Table 3). In addition to the five new species, three new families (Parachlamydiaceae, Simniaceae and Waddliaceae) were also recommended. However, the proposal to change the taxonomic nomenclature for the Chlamyadiaceae family has not been generally accepted in the field (Schachter et al. 2001).

Two of the species, C. trachomatis and C. pneumoniae, are common pathogens in humans, whereas the other species occur mainly in animals. C. trachomatis has been isolated only from humans and comprises two human biovars (trachoma and lymphogranuloma venereum, LGV), including a total of 18 serovars, whereas C. pneumoniae has one human biovar (TWAR) and two animal biovars, one infecting horses (biovar equine) and the other infecting frogs and koalas (biovar koala).

2.2.2. History

The divergence of C. trachomatis and C. pneumoniae 125 million years ago preceded the appearance of higher primates and, finally, Homo sapiens 500 000 years ago (Stephens 2002). Despite this, C. pneumoniae was only described quite recently. In the mid-1980s, an atypical strain of C. psittaci was found to be responsible for the epidemic of mild pneumonia that had occurred in Finland in 1978 (Saikku et al. 1985). The strain was called TWAR, which was an acronym of the first two Seattle isolates: TW-183, isolated in 1965 from the eye of a child during a trachoma vaccine trial in Taiwan, and AR-39, isolated in 1983 from a throat swab of a university student with pharyngitis in Seattle (Grayston et al. 1986). In 1989, the strain was identified as a separate species within the genus Chlamydia and named Chlamydia pneumoniae (Grayston et al. 1989). Since then, it has been proposed that the species should be rather placed under the new genus called Chlamydophila, as distinct from the genus Chlamydia, and be renamed as Chlamydophila pneumoniae (Everett et al. 1999). However, as it was mentioned above, the proposal has not gained extensive support in the field (Schachter et al. 2001), and the previous designation, Chlamydia pneumoniae, is still widely used in publications.

2.2.3. Developmental cycle

Chlamydiae are intracellular bacteria that have a unique biphasic developmental cycle with two distinct morphological forms. The extracellular, infectious form (0.3 µm) is called elementary body (EB), and the intracellular, replicating form (1.0 µm) is called reticulate body (RB). Infectious EBs start the cycle by attaching to a susceptible host cell membrane. They gain access into the host cell via either parasite-specified phagocytosis or receptor-mediated endocytosis. When inside the cell, the chlamydiae remain within an enlarging intracellular vacuole, a characteristic inclusion, avoiding lysosomal fusion and hence destruction. During the first few hours, EBs differentiate into metabolically active RBs. By using the host cell’s energy and nutrient resources, RBs begin to multiply by binary fission. After multiple rounds of division, RBs start to transform back to EBs. Finally, by exocytosis or host cell lysis, the infectious EBs are released into the cytoplasm, to initiate new cycles in new host cells. (Reviewed by Hatch 1999)

In cell culture conditions, the duration of the developmental cycle is between 2 and 3 days. In natural infections, the situation is more complicated, and the normal development of Chlamydia is easily disturbed. Certain circumstances (nutrient deficiency, interferon-gamma, antibiotics) may result in morphological alterations of RBs and the emergence of enlarged, atypical chlamydial forms (Beatty et al. 1993). These aberrant forms may persist inside the host cell in a viable but culture-negative state for a long time. The cycle of both normal and altered development of Chlamydia is presented in Fig. 2.

2.2.4. Structure

At all stages of development, chlamydial cells appear to be surrounded by a double membrane, a characteristic feature of gram-negative bacteria. However, unlike other gram-negative bacteria, chlamydiae do not have a peptidoglycan layer in the space between the two membranes (Barbour et al. 1982, Fox et al. 1990). On the other hand, they contain penicillin-binding proteins, and the presence of peptide crosslinks analogous to those between peptidoglycan backbones has been suggested (Barbour et al. 1982). The genomic sequence of C. trachomatis revealed the presence of genes for peptidoglycan synthesis, membrane assembly and recycling (Stephens et al. 1998). Peptidoglycan has been suggested to be needed in RB cell division (Brown & Rockey 2000).

Lipopolysaccharide (LPS), which is a general endotoxin in gram-negative bacteria, is localised on the surface of Chlamydia, both at EBs and RBs (Birkelund et al. 1989). Chlamydial LPS is structurally similar to the rough form of LPS found in enterobacteria, having both a cross-reactive epitope and a genus-specific epitope (Nurminen et al. 1983, Brade et al. 1987). In addition to the rough-type LPS, a smooth form of chlamydial LPS has also been found (Lukacova et al. 1994). The structure of LPS is not identical in all chlamydial species, and compared to the LPS of enterobacteria, chlamydial LPS has much lower endotoxin activity (Nurminen et al. 1983, Brade et al. 1987, Ingalls et al. 1995).

The outer membrane contains proteins named outer membrane proteins (Omp). The most abundant of them is the major outer membrane protein (MOMP) of 38 to 42 kDa, comprising about 60% of Omps (Caldwell et al. 1981). MOMP contains serovar-, subspecies- and species-specific epitopes that can be identified by monoclonal antibodies (Campbell et al. 1990, Perez Melgosa et al. 1991). MOMP is surface-localised not only on C. trachomatis and C. psittaci, as first thought (Knudsen et al. 1999), but also on C. pneumoniae (Wolf et al. 2001). However, the MOMP of C. pneumoniae appears to be less immunogenic and antigenically complex than that of the other chlamydiae (Campbell et al. 1990, Perez Melgosa et al. 1991).

Omp3, a small cysteine-rich protein, is synthesised late in the developmental cycle, and it is not exposed at the surface of Chlamydia (Collett et al. 1989). Omp2 is a 60-kDa cysteine-rich protein, and it has been suggested to be surface-exposed (Ting et al. 1995, Stephens et al. 2001). On the other hand, it has been suggested that Omp2 is the structural element for the hexagonally arrayed structures, and only seen at the inner surface of the outer membrane complex (Mygind et al. 1998).

Proteins named polymorphic outer membrane proteins (Pmps) have also been localised in the outer membrane (Longbottom et al. 1998, Knudsen et al. 1999). Each of the three studied Chlamydia species has a family of distantly related Pmp genes. The highest number of such genes, 21, has been found in C. pneumoniae (Kalman et al. 1999). The C. pneumoniae Pmp gene family consists of a heterogeneous group of genes with low identity but with shared characteristics. They resemble members of the autotransporter family (Christiansen et al. 2000). Most of the genes encode proteins 90 to 100 kDa in size. Pmps have been shown to be localised on the surface of C. pneumoniae (Knudsen et al. 1999).

Chlamydiae also contain heat shock proteins. The genes encoding Hsp10, Hsp60 and Hsp70 have been cloned and sequenced (Morrison et al. 1989a, Danilition et al. 1990, LaVerda & Byrne 1997). These genes are continuously expressed throughout the developmental cycle. The Hsps are highly conserved within chlamydial species, including C. pneumoniae (Kikuta et al. 1991, Kornak et al. 1991). All three Hsps can be found in the outer membrane complexes of both EBs and RBs (Brunham & Peeling 1994). Chlamydial Hsp60 and Hsp70 are highly immunogenic during natural infection (Brunham & Peeling 1994).

In the inclusion membrane, there exists a group of proteins called inclusion membrane proteins (Inc). The first of them was demonstrated in C. psittaci by Rockey et al. (1995) and named IncA. Since then, six other Incs, from IncB to IncG, have been characterised (Bannantine et al. 1998, Scidmore-Carlson et al. 1999). However, a genome search of C. trachomatis revealed 46 candidates as potential members of Incs (Bannantine et al. 2000). Six of the genes were selected for antibody production, and five of these were shown to be located in the inclusion membrane. The genome of C. pneumoniae contains an even higher number of hypothetical Inc proteins (Rockey et al. 2000). The potential to export such a high number of Incs to the inclusion membrane suggests that the inclusion membrane may have several functions in vesicle trafficking, inclusion development, avoidance of lysosomal fusion, nutrient acquisition and signalling associated with EB-RB-EB reorganisation.

2.2.5. Pathogenesis

Different chlamydial species as well as different biovars infect different cell types. The human biovar of C. pneumoniae has been shown to be able to infect and multiply in endothelial cells, smooth muscle cells, monocytes/macrophages and lymphocytes in vitro (Kaukoranta-Tolvanen et al. 1994, Gaydos et al. 1996, Fryer et al. 1997, Airenne et al. 1999, Haranaga et al. 2001). The dissemination of C. pneumoniae has been studied in mouse models. After intranasal inoculation, C. pneumoniae spreads systemically in mice, and it can be isolated from lungs, spleen and peritoneal macrophages (Yang et al. 1995). Intravenous and subcutaneous inoculations also result in disseminated infections. It has further been shown, also in mice, that C. pneumoniae has an ability to disseminate systematically via infected macrophages along hematogenous and lymphatic routes (Moazed et al. 1998).

The host defence mechanisms seem to be unable to eradicate Chlamydia or to provide protection from reinfections. Therefore, repeated infections with Chlamydia are common (Ward 1995). Repeated and persistent chlamydial infections are associated with adverse outcomes, in which Hsps seem to have a role (Beatty et al. 1994b). Chlamydial Hsp60 has been shown to elicit an ocular delayed hypersensitivity response (Morrison et al. 1989b), and its expression has been shown to be increased in persistent infection with C. trachomatis (Beatty et al. 1994a). An enhanced immune reaction against chlamydial Hsp60 is more typically associated with chronic upper genital tract conditions than with acute infections of the lower genital tract (Peeling & Mabey 1999). Serum antibodies to chlamydial Hsp60, as well as Hsp60-specific T cell responses, have been shown to be associated with blinding trachoma, salpingitis, pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility following ocular and genital C. trachomatis infections (reviewed in Kinnunen et al. 2001). In diseases associated with C. pneumoniae infection, antibody responses to chlamydial Hsp60 in asthma (Hahn et al. 2000), arteriosclerosis (Ciervo et al. 2002, Mahdi et al. 2002) and acute anterior uveitis (Huhtinen et al. 2001) have been reported. Since Hsp60 is highly conserved, an autoimmune response to human Hsp60 may have a role in chlamydial pathogenesis. Indeed, antibodies to chlamydial Hsp60 have been shown to cross-react with human Hsp60 (Domeika et al. 1998), and both chlamydial and human Hsp60 proteins are localised in atherosclerotic plaque macrophages (Kol et al. 1998). It has also been found that Hsp60 of C. pneumoniae induces foam cell formation by inducing oxidation of LDL in monocytes (Kalayoglu et al. 1999).

2.2.6. Epidemiology

C. pneumoniae is a common respiratory pathogen worldwide. Most likely, it is primarily transmitted from human to human by the respiratory tract without any animal reservoir (Saikku et al. 1985, Kleemola et al. 1988). C. pneumoniae infection spreads slowly. The incubation period is several weeks, which is longer than that for many other respiratory pathogens (Kuo et al. 1995). The time span of infection spread in families is shorter, however, ranging from 5 to 18 days (Mordhorst et al. 1992, Blasi et al. 1994).

C. pneumoniae infections appear to be most common among school-aged children (Kuo et al. 1995). In some areas, however, infections are already common in children aged 1 to 4 years (Saikku et al. 1988b, Normann et al. 1998). The prevalence increases dramatically after the age of 5, and by the age of 20, half of the population are estimated to have detectable antibody levels. Thereafter, seroprevalence continues to increase in adult age, but at a slower rate, and reaches a level of approximately 75% in the elderly (Saikku 1992, Kuo et al. 1995). Seroprevalence rates continue to be this high despite the fact that some individuals lose their antibodies over a period of several years, suggesting that the majority of people are infected during their lifetime, and that reinfections are common (Grayston et al. 1990).

Seroprevalence is almost equal in both sexes up till adolescence, but higher among adult men than adult women (Saikku 1992, Kuo et al. 1995). In addition to male sex, smoking has been shown to be associated with C. pneumoniae infection (Hahn & Golubjatnikov 1992, Karvonen et al. 1994, Laurila et al. 1997b, von Hertzen et al. 1998, Mayr et al. 2000).

C. pneumoniae infections occur annually, but cyclic variations have been demonstrated. Periods of 2 to 3 years with high incidence rates are followed by 4 to 5 years of lower incidence (Schachter & Grayston 1998). The first observations of C. pneumoniae epidemics came from Finland, where C. pneumoniae caused two outbreaks in civilian communities in 1978 and four in military garrisons in 1977–1978 and 1985–1987 (Saikku et al. 1985, Kleemola et al. 1988, Ekman et al. 1993a). It has been shown that C. pneumoniae was endemic in Finland as early as 1958 (Karvonen et al. 1992). During these epidemics, 43% of pneumonia cases were caused by C. pneumoniae (Ekman et al. 1993b, Kauppinen et al. 1995). At other times, C. pneumoniae causes approximately 10% of all community-acquired pneumonias (Saikku 1992).

2.2.7. Clinical manifestations

C. pneumoniae is a respiratory pathogen that causes both upper and lower respiratory tract diseases. The majority of C. pneumoniae infections are asymptomatic or mild upper respiratory tract infections (Saikku 1992, Miyashita et al. 2001b). Involvement of C. pneumoniae infection has been described in common cold, persistent cough, pharyngitis, sinusitis and otitis media (reviewed in Blasi 2000). Pneumonia and acute bronchitis are the most frequently recognised lower respiratory tract diseases associated with C. pneumoniae infection (Kuo et al. 1995). In addition to acute respiratory infections, several chronic respiratory tract inflammatory diseases have also been associated with C. pneumoniae infection. These include chronic bronchitis and chronic obstructive pulmonary disease (COPD) as well as sarcoidosis (reviewed in Blasi 2000, Saikku 2002). A number of studies also speak for an association between C. pneumoniae and asthma (reviewed by Hahn 1999).

In addition to respiratory tract infections, C. pneumoniae has been associated with cardiovascular diseases. Subacute inflammatory conditions, such as endocarditis, myocarditis and vasculitis, have been reported to follow C. pneumoniae infections (reviewed in Saikku 2002). The association of C. pneumoniae infection with coronary heart disease (CHD) and acute myocardial infarction (AMI) was discovered in 1988 by Saikku et al. (1988a). Although no causal association between C. pneumoniae infection and atherosclerosis has been demonstrated, up to 500 papers have been published to support the theory that C. pneumoniae infection is involved in the clinical diseases associated with atherosclerosis and its complications, such as AMI, stroke, transient ischaemic attack (TIA) and abdominal aortic aneurysm (AAA) (reviewed in Ngeh et al. 2002).

A role for C. pneumoniae in cerebrovascular diseases, such as stroke and TIA, has been suggested in a number of studies, and the organism has been reported to be present in cerebral vessels. There are some reports on an association of C. pneumoniae with acute infections of the central nervous system, such as meningoencephalitis and Guillain-Barre syndrome. Furthermore, C. pneumoniae may play a role in two major neurological disorders: MS and Alzheimer’s disease. (Reviewed in Saikku 2002)

C. pneumoniae infection has also been associated with cancer: small and squamous cell lung cancer, non-Hodgkin’s lymphoma and the rare Szezary’s syndrome. Other diseases associated with C. pneumoniae infection include erythema nodosum, ReA, Sweet’s syndrome and eye diseases (conjunctivitis, iritis and uveitis). (Reviewed in Saikku 2002).

2.2.8. Diagnosis

2.2.9. Treatment

A number of different antibiotics have been tested in search for an appropriate treatment for C. pneumoniae infection. Azithromycin and clarithromycin are two macrolides which have shown high activity against the organism in vitro (Agacfidan et al. 1993, Welsh et al. 1996). Some of the new fluoroquinolones and ketolides, a new class of macrolides, have also turned out effective (Roblin & Hammerschlag 1998, Strigl et al. 2000, Miyashita et al. 2001a, Miyashita et al. 2002). The organism is not susceptible in vitro to sulpha drugs, and penicillin and ampicillin prevent the growth of the organism, but do not destroy it (Kuo et al. 1995). Clinical experience has shown that the symptoms of C. pneumoniae infection frequently recur after short or conventional courses of appropriate antibiotics, and intensive long-term therapy is therefore highly recommended (Kuo et al. 1995). Inappropriate antibiotic treatment may lead to chronicity of the disease. The insidious nature of C. pneumoniae infection makes prevention very difficult, and the development of anti-chlamydial vaccines remains an important goal for researchers.