WO2005027961A2 - Chlamydia pmpd autotransporter and its role as adhesin - Google Patents

Abstract

The present invention relates to the use of chlamydial polymorphic membrane proteins for the manufacture of a medicament for treatment or/and prevention of infections caused by the intracellular bacteria Chlamydia and Chlamydophila. The invention further concerns a method for treatment or/and prevention of a chlamydial infection.

Description

Chlamydia PmpD autotransporter and its role as adhesin

Description

The present invention relates to the use of chlamydial polymorphic membrane proteins for the manufacture of a medicament for treatment or/and prevention of infections caused by the intracellular bacteria Chlamydia and Chlamydophila. The invention further concerns a method for treatment or/and prevention of a chlamydial infection.

The family Chlamydiaceae comprise a diversified group of obligate intracellular Gram-negative bacteria infecting a wide range of different cell types in their eukaryotic hosts, causing a variety of acute and chronic diseases (Kuo et al., 1995; Kalayoglu, Libby, and Byrne, 2002; Hahn and McDonald, 1998; Blanchard and Mabey, 1994). They share a characteristic, biphasic cycle of development with infectious, spore-like elementary bodies (EB) and intracellular dividing, metabolically active reticulate bodies (RB) that inhabit a non-fusogenic inclusion (Moulder, 1991).

Until recently, the family Chlamydiaceae was represented by only a single genus known as Chlamydia that composed of four species: Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia pecorum (Kaltenboeck, Kousoulas et al., 1993). In 1999, the chlamydial taxonomy was revised and the Chlamydiaceae family has been split into two genera (Chlamydia and Chlamydophila) encompassing three (Chlamydia trachomatis, Chlamydia suis, Chlamydia muridarum) and six (Chlamydophila pneumoniae, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila pecorum, Chlamydophila felis, Chlamydophila caviae) species, respectively (Everett, Bush et al., 1999). For simplicity, all members of the family Chlamydiaceae are referred, here, to as chlamydiae.

For a productive chlamydial infection, adhesion of EB leading to invasion must take place. Adhesion is often mediated via receptor-ligand interactions, where a receptor on a host cell surface is bound by a ligand on the bacterial surface. Presence or absence of receptors and ligands facilitates bacterial entry into specific tissues, being responsible for tissue tropism (Meyer, 1999). In chlamydial infections, host cell membrane components like mannose-receptor or the estrogen receptor complex were shown to support the infection (Mamelak et al., 2001; Su et al., 1996; Taraktchoglou et al., 2001; Davis, Raulston, and Wyrick, 2002). In addition, inhibition of infection after proteolytic treatment further pointed at protein components to function as cellular receptor(s) (Byrne and Moulder, 1978; Vretou, Goswami, and Bose, 1989). Differences in pathogenicity between chlamydial species and biovars are linked to a diverse tissue tropism. This can partly be explained by different abilities to cope with the challenges in the respective microenvironment but also with the prerequisite to attach to and invade susceptible cell types (Belland et al., 2001; Fehlner-Gardiner et al., 2002; Kuo et al., 2002; Rasmussen-Lathrop et al., 2000).

Evaluation of the chlamydial attachment mechanism(s) revealed a stepped effect by treating EB with mild heat and heparin or heparan sulfate or by preincubating host cells with polycationic chemicals (Kuo and Grayston, 1976; Chen and Stephens, 1997; Zhang and Stephens, 1992; Wuppermann, Hegemann, and Jantos, 2001). No single adhesin could be identified on EB, but a variety of molecules like MOMP, Hsp70 or OmcB have been suggested to be involved in adhesion (Su et al., 1990; Kuo et al., 1996; Raulston et al., 2002; Stephens et al., 2001 ; Raulston, 1995).

In this context, the polymorphic membrane proteins (Pmps) form an interesting group of chlamydial proteins defined by the presence of highly repetitive motifs of four amino acids (GGAI and FxxN) (Everett and Hatch, 1995). The importance of the Pmp superfamily is underscored by the fact that intracellular bacteria collapse the size of their genome in a process called "evolution by reduction". Nevertheless, more than five percent of the total coding capacity in C. pneumoniae and almost 22% absent in C. trachomatis consist of pmp-genes with C. pneumoniae having 21 members as compared to only 9 in C, trachomatis (pmpA-l). Functionally, Pmps were suggested to take part in binding or docking to eukaryotic cells, a task attributed to proteins containing more than one of GGAI and FxxN motifs [i.e. bacterial rOmpA (Rickettsia spp.) or eukaryotic zonadhesin (Mus musculus)] (Grimwood and Stephens, 1999; Kalman et al., 1999; Dobrindt and Hacker, 2001; Read et al. , 2000).

Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacteria proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known.

Bioinformatically, all members of the heterogeneous Pmp superfamily were classified as autotransporters (Grimwood and Stephens, 1999; Henderson and Lam, 2001). Autotransporter use a secretion mechanism, in which they pass the outer membrane (OM) of Gram-negative bacteria and reach the surface without an assistance of other proteins. In detail, after Sec- dependent delivery into the periplasm, conserved C-terminal transporter domain spontaneously forms a pore-like beta-barrel in the OM through witch the N-terminal passenger domain leaves the bacterium (Pohlner et al., 1987; Henderson, Navarro-Garcia, and Nataro, 1998). In case of C. trachomatis and C. psittaci, several Pmps [also called (P)Omps] were shown to be expressed late during the infection cycle and were present in OM-complexes or on the bacterial surface, eliciting strong immune response in the course of natural infection (Vretou et al., 2003; Tanzer, Longbottom, and Hatch, 2001; Tanzer and Hatch, 2001; Knudsen et al., 1999).

In C. pneumoniae, all 21 Pmps were shown to be transcribed (Grimwood, Olinger, and Stephens, 2001). Strain and even single clone diversity caused by a different number of tandem repeats in pmp6 and a potential frame shift provided by a slip(ped) strand mechanism with a poly(G) stretch in pmp10, respectively, was considered to provide a high level of functional and antigenic diversity on the surface of C. pneumoniae (Christiansen et al., 1999; Grimwood, Olinger, and Stephens, 2001; Shirai et al., 2000; Pedersen, Christiansen, and Birkelund, 2001; Rocha et al., 2002).

Pmp21 (PmpD) was shown to be post-translationally cleaved/processed resulting in N-terminal surface exposure, which is in agreement with the proposed autotransporter-export mechanism (Vandahl et al., 2002). Recently, NF-κB-mediated induction of the inflammatory mediators IL-6, IL-8 and MCP-1 was observed in human endothelial cells incubated with recombinant Pmp20 or Pmp21 (Niessner et al., 2003).

Two distinguishing characteristics of chlamydiae are its developmental cycle and predilection for causing a persistent (chronic or latent) infections (Moulder, 1991), during which the normal developmental cycle is altered, producing aberrant RB-like forms. Persistency can be established in vitro using several methods, including treatment with cytokines or antibiotics or by deprivation of certain nutrients, such as amino acids (Beatty, Byrne et al., 1994) and iron (Al-Younes, Rudel et al., 2001). Persistent infections produced can revert to normally growing organisms when the suppressor is removed or nutrients are replaced (Allan and Pearce, 1983; Al-Younes, Rudel et al., 2001). In humans, acute chlamydial infections can progress to persistent infections, which may lead to a pathogenic process that leads to chronic diseases including blindness, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, arthritis, Alzheimer's disease and atherosclerosis (Hammerschlag, 2002; Villareal, Whittum-Hudson et al., 2002; Stephens, 2003).

Although they have a similar unique developmental cycle, chlamydiae cause a variety of human and animal diseases. Chlamydia trachomatis, primarily a pathogen of humans, is one of the most common bacterial pathogens that primarily infects columnar epithelial cells of the ocular and genital mucosae, causing sexually transmitted and ocular diseases in humans. These diseases have a significant impact on human health worldwide, causing trachoma, the leading cause of preventable blindness, and sexually transmitted diseases (STD) that include tubal factor infertility, life-threatening ectopic pregnancy, and pelvic inflammatory disease that often result in involuntary sterility (Stephens, 2003). Chlamydial STDs are also risk factors in cervical squamous cell carcinoma (Anttila, Saikku et al., 2001) and HIV infection (Chesson and Pinkerton, 2000). Infants are at risk for chlamydial eye infection and pneumonia if they pass through an infected cervix (Stephens, 2003). Chlamydia trachomatis strains (or serovars) L1, L2 and L3 are the etiological agents of the sexually transmitted systemic syndrome Lymphogranuloma venereum (LGV). Serovars A to C are primarily the agents responsible for the endemic blinding trachoma, while serovars D to K are associated with STDs (Guaschino and De Seta, 2000).

Chlamydophila pneumoniae is an important cause of human respiratory tract diseases, such as pneumonia, pharyngitis, sinusitis, otitis, asthma, acute bronchitis (Grayston, Campbell et al., 1990), persistent cough, chronic obstructive pulmonary disease (COPD), flu-like syndrome (Blasi, Arosio et al., 1999) and lung carcinoma (Laurila, Anttila et al. 1997). In addition, this pathogen is correlated with other non-pulmonary diseases, such as erythema nodosum (Erntell, Ljunggren et al., 1989), Guillain-Barre syndrome (Haidl, Ivarsson et al., 1992), endocarditis (Grayston, Campbell et al., 1990), Alzheimer's disease (Balin, Gerard et al., 1998), reactive arthritis (Villareal, Whittum-Hudson et al., 2002), meningoencephalitis (Koskiniemi, Gencay et al., 1996) and the blood vessel disease atherosclerosis (Campbell and Kuo, 2003).

Other species, such as C. psittaci, C. abortus and C. pecorum, are responsible for several major diseases in animals, mainly spontaneous abortion in livestock and systemic disease in birds, and can also infect rodents and cats (Schachter, 1999).

The biological mechanisms responsible for these differences in vertebrate host, tissue tropism and spectrum of diseases are unknown.

Of all the infectious diseases reported to the U.S. state health departments and the U.S. Centers for Disease Control and Prevention, C. trachomatis genital tract infections are the most common, with an estimated 4 to 5 million cases occurring annually in the United States and 3 million cases occur in Europe (Marrazzo and Stamm, 1998; Schachter, 1999). In 1995, infections with C. trachomatis were the most commonly reported bacterial disease in the U.S. (Marrazzo and Stamm, 1998), and the World Health Organization estimated that 89 million new cases would arise worldwide (Marrazzo and Stamm, 1998).

Serological surveys have shown that virtually every human has been infected with C. pneumoniae (Grayston, 2000). This prevalent pathogen causes 6 to 25% of community-acquired pneumonia. Further, this pathogen is also associated with other respiratory diseases as well as non-respiratory diseases, such as cancer and Alzheimer's disease. Importantly, increasing evidence demonstrated that C. pneumoniae is present and persistent at sites of arterial disease and, thus, contributes to coronary artery disease (Atherosclerosis). The presence of C. pneumoniae in atheromatous plaques has been demonstrated by several methods, such as polymerase chain reaction (PCR), immunocytochemistry, in situ hybridization, electron microscopy and by recovery of bacteria in tissue cultures (Ramirez, 1996). Furthermore, respiratory inoculation of C. pneumoniae in experimental animal models induced or accelerated the formation of atherosclerotic lesions (de Boer, van der Wal et al., 2000). This disease continues to be the principal cause of death in the U.S. and in most Western countries (Braunwald, 1997). For example, it causes nearly 25% of. all deaths each year in the UK, whereas it causes about 40% of annual deaths in the U.S. (Gupta and Camm, 1998).

After entry into a host cell, the EB is localized to a phagosome. At the very early stage of infection (1 to 3 h), the parasite exerts profound effects on the host. Through an unknown mechanism, dependent on both bacterial transcription and translation (Scidmore, Rockey et al., 1996; Al-Younes, Rudel et al, 1999), chlamydiae modify the properties of the phagosome and prevent its entry into the lysosomal pathway (Heinzen, Scidmore et al., 1996; Al-Younes, Rudel et al., 1999). Many obligate and facultative intracellular pathogens use this approach to avoid intracellular killing by using different means to interfere with cellular trafficking (Duclos and Desjardins, 2000). This unique parasite strategy provides a continuously protected intracellular niche in which chlamydiae then replicate.

Because of the unique biology of chlamydiae, specific requirements are imposed on antimicrobial agents employed for therapy of chlamydial infections. The extracellular EBs are metabolically inactive and resistant to killing. Therefore, antichlamydial agents must efficiently penetrate tissues and then cellular and inclusion membranes in order to inhibit growth of the metabolically active and dividing RBs. Chlamydiae have a relatively long developmental cycle, thus, prolonged course of therapy must be adopted or an antibiotic with a long half-life must be selected.

Several antibiotics such as doxycycline, azithromycin and rifampin, were considered as first-line choices in treatment of C. pneumoniae infections and uncomplicated human genital infections with C. trachomatis (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). These antibiotics are characterized by long half-life and good tissue and cell penetration (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). Several quinolones (ofloxacin and ciprofloxacin) are also recommended as an alternative therapy for chlamydial infections in humans (Marrazo and Stamm, 1998). Therapies with other antibiotics, such as amoxicillin, erythromycin and sulfa drugs are less effective with efficacies between 60% and 80% (Marrazo and Stamm, 1998; Guaschino and Ricci, 2003). Other antibiotics were recommended, including ceftriaxone, cefoxitin, probenecid, mitronidazole, cefotetan, gentamicin (Mazzarro and Stamm, 1998, Guaschino and Ricci, 2003), levofloxacin (Baltch, Smith et al., 2003), garenoxacin (Roblin, Reznik et al., 2003a) and rifamycin derivatives ABI-1648, ABI-1657 and ABI-1131 (Roblin, Reznik et al., 2003b).

Many disadvantages were reported on the use of antibiotics. For instance, some antibiotics should not be used by pregnant and lactating women and in individuals younger than 16 years of age. Some antimicrobial agents have been associated with an unacceptable rate of chlamydial relapse. Use of antibiotics is sometimes associated with significant side effects, such as gastrointestinal intolerance (Marrazzo and Stamm, 1998; Guaschino and Ricci, 2003) and up to 20% discontinue therapy because of these adverse effects (Guaschino and Ricci, 2003). Some antibiotics have to be given for longer than one week (2 to 3 weeks) to avoid recurrence of infection, which is common (Roblin, Montalban et al., 1994). Recently, there have been reports of multi-drug resistant chlamydial infections causing relapses or persistent infections (Hammerschlag, 2002; Guaschino and Ricci, 2003).

Another more important disadvantage on the use of antibiotics is that chronic infections are less responsive to antibiotic therapy, compared to the acute infection with chlamydiae or to the in vitro infection (Beatty, Byrne et al., 1994). In addition, chlamydial infection in certain cell types were reported not responsive to antibiotic treatment. For instance, infections of C. pneumoniae in human monocytes and lymphocytes are not responsive to treatment of antibiotics usually efficacious in treatment of infection in other cell types. Thus, the reduced antimicrobial susceptibility might probably allow circulating monocytes and lymphocytes to transfer the pathogen from the respiratory tract (primary site of infection) to the cells of the vascular wall and other sites, where reinfection is initiated and, thus, chronic disease formation is promoted (Boman, Soderberg et al., 1998; Gieffers, Fullgraf et al., 2001 ; Yamaguchi, Friedman et al., 2003). In vitro experiments showed that chlamydiae in monocytes and lymphocytes showed reduced antibiotic susceptibility in the presence of rifampin, the most effective anti-C. pneumoniae drug in vitro (Gieffers, Solbach et al., 1998), and azithromycin a macrolide widely used in current treatment trials (Grayston, 1999). Beside in vitro studies, C. pneumoniae were cultured from monocytes of coronary artery disease patients undergoing experimental azithromycin treatment for coronary sclerosis. This finding proves the presence of viable chlamydiae in the bloodstream, despite antichlamydial therapy (Gieffers, Fullgraf et al., 2001) and indicates the not sufficiently successful antibiotic therapy of in vivo infection, compared to more efficacious in vitro treatment trials.

Antibiotic-resistant C. pneumoniae was observed not only in blood cells but also in tissues of atheromas and infected tissues of the respiratory system and joints. Treatment failures were seen in respiratory infections with chlamydial strains that seemed susceptible in acute infections in vitro (Hammerschlag, Chirgwin et al., 1992). In addition, using standard antibiotic therapeutic approaches against chlamydiae may not be successful in alleviating clinical coronary artery disease symptoms (Meier, Derby et al., 1999; Muhlestein, Anderson et al., 2000). Reduced antibiotic susceptibility of chlamydiae in tissues to antibiotic intervention is likely due to the presence of chlamydiae in a persistent state. For instance, Hammerschlag, Chirgwin et al. (1992) described persistent nasopharyngeal infection after acute respiratory illness caused by C. pneumoniae in 5 patients for periods up to 11 months, despite treatment with multiple and prolonged courses of antibiotics. Follow-up studies of 2 of these patients documented persistence for 7 to 9 years (Hammerschlag, 2002). One patient was culture-positive on 14 separate occasions over the course of 9 years. In vitro susceptibility testing of these isolates did not demonstrate development of antibiotic resistance. This in vivo multiple drug resistance was also observed in C. trachomatis infections and was associated with clinical treatment failures (Guaschino and Ricci, 2003). Taken together, chlamydial infection in tissues cannot be completely eliminated by antibiotic trials, and thus, forming persistent multidrug-resistant infections in tissues that can be reactivated and reinfection may occur, especially when antibiotic levels in tissues declined with time, causing promotion of chronic diseases in those tissues.

The extremely high prevalence of C. pneumoniae and C. trachomatis infections in humans and the inefficacy of presently used drugs (antibiotics) to completely resolve the chronic infection and the eventual role of antibiotics in establishing persistent chlamydial infections, associated with many serious chronic diseases, necessitate a more reliable and effective 5 therapeutic approach which could help in complete resolving of chlamydial infections. Further, an effective antichlamydial vaccine is still not present.

It is therefore the object of the present invention to provide a method of prevention or/and treatment of chlamydial infections which at least partiallyo overcomes the disadvantages of the state of the art treatments of chlamydial infections. In particular, medicaments are needed which are able to completely eradicate chlamydiae in order to prevent relapsing infections.

Despite existing in silico predictions, experimentally verified function of5 PmpD remained to date unclear. Here, we wanted to determine the detailed processing and subcellular localization of PmpD and the functional properties of this protein. To address this, we raised a polyclonal serum against PmpD and performed series of experiments to determine its temporal expression pattern during infection, mechanism of transport and theo processing and localization along with its function as a chlamydial adhesin.

Surprisingly, it was found in the context of the present invention that PmpD from C. pneumoniae is a cleaved, surface exposed protein mediating the early interaction of EB with the host cell and inducing activation and cytokine5 release from monocytes. The experiments further demonstrated a surprising reduction in the chlamydial infectivity by anti-N-pmpD antibodies for up to 95% in a concentration-dependent manner.

Here, using 1D and 2D PAGE we demonstrate that PmpD is processed intoo two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C- terminal part (C-pmpD). Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane. We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells. In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner. These results demonstrate that N-pmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation. Chlamydial polymorphic membrane proteins, in particular PmpD, or immunogenic fragments thereof were found to be important targets for anti-chlamydial vaccination.

The solution to the problem outlined above are thus chlamydial polymorphic membrane proteins, in particular PmpD, or immunogenic fragments thereof, in particular N-pmpD or M-pmpD, or antibodies against chlamydial polymorphic membrane proteins or immunogenic fragments thereof, which can be used for the manufacture of a medicament for treatment or/and prevention of chlamydial infections in warm blooded animals, including humans.

A first aspect of the present invention ist therefore the use of (i) a chlamydial polymorphic membrane protein or/and an immunogenic fragment thereof, or/and (ii) antibodies against a chlamydial polymorphic membrane protein or/and against an immunogenic fragment thereof, for the manufacture of a medicament for the treatment or/and prevention of a chlamydial infection.

In a preferred embodiment, the medicament causes stimulation of a humoral response against the chlamydial polymorphic membrane protein or/and the immunogenic fragment thereof. Administration of a chlamydial polymorphic membrane protein, in particular PmpD, or an immunogenic fragment thereof to a subject in need thereof optionally together with suitable adjuvants, could induce formation of antibodies in the subject which are effective against chlamydiae in treatment or/and prevention.of chlamydial infections.

The antibody of the present invention contacts chlamydiae (EBs) before entry into a host cell. Therefore, the antibody impedes/prevents propagation of a chlamydial infection once chlamydiae have entered the body. This can either take place by inhibiting attachment and internalization of the infectious EBs to the host cells or by destruction of the antibody-labelled chlamydiae through the immune system.

In yet another preferred embodiment, the polymorphic membrane protein is selected from the group consisting of PmpG, PmpA/l, PmpH, PmpE, PmpE/F, PmpA, PmpB, PmpD, CPJ0015, CPJ0017 and Cpj0019. PmpG may be characterized by the homologues CpnOOOδ (GenBank-identifier gi14195071), Cpn0013 (gi14195068), Cpn0444 (gi14195070), Cpn0445 (gi 14195069), Cpn0446 (gi 14195066), Cpn0447 (gi 14195067), Cpn0449 (gi14195016), Cpn0451 (gi14195006), or/and Cpn0453 (gi14195030). PmpA/l may be characterized by Cpn0452 (gi14195022). PmpH may be characterized by Cpn0454 (gi14195029). PmpE may be characterized by the homologues Cpn0466 (gi14195028), Cpn0467 (gi14195027), or/and Cpn0470 (gi15618381). PmpE/F may be characterized by Cpn0471 (gi14195026). PmpA may be characterized by Cpn0539 (gi14195025). PmpB may be characterized by Cpn0540 (gi14195024). PmpD may be characterized by Cpn0963 (gi14195023). CPJ0015 may be characterized by the sequence gi15835551. CPJ0017 may be characterized by the sequence gi15835554. Cpj0019 may be characterized by the sequence gi15835556. Further details of the sequences are described in Table 1. Cpj0015, CP0017, CP0019, and Cpn0470 may contain a frameshift mutation in strain CWL029 compared with other strains.

An especially prefered polymorphic membrane protein is PmpD. Table 1: Polymorphic membrane proteins auf C. pneumoniae strain CWL029

Ppm No. Ppm Name GenBank ID Cpn No. Omp-No. Notes

Pmp1 PmpG gi|14195071 CpnOOOδ Omp6

Pmp2 PmpG gi|14195068 Cpn0013 Omp7

Pmp3 gi|15835551 CPJ0015 frameshift

Pmp4 gi|15835554 CPJ0017 frameshift

Pmp5 gi|15835556 CPJ0019 frameshift

Pmp6 PmpG gi|14195070 Cpn0444

Pmp7 PmpG gi|14195069 Cpn0445 Omp12

Pmp8 PmpG gi|14195066 Cpn0446 Omp11

Pmp9 PmpG gi|14195067 Cpn0447 Omp10

Pmp10 PmpG gi|14195016 Cpn0449 Omp5

Pmp11 PmpG gi|14195006 Cpn0451 Omp4

Pmp12 PmpA/l gi|14195022 Cpn0452 Omp13

Pmp13 PmpG gi|14195030 Cpn0453 Omp14

Pmp14 PmpH gi|14195029 Cpn0454

Pmp15 PmpE gi|14195028 Cpn0466

Pmp16 PmPE gi|14195027 Cpn0467 (OmpE)

Pmp17 PmpE gi|15618381 Cpn0470 frameshift

Pmp18 PmpE/F gi|14195026 Cpn0471

Pmp19 PmpA gi|14195025 Cpn0539 (OmpA)

Pmp20 PmpB gi|14195024 Cpn0540

Pmp21 PmpD gi|14195023 Cpn0963

SEQ.ID.NO:1 describes the nucleotide sequence encoding PmpD (see Figure 1D). SEQ.ID.NO:2 describes the amino acid sequence of PmpD (see Figure 1B).

In a further preferred embodiment, the polymorphic membrane protein or the immunogenic fragment thereof is therefore encoded by a nucleic acid comprising

(a) the nucleotide sequence of SEQ.ID.NO:1 , (b) a nucleotide sequence corresponding to the sequence of (a) within the scope of the degeneracy of the genetic code, (c) a nucleotide sequence which is at least 70 % homologous to the sequence of (a) or (b), preferably at least 80 %, more preferably at least 90 %, most preferably at least 95 %, or (d) a fragment of the sequences of (a), (b) or (c).

In another preferred embodiment, the polymorphic membrane protein or the 5 immunogenic fragment thereof may comprise

(a) the amino acid sequence of SEQ.ID.NO:2, (b) an amino acid sequence which is at least 70 % homologous to the sequence of (a), preferably at least 80 %, more preferably at leasto 90 %, most preferably at least 95 %, or (c) a fragment of the sequences of (a) or (b).

The immunogenic fragment of the present invention has a length of a least 6 amino acids, preferably at least 10 amino acids, more preferably at least 50s amino acids, most preferably at least 100 amino acids.

The immunogenic fragment of the present invention has a length of at the maximum 100 amino acids, preferably at the maximum 250 amino acids, more preferably at the maximum 600 amino acids, most preferably at theo maximum 800 amino acids.

It turned out that a lysate of RB contained peptides from the N-terminal part of PmpD, N-pmpD (amino acid 122-655 in SEQ.ID.NO:2) and M-pmpD (amino acid 670-1114 in SEQ.ID.NO:2) matching to the theoretical trypsin5 cleavage-products from the middle part of PmpD (Fig. 1B). The most preferred immunogenic fragments of the polymorphic membrane protein are therefore a polypeptide comprising N-pmpD (amino acids 122 to 655 in SEQ.ID.NO:2), a polypeptide comprising M-pmpD (amino acids 670 to 1114 in SEQ.ID.NO:2), a polypeptide comprising the sequence of amino acids 16o to 670 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 670 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1114 in SEQ.ID.NO:2, or/and a polypeptide comprising a sequence starting at amino acid 31 in SEQ.ID.NO:2, which is the first amino acid of the mature PmpD protein (see Fig. 1B), amino acid 16 in SEQ.ID.NO:2, or amino acid 122 in SEQ.ID.NO-.2, and terminating at amino acid 660 in SEQ.ID.NO:2 (see Fig. 1C), at amino acid 655 in SEQ.ID.NO:2, or at amino acid 647 in SEQ.ID.NO:2. The two serine residues at positions 648 and 649 indicate a cleavage site between the positions 647 and 648.

Alternatively, the immunogenic fragment as described above may contain alone at either the N-terminal or C-terminal end or at both ends at least 1 , at least 5, at least 10, or at least 20 additional amino acid residues, which may be derived from SEQ.ID.NO:2. The immunogenic fragment as described above may contain alone at either the N-terminal or C-terminal end or at both ends, at the maximum 1, at the maximum 5, at the maximum 10, at the maximum 20 or at the maximum 50 additional amino acid residues, which may be derived from SEQ.ID.NO:2.

The above described immunogenic fragments of SEQ.ID.NO:2 may be encoded by a nucleic acid comprising

(a) a fragment of SEQ.ID.NO:1, (b) a sequence corresponding to the fragment of SEQ.ID.NO:1 of (a) within the scope of the degeneracy of the genetic code, or/and (c) a nucleotide sequence which is at least 70 % homologous to sequence of (a) or (b), preferably at least 80 %, more preferably at least 90 %, most preferably at least 95 %.

Suitable hosts and vectors for recombinant expression of the polymorphic membrane protein or immunogenic fragments thereof as described above by a nucleic acid as described above are known by a person skilled in the art.

A BLAST search of the PmpD sequence against the known sequences of chlamydiae revealed that there is only a homology of at the maximum 25 % between PmpD and other Pmp proteins. In further BLAST searches of N- pmpD and M-pmpD (in total amino acids 1 to 1145 of SEQ.ID.NO:2), it turned out that no proteins exists having a homology of at least 70% to either N-pmpD or M-pmpD.

In the present invention, homology refers to the proportion of identical amino acids or nucleotides in two polypeptides or nucleic acids.

The chlamydial infection may be an infection with microorganisms from the genus Chlamydia, preferably Chlamydia trachomatis. Diseases caused by Chlamydia, in particular C. trachomatis, which can be treated by the medicament/pharmaceutical composition of the present invention can be diseases in humans, including infections of columnar epithelial cells of the ocular and genital mucosae, ocular diseases, trachoma, endemic blinding trachoma transmitted by Chlamydia trachomatis serovars A to C, chlamydial eye infection and pneumonia in infants, Lymphogranuloma venereum transmitted by Chlamydia trachomatis strains (or serovars) L1 , L2 and L3, sexually transmitted diseases (STDs) including tubal factor infertility, life- threatening ectopic pregnancy, pelvic inflammatory involuntary sterility, STDs transmitted by Chlamydia trachomatis serovars D to K, and chlamydial STDs associated with cervical squamous cell carcinoma or HIV infection.

The chlamydial infection may also be an infection with microorganisms from the genus Chlamydophila, preferably Chlamydophila pneumoniae. Diseases caused by Chlamydophila, in particular C. pneumoniae, which can be treated by the medicament/pharmaceutical composition of the present invention are human respiratory tract diseases including pneumonia, pharyngitis, sinusitis, otitis, asthma, acute bronchitis, persistent cough, chronic obstructive pulmonary disease (COPD), flu-like syndrome, lung carcinoma, and non- pulmonary human diseases including erythema nodosum, Guillain-Barre syndrome, endocarditis, Alzheimer's disease, reactive arthritis, meningoencephalitis and atherosclerosis. Animal diseases caused by Chlamydophila, in particular C. psittaci, C. abortus or C. pecorum, which can be treated by the medicament/pharmaceutical composition of the present invention are spontaneous abortion in livestock and systemic disease in birds, rodents and cats.

The antibody of the present invention may be an antibody against a chlamydial polymorphic membrane protein, preferably PmpD. It is preferred that antibodies are directed against an immunogenic fragment of a polymorphic membrane protein, in particular against an immunogenic fragment of PmpD. The most preferred antibodies are antibodies directed against a polypeptide comprising N-pmpD (amino acid 122-655 in SEQ.ID.NO:2) (anti-N-pmpD antibodies), a polypeptide comprising M-pmpD (amino acid 670-1114 in SEQ.ID.NO:2) (anti-M-pmpD antibodies), a polypeptide comprising the sequence of amino acids 16 to 670 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 670 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1114 in SEQ.ID.NO:2, or/and a polypeptide comprising a sequence starting at amino acid 31 in SEQ.ID.NO:2, amino acid 16 in SEQ.ID.NO:2, or amino acid 122 in SEQ.ID.NO:2, and terminating at amino acid 660 in SEQ.ID.NO:2, at amino acid 655 in SEQ.ID.NO:2, or at amino acid 647 in SEQ.ID.NO:2.

The antibodies of the present invention may be used in a pharmaceutical composition, which may be a vaccine, for treatment or/and prevention of a chlamydial infection or/and for diagnosis of a chlamydial infection.

The antibody of the present invention may be a monoclonal or polyclonal antibody, a chimeric antibody, a chimeric single chain antibody, a Fab fragment or a fragment produced by a Fab expression library.

Techniques of preparing antibodies of the present invention are known by a skilled person. Monoclonal antibodies against polymorphic membrane proteins or immunogenic fragments thereof may be prepared by the human B-cell hybridoma technique or by the EBV-hybridoma technique (Kόhler et al., 1975, Nature 256:495-497, Kozbor et al., 1985, J. Immunol. Methods 81,31-42, Cote et al., PNAS, 80:2026-2030, Cole et al., 1984, Mol. Cell Biol. 62:109-120). Chimeric antibodies (mouse/human) against polymorphic membrane proteins or immunogenic fragments thereof may be prepared by carrying out the methods of Morrison et al. (1984, PNAS, 81:6851-6855), Neuberger et al. (1984, 312:604-608) and Takeda et al. (1985, Nature 314:452-454). Single chain antibodies may be prepared by techniques known by a person skilled in the art.

Recombinant immunoglobulin libraries (Orlandi et al, 1989, PNAS 86:3833- 3837, Winter et al., 1991, Nature 349:293-299) may be screened to obtain an antibody of the present invention which are specific against polymorphic membrane proteins or immunogenic fragments thereof. A random combinatory immunoglobulin library (Burton, 1991, PNAS, 88:11120-11123) may be used to generate an antibody with a related specifity having a different idiotypic composition.

Another strategy for antibody production is the in vivo stimulation of the lymphocyte population.

Furthermore, antibody fragments (containing F(ab')₂ fragments) of the present invention can be prepared by protease digestion of an antibody, e.g. by pepsin. Reducing the disulfide bonding of such F(ab')₂ fragments results in the Fab fragments. In another approach, the Fab fragment may be directly obtained from an Fab expression library (Huse et al., 1989, Science 254:1275-1281).

Polyclonal antibodies of present invention may be prepared employing the polymorphic membrane proteins or immunogenic fragments thereof as antigen by standard immunization protocols of a host, e.g. a horse, a goat, a rabbit, a human, etc., which standard immunization protocols are known by a person skilled in the art. ln yet another embodiement, the present invention concerns a pharmaceutical composition comprising as an active agent for the prevention or/and treatment of infections with chlamydiae (i) a polymorphic membrane protein or/and an immunogenic fragment thereof as defined above, or/and (ii) an antibody as defined above, optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives.

The pharmaceutical composition of the present invention may be used for treatment and prevention of a warm blooded animal, preferably a mammal including a human. In a preferred embodiment, the pharmaceutical composition is for use in human medicine.

The pharmaceutical composition of the present invention may be a vaccine in which the chlamydial polymorphic membrane protein or immunogenic fragment thereof acts as an antigen, for treatment or prevention of a subject in need thereof. The pharmaceutical composition of the present invention may also by used for antigen production in a suitable host. The antigen may be formulated together with suitable carriers, adjuvants, e.g. Freund's adjuvant, diluents or/and additives. The antigen may be prepared in a live vaccine, which is a recombinant vector, e.g. a gram negative bacterium, such as E. coli, capable of expressing the antigen. Expression of the antigen in a recombinant live vector is known by a person skilled in the art. The homologous autotransporter domain of the polymorphic membran protein may be replaced by an autotransporter domain suitable for expression in the recombinant live vector, which preferably is an autotransporter domain homologous to the recombinant live vector. A prefered autotransporter is AIDA-I from E. coli (Maurer et al., 1999, J. Bacteriol. 181:7014-20), and a preferred host is E. coli. If an immunogenic fragment is to be expressed in a recombinant live vector, it may be fused to a suitable autotransporter domain. As pointed out above, eradication is a major problem in patients with chronic chlamydial infections. It is thus preferred that the pharmaceutical composition of the invention is for treatment of patients with chronic infections with chlamydiae, especially those that are associated with chronic respiratory system and heart diseases.

The amount of the chlamydial polymorphic membrane protein, the immunogenic fragment thereof, the antibody against a chlamydial polymorphic membrane protein or/and the antibody against the immunogenic fragment thereof present in the pharmaceutial composition of the present invention effective for treatment or/and prevention of chlamydial infection can be determined by a person skilled in the art, in particular by a physician.

In yet another embodiment, the pharmaceutical composition of the present invention comprises at least one further active ingredient for the prevention or/and treatment of chlamydial infections. The at least one further active ingredient may be any known agent suitable for treatment or/and prevention of chlamydial infections. It is prefered that the at least one further active ingredient is selected from antibiotics or/and amino acids.

In a preferred embodiment, the pharmaceutical composition of the present invention comprises at least one amino acid selected from naturally occurring L-amino acids, analogues and derivatives thereof. It was surprisingly found that increasing concentrations of amino acids, e.g. L- leucine, L-isoleucine, L-methionine or L-phenylalanine could dramatically suppress chlamydial growth. Supplementation of human cell cultures infected with chlamydiae with exogenous amounts of individual amino acids markedly affected at least one of the following: the inclusion size, morphology of chlamydial forms and development of infectious progeny. More importantly, L-methionine, L-isoleucine and L-leucine (at concentrations of 10 mM each) completely inhibited multiplication of entered bacteria, leading to total arrest of inclusion maturation and to complete suppression (100%) of the production of infectious chlamydiae. 50% of inhibition of production of infectious progeny (EBs) was obtained in C. trachomatis at a concentration of 0.25-0.5 mM L-leucine, L-isoleucine or L- methionine. C. pneumoniae was found to be slightly more sensitive to amino acid treatment. In C. pneumoniae, 50% inhibition of production of infectious progeny was obtained at a concentration of 0.1-0.25 mM L-leucine, < 0.1 mM L-isoleucine or 0.25-0.5 mM L-methionine. 10 mM L-phenylalanine, which also markedly inhibited the growth of the bacterial vacuole, very strongly reduced the production of infectious chlamydial progeny by more than 99%, compared to the control untreated infected cell cultures. Details of the experimental procedure are described in patent application PCT/EP2004/009926, which is included herein by reference.

It is more preferred that the pharmaceutical composition comprises at least one amino acid selected from essential amino acids, analogues and derivatives thereof. It is most preferred that the amino acid effective as further active ingredient is selected from the group consisting of L-leucine, L- isoleucine, L-methionine, L-phenyialanine, analogues and derivatives thereof.

A person skilled in the art, in particular a physician, is able to determine the dosage of amino acids or/and analogues or/and derivatives thereof, which have to be administered to reach plasma concentrations sufficient for effective treatment or/and prevention. Suitable amounts of an amino acid or/and an analogue or/and a derivative thereof are preferably at least 1 mmol/kg body weight up to 10 mmol/kg body weight, more preferably up to 5 mmol/kg body weight, most preferably up to 2,5 mmol/kg body weight. Amino acids may be taken, for example, orally as tablets, capsules or as a drink three times daily for preferably at least 2 days, more preferably at least 1 week.

The antibiotic effective as further active ingredient in the pharmaceutical composition of the present invention may be any known antibiotic suitable for treatment or/and prevention of chlamydial infections. It is therefore preferred that the further active ingredient is an antibiotic selected from macrolides, quinolones and combinations thereof.

The pharmaceutical composition of the present invention comprising the antibiotic or/and the amino acid could improve the clinical condition of patients with coronary heart disease by eradication of chlamydiae from lesions in the blood vessel wall (atheromas), where antimicrobial resistance is common. Due to the combination of a polymorphic membrane protein, an immunogenic fragment thereof or/and an antibody against the polymorphic membrane protein or/and an immunogenic fragment thereof with an antibiotic or/and an amino acid, a complete eradication of the chlamdyiae may be achieved, which is difficult to achive with an antibiotic treatment alone.

The amount of antibiotic in the pharmaceutial composition of the present invention effective for treatment or/and prevention of chlamydial infection can be determined by a person skilled in the art, in particular by a physician.

A further aspect of the present invention is a method for treating or/and prevention of a chlamydial infection, the method comprising the administration of (i) a chlamydial polymorphic membrane protein or an immunogenic fragment thereof, or/and (ii) antibodies against a polymorphic membrane protein or against an immunogenic fragment thereof, in a amount effective in therapy or/and prevention to a subject in need thereof. Effective amounts for therapy or/and prevention of chlamydial infections can be determined by a person skilled in the art, in particular by a physician. Common administration routes may be used, in particular the oral, subcutaneous, or/and intramuscular route.

By the method of the present invention, a humoral response against the chlamydial polymorphic membrane protein or/and the immunogenic fragment thereof may be stimulated, when the chlamydial polymorphic membrane protein or/and the immunogenic fragment thereof is administered in an amount effective to elicit a humoral response.

It was surprisingly found that PmpD both upregulates mitochondrial activity and stimulates cytokine secretion in monocytes. Therefore, the presence of chlamdyiae may be determined by cytokine secretion or by an increased metabolic activity. Another aspect of the present invention is therefore a screening method for identification of a compound suitable for treatment, prevention or/and diagnosis of chlamydial infections, comprising the steps (a) providing a cell capable of secreting cytokines, (b) contacting a compound with chlamydiae or/and the cell, (c) determining the infectivity of the chlamydiae by cytokine secretion of the cell, and (d) selecting a compound which reduce the infectivity of the chlamydiae.

Preferred cells capable of secreting cytokines are monocytes, e.g. the THP-1 cell line, the monocytic cell line Mono Mac 6, freshly isolated peripheral blood mononuclear cells (PBMCs), endothelial cells (e.g. primary cultured HUVECs), alveolar macrophages, or/and mouse macrophages. The most preferred cell is THP-1.

Preferred cytokines are IL-8, IL-1β, TNF-α, IL-6, IFN-α, or/and MCP-1. The most prefered cytokine is IL-8.

A further aspect is a screening method for identification of a compound suitable for treatment, prevention or/and diagnosis of chlamydial infections, comprising the steps (i) incubating a eukaryotic cell, e.g. a cell of the immune system, in the presence of a chlamydial polymorphic membrane protein or/and an immunogenic fragment thereof with a compound, (ii) measuring the interaction of the polymorphic membrane protein or/and the immunogenic fragment thereof with the cell, and (iii) selecting a compound which is able to suppress or reduce the interaction of the polymorphic membrane protein or/and the immunogenic fragment thereof with the cell.

It is preferred that in the screening method of the present invention, the polymorphic membrane protein is PmpD or/and the immunogenic fragment thereof is a polypeptide comprising N-pmpD, a polypeptide comprising M- pmpD, a polypeptide comprising the sequence of amino acids 16 to 670 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 670 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1114 in SEQ.ID.NO:2, or/and a polypeptide comprising a sequence starting at amino acid 31 in SEQ.ID.NO:2, amino acid 16 in SEQ.ID.NO:2, or amino acid 122 in SEQ.ID.NO:2, and terminating at amino acid 660 in SEQ.ID.NO:2, at amino acid 655 in SEQ.ID.NO:2, or at amino acid 647 in SEQ.ID.NO:2.

It is also preferred that the interaction in step (ii) of the screening method is measured by the metabolic activity of the cell, e.g. by measuring the mitochondrial activity.

It is further preferred that in step (iii) of the screening method compounds are selected which suppress metabolism-enhancing effect of PmpD, N- pmpD, or/and M-pmpD by competition with the cell for binding with PmpD, N- pmpD, or/and M-pmpD.

Yet another subject of the present invention is an inhibitor of polymorphic membrane proteins or immunogenic fragments thereof, preferably of PmpD, N-pmpD, or/and M-pmpD, for treatment, prevention or/and diagnosis of chlamydial infections. The inhibitor may be identified by the screening method as described above, or may be an antibody of the present invention. The invention is further illustrated by the following figures and examples:

FIGURE LEGENDS

Fig. 1. PmpD is post-translationaliy modified - analysis using 2D-gels.

C. pneumoniae RB were harvested at 2 and 4 days p. i., purified over an Urografin density-step gradient and separated using two-dimensional gel electrophoresis. Positions of the spots identified as N-terminal (amino acid 122-655) and middle (amino acid 670-1114) parts of PmpD by MALDI-PMF are indicated in a small part of the acidic region of a silver-stained gel (panel A).

Panel B represents the amino acid sequence of PmpD (SEQ.ID.NO:1) Identified peptides matching the N-terminus and the middle part are shown underlined and in bold/italic, respectively. Positions of the signal sequence (small caps) and the C-terminal part (box) as shown earlier (Vandahl et al., 2002) are highlighted.

Panel C illustrates schematic representation of the whole PmpD molecule with its possible cleavage sites and respective molecular weights.

Panel D represents the nucleotide sequence encoding PmpD (SEQ.ID.NO:2).

Fig. 2. PmpD is processed in the course of an in vitro infection.

HEp-2 cells were infected with C. pneumoniae (MOI = 1) and incubated for 1 , 2, 3 and 4 days under standard conditions. Total cell lysates were separated on a 6% SDS-gel and immunoblotted. Using the rabbit polyclonal serum against N-pmpD, three bands of about 170, 130 and 70 kDa were detected in the infected cells (Cpn +) (panel A). All bands were present as of 2 days p.i. with the 70 kDa isoform being the predominant one during whole infection and 120 kDa giving the most intense staining on day 2 p.i. The blot was stripped and re-probed with a mouse antiserum against chlamydial Hsp70 for normalization (panel B). Fig. 3. PmpD is surface exposed - staining with and without Triton X- 100 permeabilization.

After 2 days of infection, C. pneumon/ae-infected HEp-2 cells were either fixed using STF and permeabilized with glass beads (425-600 μm) leaving the bacteria intact (panel A and B) or fixed with 2% PFA and incubated in 0.5% Triton X-100 for total permeabilization (panel C and D). The samples were then blocked in 0.2% BSA and stained with the anti-N-pmpD serum (red, panel A and C) or an anti-E. coli Fur protein serum, which recognizes an intracellular factor also in C. pneumoniae (red, panel B and D). Only N- pmpD could be detected without Triton X-100 permeabilization (panel A) suggestive of its surface localization. After permeabilization, both proteins could be detected (panel C and D).

Fig. 4. PmpD is surface exposed - limited trypsin digestion of EB. Twenty μl of highly concentrated EB (1x10⁷ IFU) from C. pneumoniae harvested 4 days p. i. were incubated with the indicated concentrations of trypsin in PBS at 37°C for 30min. EB were collected by centrifugation, washed and resuspended in Laemmli SDS-sample buffer. After separation, the samples were immunoblotted with rabbit serum against N-pmpD (panel A) and next re-probed with mouse serum against chlamydial Hsp70 (panel B). Starting at 10 μg/ml, trypsin removed N-pmpD but not Hsp70 from the surface of EB confirming its surface localization.

Fig. 5. N-pmpD interacts with the components of OM on EB. Twenty μl EB stock (1x10⁷ IFU) from C. pneumoniae harvested 4 days p. i. were incubated in a volume of 1 ml at 37°C for 60min (or at 60°C in PBS for 20min as indicated) in different buffers [200 mM KH₂C0₃ (pH 9.5), 100 mM glycine (pH 3.0), 60 mM EDTA + 3 M NaCl, PBS and 100 mM NaOH (pH 12.5)], centrifuged and processed by immunoblotting with anti-N-pmpD (Panel A). Only basic pH disrupted the connection of N-pmpD with the surface of bacteria.

Chlamydial membrane fractions insoluble in 2% Sarkosyl or in 2% SDS were incubated in presence or absence of the reducing agents (10 mM DTT + 10% 2-ME). The remaining insoluble fractions were then processed by immunoblotting with anti-N-pmpD (Panel B). Addition of reducing agents disrupted the association of N-pmpD with Sarkosyl and SDS-insoluble membrane fractions.

Fig. 6. PmpD associates with bacteria - confocal microscopy study.

HEp-2 cells were infected with C. pneumoniae and incubated for 1 , 2 and 3 days (panel A/B, C/D and E/F, respectively). Next, the cells were washed, fixed with STF, permeabilized with 0.5% Triton X-100 and stained using the polyclonal rabbit serum raised against N-pmpD (red) and the monoclonal mouse antibody against Hsp60 (green). N-pmpD could not be found in cellular compartments outside the chlamydial inclusion (fluorescence + phase contrast in panel A, C, D). Staining pattern indicates co-localization of N-pmpD with individual bacteria in a clustered structure surrounding larger forms stained by Hsp60 and reminiscent of an honeycomb (panel B, D, F).

Fig. 7. PmpD localizes to the surface of bacteria - EM immunogold labeling. HEp-2 cells were infected with C. pneumoniae and incubated for 3 days.

Specimens were fixed (4% paraformaldehyd/0.1% glutaraldehyde), infiltrated with 1.6 M saccharose/25% polyvinylpyrollidone, mounted on aluminum stubs and frozen. Ultrathin cryόsections were incubated with the rabbit antiserum against C. pneumoniae N-pmpD followed by a goat anti-rabbit antibody coupled to 12 nm gold colloids. Gold particles indicating presence of N-pmpD localized to RB, EB and intermediate forms. Panel A shows the whole infected cell, panel B is a magnification of an area inside the inclusion and panel C is a further magnification of panel B (positions indicated by white boxes). Panel D shows part of an inclusion stained with pre- immunization serum as a negative control.

The predominant staining was on the surface of bacteria (arrowheads) or associated with vesicle-like structures of unknown nature in the inclusion lumen (arrow) (panel C). Fig. 8. Neutralization assay with antibodies against N-pmpD.

Different volumes (as indicated by percentage; V/V) of antigen-purified rabbit-antibodies or total rabbit IgG fraction (5 mg ml"¹ each) were incubated with 5x10⁵ IFU for 1h at 37°C in PBS without addition of complement. Aliquots were taken to infect HEp-2 cells seeded on coverslips in 24-well plates and inclusions stained and counted at 2 days p.i. Mouse monoclonal α-MOMP or genus-specific α-LPS antibodies were used as controls and the results normalized for infection with EB incubated with PBS only (=100% infectivity). Incubation of EB with 0.5 mg ml^"1 of the α-N-pmpD antiserum ("10%" V/V) reduced the infectivity by about 50%, whereas incubation of EB with 2.5 mg ml^"1 ("50%") or 4.5 mg ml^"1 ("90%") of the anti-N-pmpD antiserum reduced the infectivity by about 80% and 95%, respectively. Incubation of EB with the respective amounts of control antibodies has not decreased infectivity but induced greater uptake and development of bacteria, instead.

Fig. 9. N-pmpD activates human monocytes as indicated by upregulated mitochondrial activity and IL-8 release assays.

THP-1 cells (4x10⁴) were synchronized for 40h in medium containing 0.2% FBS, resuspended in 10% FBS and incubated in 200 μl with: medium only (0 μg), medium that contained 2.5 and 25 μg mr¹of the recombinant N-pmpD,

2.5 and 25 μg ml^"1 of the recombinant N-pmpD pre-treated with 100 μg ml^"1 polymyxin B for 30min at 37°C and 100 nM PMA. Cells were centrifuged at

920 x g for 1 h and incubated for 24h at 35°C. Metabolic activity was determined using the colorimetric assay with WST-1

( _KSO after 120min incubation at 37°C) (panel A). Supematants from the cell culture were subjected to IL-8 sandwich ELISA (panel B). We observed increased metabolic activity in THP-1 cells incubated with 0.5 μg and 5 μg of the recombinant N-pmpD which had a more pronounced effect than PMA as an activating agent (positive control). The N-pmpD-induced activity correlated with the IL-8 release in a dose-dependent manner. EXAMPLE

MATERIALS AND METHODS

Media

Growth medium for eukaryotic cells was RPMI 1640 (Gibco BRL) supplemented with 300mg ml^"1 L-glutamine, 10% FBS (heat-inactivated, Biochrome, Berlin, Germany), 25 mM HEPES, and 10 μg ml^"1 gentamicin. Unless otherwise specified, during the infection FBS concentration was reduced to 5% and 1 μg ml^-1 cycloheximide was added.

Cell line, bacterial strains and propagation.

The human epithelioid cell line HEp-2 (ATCC-CCL23) derived from a larynx carcinoma was used as host cells. C. pneumoniae strain CWL029 (ATCC strain VR1013, a kind gift from Dr. Gunna Christiansen) was propagated in HEp-2 cell line by centrifugation at 920 x g for 1h at 35°C (Al Younes, Rudel, and Meyer, 1999). Presence of Mycoplasma spp. was excluded using PCR, mycoplasma detection system (Roche Diagnostics GmbH, Mannheim, Germany) and DAPI staining. The infection was done using one infectious EB/host cell (MOI=1).

Purification of C. pneumoniae

EB and RB were purified from HEp-2 cells grown in six-well plates. The infected HEp-2 cells were harvested at 2 or 4 days post infection (p.i.) and disrupted using glass beads (3mm, Roth, Germany). HEp-2 cell debris was removed by centrifugation (10min at 500 * g). The supernatant was then centrifuged at 4°C for 40min at 48 000 x g and the pellet was resuspended in 5 ml PBS-buffer containing 0.25 M sucrose (SPG). EB and RB were separated and purified from host organelles by ultracentrifugation at 50 000 * g for 1h through a discontinuous gradient consisting of 30, 35, 40 and 45% Urografin (Schering, Germany). Upon centrifugation, the two layers (an EB layer at the 40-45% and an RB layer at the 35-40% interface) were transferred to separate vials, washed in SPG and pelleted.

Purity of the preparations was confirmed by electron microscopy (data not shown).

Two-dimensional gel electrophoresis

EB- or RB-pellets were solubilized in five volumes of a buffer containing 9 M urea, 25 mM Tris/HCI, pH 7.1 , 50 mM KCI, 3 mM EDTA, 70 mM DTT, 100 nM pepstatin, 1 mM PMSF, 2% CHAPS and 2% carrier ampholytes (Servalyte pH 2-4; Serva, Heidelberg, Germany). After 30-60min of stirring and vortexing at room temperature (RT), the samples were ultracentrifuged at 100 000 x g (Optima TLX; Beckman, Palo Alto, CA, USA) for 30min at RT. The clear supernatant was frozen at -70°C. The proteins were separated by a large gel 2-D technique (gel size 30 cm x 23 cm) (Klose and Kobalz, 1995). The IEF rod gels (diameter of 1.5 or 2.5 mm for preparative gels, 0.9 mm for analytical gels) contained 3.5% acrylamide, 0.3% piperazine diacrylamide (Bio-Rad, Richmond, CA, USA) and a mixture of 4% w/v carrier ampholytes (Klose and Kobalz, 1995). For preparative gels and analytical gels 250 μg, and 60 μg of protein, respectively, were applied to the anodic side of the gel and focused at 8870 Vh. After focusing, the gels were equilibrated for 10min in a buffer containing 125 mM Tris/phosphate, pH 6.8, 40% glycerol, 70 mM DTT , and 3% SDS. The equilibrated gels were frozen at -70°C (or directly run in the second dimension). After thawing, the IEF gels were immediately applied to SDS-PAGE gels, which contained 15% w/v acrylamide and 0.2% bisacrylamide. The SDS-PAGE system of Laemmli was used, replacing the stacking gel by the equilibrated IEF gel. Electrophoresis was performed using a two-step increase of current, starting with 15min at 120 mA and 65 mA for preparative gels and analytical gels, respectively, followed by a run of about 6h at 150 mA and 85 mA for preparative gels and analytical gels, respectively, until the front reached the end of the gel.

Analytical gels were stained with silver nitrate and dried for 2h at 75°C between cellophane membranes using a gel dryer (Model 585; Bio-Rad) (Jungblut and Seifert, 1990). Preparative gels were stained with Coomassie Brilliant Blue G-250 (Serva), equilibrated in water and stored sealed in plastic bags (Doherty et al., 1998).

Tryptic digestion The in-gel direct measurement procedure was used (Lamer and Jungblut, 2001). The Coomassie Brilliant Blue G-250 stained single gel spots were excised with a scalpel and equilibrated by addition of 75 μl 200 mM ammonium bicarbonate (pH 7.8) for 30min at 30°C while shaking. By addition of 105 μl acetonitrile gel pieces were shrunk for another 30min Subsequently the solution was exchanged with 75 μl 50 mM ammonium bicarbonate (pH 7.8), for re-swelling of the gel piece for 30min at 30°C while shaking. After shrinking, equilibrating and removing the buffer the gel spots were dried in a SpeedVac Concentrator (Eppendorf, Hamburg, Germany). Trypsin (0.1 μg; Promega, Madison, Wl, USA) dissolved in I μl 50 mM acetic acid and 19 μl 50 mM ammonium bicarbonate (pH 7.8) was added and incubated at 37°C overnight. The supernatant was collected and the gel pieces were washed with 25 μl 0.25% aqueous TFA/acetonitrile (mixed 2/1 , respectively) and again the supernatant was collected. The combined supematants were evaporated in the SpeedVac Concentrator and dissolved in 2 μl 0.5% aqueous TFA/acetonitrile (mixed 2/1, respectively) for mass spectrometric analysis.

Peptide mass fingerprinting by MALDI-MS and identification of the proteins The mass spectra were recorded by using a time-of-flight MALDI mass spectrometer (Voyager-Elite; PerSeptive Biosystems, Framingham, MA, USA). The samples were mixed in an Eppendorf tube with equal volume (0.4 μl each) of the matrix solution: 20 mg ml^"1 of α-cyano-4- hydroxycinnamic acid (CHCA) in 0.3% aqueous TFA/acetonitrile (mixed 1/1, respectively) or 50 mg ml^-1 of 2,5-dihydroxybenzoic acid (DHB) in 0.3% aqueous TFA/acetonitrile (mixed 2/1, respectively) were used as matrices. The mixtures were applied to a gold-plated sample holder and introduced into the mass spectrometer after drying. The spectra were obtained in the reflectron mode by summing 100-200 laser shots with an acceleration voltage of 20 kV, 70% grid voltage, 0.05 guide wire voltage, 100ns delay, and low mass gate at 500 m/z.

The proteins were identified by using the peptide mass fingerprinting analysis software MS-Fit (http://prospector.ucsf.edU/ucsfhtmL4.0/msfit.htm), ProFound (http://129.85.19.192/prowl-cgi/ProFound.exe?FORM=1) or Mascot (http://www.matrixscience.com/cgi/index.pl?page=../home.html). The NCBInr database was used for the searches, considering maximum one missed cleavage site, pyro-glutamate formation at N-terminal residues, oxidation of methionine, acetylation of N-terminus, and modification of cysteines by acrylamide.

Chemicals, fluorescent probes and antibodies

Genus-specific rabbit polyclonal antibodies were from Milan Analytica AG

(La Roche, Switzerland) and used in a dilution of 1 :60. Anti-C. pneumoniae HspδO-antibodies from Alexis Biochemicals (San Diego, USA) were diluted 1 :5 000 for immunoblot analysis and 1:500 for immunofluorescence experiments, respectively.

The polyclonal rabbit antiserum against N-pmpD (amino acid 16-670) was produced by BioGenes (Berlin, Germany) and diluted 1 :300 for immunoblot analysis and 1 :100 for confocal microscopy. Antisera were raised against the recombinant, denatured protein (amino acid 16-670 overexpressed in E. coli BL21) purified from preparative SDS-PAGE gels; antibodies for neutralization experiments were either affinity-purified in a batch procedure using the recombinant native N-terminal part of PmpD coupled to CNBr- activated sepharose 4B (Sigma, Germany) or total IgG was isolated by protein A-affinity chromatography following the manufacture^'s instructions. Hsp70 in immunoblot analysis was detected with a polyclonal mouse serum raised against recombinant (His)₆-tagged protein (BioGenes) and diluted 1 :5000.

Secondary antibodies coupled with fluorochromes were from Molecular

Probes (Leiden, the Netherlands) and diluted 1:100-1:200.

The secondary antibody used in western blots in the peroxidase linked species-specific whole antibodies from Amersham Biosciences (UK) (dilutions of 1: 4000 was used).

Cloning and purification of C. pneumoniae N-pmpD The N-terminal part of PmpD without the signal sequence (amino acid 16-

670) was cloned in the IPTG-inducible vector pET43a using the primers

5'-TTG ATG CAT TCC GTA ATA GTA GCA ATA TTG TCA G (SEQ.ID.NO:3,

5' end) and 5'- CTG CTA AGT TTT AGG AGG ATA ATG ATC TCC ATG

(SEQ.ID.NO:4, 3'end) using standard procedures. The (His)e-tagged native N-terminus was overexpressed in E. coli BL21 and purified using Ni-NTA Agarose (Qiagen, Germany) following the manufacture^'s instructions.

Cell staining and immunoblot analyses: For confocal microscopy, cells were seeded and infected on glass coverslips in 24-well plates (Al Younes, Rudel, and Meyer, 1999). At indicated days post infection, the cells were washed with PBS, fixed for 30min with 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany), permeabilized if indicated using 0.5% Triton X-100 (Merck) for 5min and blocked for 20min with 2% BSA in PBS. For differential staining of surface-localized N-pmpD infected cells at 2 days p.i. were fixed with STF (Streck Laboratories, Inc., Omaha, U.S.A) for 20min. and permeabilized with glass beads (425-600 μm, Sigma, Germany).

The respective antibodies were diluted in 2% BSA, incubated for 1h at RT and washed 3 x with PBS. Incubation with the secondary antibodies diluted in PBS was also for 1h at RT. After washing, the glass slides were dried and mounted in Mowiol mounting media on glass microscopic slides. The labeled preparations were analyzed using a Leica TCS NT laser scanning confocal microscope equipped with krypton-argon mixed gas laser. Images were obtained and processed using Adobe Photoshop 6.0. Immunoblotting was done according to standard procedures with ECL detection system (Amersham and Perkin Elmer Life Sciences, Inc.). EM and immunogold labeling

Infected HEp-2 cells (C. pneumoniae or C. trachomatis as indicated) were fixed with 4% paraformaldehyde/0.1% glutaraledhyde, infiltrated with 1.6 M saccharose/25% polyvinylpyrollidone, mounted on aluminum stubs and frozen. Ultrathin cryosections were incubated with the rabbit antiserum against C. pneumoniae N-pmpD followed by a goat anti-rabbit antibodies coupled to 12 nm gold colloids (Jackson). For the evaluation of staining, a Leo 906 transmission electron microscope was used.

Preparation of detergent-insoluble complexes and outer membrane Chlamydial Outer Membrane Complexes (COMC) and OM fractions were isolated based on their insolubility in 2% SDS under non-reducing conditions and 2% Sarkosyl, respectively (Caldwell, Kromhout, and Schachter, 1981). EB from C. pneumoniae strain VR1310 (1x10⁷ IFU) harvested 4 days post infection were incubated in 1 ml of either 2% Sarkosyl (Sigma, Germany), 2% SDS, 2% Sarkosyl + 10mM DTT + 10% β-mercaptoethanol (2-ME) and 2% SDS + 10mM DTT + 10% 2-ME. OM and COMC fractions were pelleted at 250 000 x g for 30min and resuspended in Laemmli sample buffer for SDS-PAGE.

Interaction studies of PmpD with the EB-OM

Binding of the N-pmpD to EB was evaluated by incubating EB (1x10⁷ IFU) at different conditions; either for 20min at 60°C in PBS or for 60min at 37°C in PBS, 200 mM KH₂C0₃ (pH 9.5), 100 mM glycine (pH 3.0), 60 mM EDTA + 2 M NaCl, 2% Sarkosyl (Sigma), 2% Zwittergent (Sigma), 2% SDS (Biomol), Tween 20 and -80 (Merck), 2% Triton X-100 (Calbiochem) and 2% saponin (Sigma) in PBS. Intact EB and insoluble complexes were collected by centrifugation at 20 000 or 250 000 x g, respectively, and resuspended in Laemmli sample buffer for SDS-PAGE and immunoblotting.

Limited trypsin and proteinase K digestion

To determine the surface exposure of N-pmpD EB (1x10⁷ IFU) were incubated with 0, 0.5, 2, 10, 50 and 200 μg ml^"1 trypsin (Difco Laboratories, USA) at 37°C for 30min. Intact EB were collected by centrifugation, washed and resuspended in Laemmli sample buffer for SDS-PAGE. EB were also incubated with proteinase K (Merck, Germany) in the concentration range of 0.01 - 1 μg ml^"1 at RT for 10min and processed the same way for immunoblotting.

Neutralization studies

Different volumes (as indicated by percentage; V/V) of antigen-purified rabbit antibodies or total rabbit IgG fraction (5 mg ml^"1 each) were incubated with EB (5x10⁵ IFU) in a volume of 50 or 100 μl for 1h at 37°C in PBS without addition of complement. Aliquots were taken to infect HEp-2 cells seeded on coverslips in 24-well plates overnight. HEp-2 cells were centrifuged (920 x g for 1h at 35°C) and incubated for 2 days in infection medium containing 1 μg ml^"1 cycloheximide. Inclusions were visualized and counted at 2 days p.i. Mouse monoclonal α-MOMP (Dako Ltd., Ely, UK) or genus-specific α-LPS antibodies (Progen GmbH, Heidelberg, Germany) were used as controls and the results normalized for infection with EB incubated with PBS only (=100% infectivity). Three different dilutions were used for each condition giving approximately 100, 50 and 25 inclusions per microscopic field (magnification: x 40). Ten or more fields were counted.

WST-1 activity assay

The monocytic cell line THP-1 was placed in RPMI medium containing 0.2% FBS for 40h before the experiment. For the proliferation assay the cells were washed and resuspended in medium containing 10% FBS. In 96-well plates 200 μl containing 4x10⁴ cells each were with incubated with different amounts of the recombinant N-pmpD (untreated or incubated with 100 μg ml"¹ polymyxin B (Sigma-Aldrich Chemie, Germany) for 30min at 37°C) or 100 nM PMA as indicated, centrifuged at 920 x g for 1h and incubated for further 24h at 35°C. Metabolic activity was determined using a colorimetric assay with the cell proliferation reagent WST-1 (Roche, Germany) following the manufacture^'s instructions. Shortly, after aspirating the supernatant for IL-8 measurement 10 μl of the reagent and 40 μl medium was added to the remaining THP-1 cells (in 40 μl) in each well, incubated for 120min and the absorbance at 450 nm determined with an ELISA-reader (Molecular Devices, CA, USA). Probability levels of p <0.05 calculated with the Student^'s t test were considered to be significant.

IL-8 ELISA lnterleukin-8 (IL-8) was measured in the supematants after 24h with a sandwich enzyme-linked immunosorbent assay (ELISA, Biosource) according to the manufacture^'s instructions. In this assay a monoclonal mouse goat anti-IL-8 antibody was bound to the wells of 96-well microtiter plates (Nunc, Maxisorp) at 4°C for 20h. Nonspecific binding sites were blocked with 0.5% BSA. Supematants and recombinant human IL-8 standard (BioSource, Inc.) were added and incubated for 2h, followed by incubation for 90min with the biotinylated monoclonal mouse anti-IL-8 detection antibody. After incubation with peroxidase-conjugated streptavidin for 30min, plates were developed using freshly prepared tetramethyl benzidine for 20- 40min and read at 450 nm. Results were obtained by interpolation from the standard curve.

RESULTS

Posttranslational processing of C. pneumoniae PmpD First, we wanted to correlate a pattern of protein species with a certain stage in the developmental cycle of C. pneumoniae. To achieve this we performed bacterial proteome analyses at different times during chlamydial infection in HEp-2 cells. Total lysates of urografin-purified EB and RB were separated using 2D-gel technology. Next, large 2D-gels were stained with Coomassie G-250, individual spots were excised and proteins were identified by MALDI- Peptide Mass Fingerprinting (PMF).

Two prominent features present in the lysate of RB at 4d p.i. in the molecular weight range of <70 kDa contained peptides from the N-terminal part of PmpD (Pmp21), N-pmpD, and peptides from a series of 2 x 3 features in the size of about 55 kDa matched to theoretical trypsin cleavage-products from the middle part of PmpD, M-pmpD, with peptides from amino acid 122-655 and 670-1114, respectively (Fig.1, A and B). The predicted pi for the N- terminal fragment is 4.6-4.7 and for the middle part 4.4. The occurrence of 2 and 2 x 3 spots indicates post-translational modification of the two fragments (Fig. 1A). Schematic representation of the possible cleavage sites and deduced molecular weight for PmpD protein fragments is shown in Fig. 1C.

In order to exclude artificial cleavages by the density-step ultracentrifugation procedure or sample preparation for 2D-analysis, we determined the molecular size of PmpD in total lysates of infected HEp-2 cells. Samples were placed directly into Laemmli SDS lysis-buffer and processed by immunoblotting with the rabbit polyclonal antiserum raised against the denatured, purified, (His)e-tagged recombinant N-terminal part of PmpD (without the signal sequence, amino acid 17-670). In the infected HEp-2 cells ("+") collected sequentially over 4 days of infection, three protein bands reacted with the antiserum (Fig. 2A): a band of 170 kDa corresponding to the size of full-length PmpD, one of about 120-130 kDa correlating in size with the N-terminal plus the middle part connected together and cleaved from the C-terminal part and a band of 70 kDa in the size of the N-terminus only. Fig. 2B shows the same blot stripped and incubated with a mouse antiserum against C. pneumoniae Hsp70 as a loading control. Lower amount of Hsp70 on day 1 p.i. could be explained by less bacteria present at this time of infection. Interestingly, regardless of the time p.i., the strongest band was that of 70 kDa (Fig. 2A). The 130 kDa band was most abundant at 2 days p.i., as compared to the later phases of the infection cycle. Characteristic for the majority of polyclonal antibodies, non-specific bands suggestive of cross- reaction with proteins in uninfected HEp-2 cells ("-") could be observed, but clearly distinguished from the pattern in infected cells. This was also true for the region of 60-70 kDa where a cross-reactive protein runs beneath the 70 kDa fragment of PmpD. Isolation of total lgG-fraction from the rabbit serum or affinity purification of antibodies with the native recombinant N-terminal part of PmpD coupled to CNBr-activated sepharose 4B had not improved the reaction pattern (data not shown).

In addition, we determined the expression of PmpD during persistent infection induced by the iron chelator DAM (50 μg ml^"1). However, there were no quantitative differences between acute and persistent infection at 3 days p.i. after normalization with Hsp70 as an internal control (data not shown).

Localization and surface accessibility ofN-omoD Based on structural composition and homology, two groups predicted PmpD to be a member of the autotransporter system (also called type V secretion system) using in silico analysis (Yen et al., 2002; Henderson, Navarro- Garcia, and Nataro, 1998). We therefore tested whether N-pmpD is indeed located to the outside of bacteria, suggestive of transport across both bacterial membranes. C. pneumoniae was grown for 2 days and infected HEp-2 cells were fixed and incubated with glass beads (diameter 425-600 μm) in order to permeabilize eukaryotic membranes while leaving the bacterial OM intact. PmpD was accessible to the antibodies without permeabilization using Triton X-100 (in red, Fig. 3A), proving that it must be exposed to the surface of bacteria. As a control, we used a polyclonal serum raised against the intracellular Fur transcription factor (ferric uptake regulator) from E. coli that also recognizes an intracellular factor in C. pneumoniae (our unpublished data) (kindly provided by Michael Vasil, University of Colorado Health Sciences Center, Denver, Colorado). Unlike serum reacting with PmpD, anti-Fur serum gave strong positive staining only after permeabilization with Triton X-100, confirming the reliability of our experimental system thus unequivocal localization of PmpD to the external surface of chlamydial cells. (Fig. 3, B and D).

To verify the immunofluorescence data, we performed limited trypsin digestion. In this experiment, purified EB were incubated with increasing concentrations of trypsin and next washed and analyzed by immunoblotting. We observed degradation of N-pmpD starting at 10 μg ml^"1 of trypsin, indicative of its external localization on EB (Fig. 4A). At the same time, levels of Hsp70 remained intact (Fig. 4B). Additionally, a gradual degradation of N- pmpD observed using proteinase K confirmed the results of limited trypsin digestion (data not shown).

Despite cleavage from the full-length molecule, the N-terminal part of PmpD remained attached to the chlamydial outer membrane. The topology predictions have not allocated N-pmpD as an integral part of the membrane, thus we hypothesized that it must interact with other OM structures. For Antigen49 from E. coli, a non-covalent binding with its previously connected

C-terminal β-barrel was described. This binding could be broken by mild heat treatment (incubation at 60°C for 10min) (Caffrey and Owen, 1989). In our experiments, N-pmpD could not be released from the bacterial surface by heat treatment, neither by incubation in phosphate buffer with high ionic strength (3 M NaCl) or using the reducing medium alone (10 mM DTT and β- mercaptoethanol, data not shown). Fig. 5 shows the amount of N-pmpD attached to EB (panel A) or membrane pellets (panel B) after incubation with different conditions. The interaction with EB seemed not to be mediated by divalent cations (no effect with 60 mM EDTA) and could not be disturbed by a pH shift to pH 3 (incubation in 100 mM glycine) or pH 9.5 (200 mM KH₂C0₃) but only at a pH of more than 12 (in 100 mM NaOH) (Fig. 5A).

To examine whether the binding partner is an integral outer membrane protein, we isolated the OM-fraction (insoluble in 2% Sarkosyl) as well as the Chlamydial Outer Membrane Complexes (COMC) (insoluble in 2% SDS). Without reducing agents, a small portion of N-pmpD was found in the pellet (<5%) in both conditions, but adding β-mercaptoethanol and DTT resolved N-pmpD completely, suggestive of association of N-pmpD with OM and COMC (Fig. 5B).

Many autotransporters are cleaved off and released from their C-terminal beta-barrel in the OM, even only under certain growth conditions or during an infection in vivo (Goldberg etal., 1993). We asked whether N-pmpD could be released and secreted to the inclusion lumen or to the cytoplasm of the host cell. Confocal analysis after immunofluorescence staining at 1, 2 and 3 days p.i. indicated distribution of PmpD strictly inside the inclusions of C. pneumoniae (Fig. 6A, C, E). Interestingly, the honeycomb-like pattern was different than the staining pattern of HspδO (large bodies) and PmpD seemed to form a cluster surrounding the bacteria (Fig. 6B, D, F).

To determine the subcellular localization of N-pmpD we also performed immunogold EM studies. The samples were stained on fourth day p.i. The rabbit antiserum reacted with RB, EB and intermediate forms. Interestingly, N-pmpD was localized preferentially on the surface of bacteria and on vesicular structures in the inclusion lumen that seemed to be pinched off from chlamydial cells or membranes (Fig. 7). There was no staining in the cytoplasm of infected cells. Thus, corroborating the confocal analyses, PmpD was not found to be secreted into the host cell or inserted in the inclusion membrane but remained associated with bacteria and their membranes.

Bioactivity ofN-pmpD - infection and activation of human cells

In addition to homology with known autotransporters and despite the long phylogenetic distance between Chlamydiaceae and the proteobacteria, PmpD is homologous to a variety of bacterial adhesins. Members of the unique polymorphic family of Pmps have in common characteristic repeats of four amino acids (GGAI/L/V, 2-12 times and FxxN, 4-23 times). To date, all proteins containing more than one GGAI/L/V repeat were shown to be involved in adhesion [i.e. binding to eukaryotic cell surfaces either as bacterial adhesins (rickettsiae rOmpA) or as eukaryotic docking- and recognition partners (mouse zonadhesin) (Grimwood and Stephens, 1999)]. We therefore assumed that if the physiological function of N-pmpD would be to mediate binding to and/or entry into eukaryotic host cells, antibodies against N-pmpD should neutralize chlamydial infectivity. To investigate this, affinity purified serum or IgG fraction from the polyclonal rabbit serum against N-pmpD were incubated with EB at 37°C 1h prior to infection of HEp- 2 cells. After centrifugation for 1h and incubation for 2 days, the inclusions were visualized by immunostaining. Infectivity was calculated as the mean value of inclusion numbers in 3 different dilutions for each condition. Already O.δ mg ml^"1 (10% v/v) α-N-pmpD serum had a decreasing effect on chlamydial infectivity, 2.δ mg ml^"1 α-N-pmpD had a dramatic effect lowering infectivity down to about 40% whereas 4.5 mg ml^"1 α-N-pmpD abolished the infection almost completely (Fig. 8). No difference was seen in the blocking activity between the two types of antibodies used (affinity purified or IgG fraction). Control antibodies against MOMP or LPS had no decreasing effect on chlamydial infectivity.

To determine if the attachment of PmpD could activate immune host cells we determined the metabolic activity of human monocytes (cell line THP-1) in response to the recombinant protein. The THP-1 cells were arrested in growth by starvation for 40h in 0.2% FBS and next incubated with the recombinant N-pmpD. Twenty four hours later metabolic activity was measured. Recombinant N-pmpD had a strong metabolism-enhancing effect in a concentration-dependent manner with values of almost twice of the background activity exceeding the effect of THP-1 incubation with PMA as a positive control for differentiation and hence stimulation of monocytic cells (Fig. 9A). In addition, the supematants from THP-1 incubated with N-pmpD contained IL-8 as measured by ELISA. The concentration of secreted IL-8 increased in a dose dependent manner in response to the recombinant N- pmpD (Fig. 9B). The response to rN-pmpD was not abolished by the addition of polymyxin B (100 μg ml^"1, incubated for 30min at 37°C), therefore excluding the possibility of artificial activation by contaminating LPS from E.coli.

DISCUSSION

The main finding of the presented study is that PmpD from C. pneumoniae is a cleaved, surface exposed protein mediating the early interaction of EB with the host cell and inducing activation and cytokine release from monocytes. Our data are consistent with observations and predictions of previous investigators and significantly extend our understanding of the processing and the function of this chlamydial protein.

Our observations suggest extensive processing of PmpD (Fig. 1C). In the first step, the full length PmpD is exported to a periplasmic space possibly by the Sec-machinery. Next, the signal sequence is most likely cleaved off in agreement with a recent finding by Vandahl and colleagues (2002) who identified the N-terminal amino acid as ³⁰Ala from their 66 kDa fragment corresponding to our fragment in the size of <70 kDa Then, a β-barrel may be formed by the C-terminal part and the N-terminal passenger domain is most likely exported outside and cleaved from its translocation unit (~45 kDa) referring to the model autotransporter. The last step, in which presence of two fragments of 70 and 55 kDa was observed in our 2D-analysis, would be a cleavage in the N-terminal passenger domain. Immunoblot analysis of the infected HEp-2 cells confirmed these processing events from full length PmpD (170 kDa) via the intermediate pro-protein (N-pmpD + M-pmpD, 125 kDa) finally leading to N-pmpD (70 kDa).

Processing of PmpD is supported by immunoblotting of the infected cell lysate with antiserum gained by immunization with synthetic PmpD peptide, which revealed two weakly reacting bands (40 and 60 kDa) that could correspond to the C-terminal and middle part (Grimwood, Olinger, and Stephens, 2001). Another PmpD fragment of 66 kDa was identified with peptides matching the N-terminal part. In addition, the first amino acid of the C-terminal part was determined by N-terminal sequencing (¹¹⁴⁶Ser, Fig.lB) (Vandahl et al., 2002). The authors suggested a further cleavage consistent with the idea of PmpD to be a member of the autotransporter family but in contrast to the serum we used, their polyclonal antiserum raised against a larger portion of PmpD (recombinant protein with AA 62-1129) could neither detect the middle part or full length PmpD. The reason might be a different epitope recognition by our antiserum that was raised specifically against the recombinant N-pmpD (AA 16-670). However, both antibodies reacted with the surface of C. pneumoniae.

In our 2D-analysis, we could not identify a spot with a PMF corresponding to C-pmpD in the range of 4δ kDa. On the other hand, Vandahl and colleagues (2001) identified only the C-terminal fragment of PmpD (47 kDa, pi δ.δ) in the proteome of C. pneumoniae EB. This could be explained by using different procedures in purification, sample preparation and staining of 2D- gels (silver and Coomassie vs. radioactive labeling by ³⁵S-Met/Cys incorporation). In addition, we used purified RB while the previous proteome map (Vandahl et al, 2001) was obtained by using purified EB. C-pmpD being an integral part of the OM could have been better preserved than the non-covalently bound fragments. Thus, our data are complementary rather than contradictory to previous results and the presence of cleavage products and unprocessed PmpD observed in total cell lysates during the course of infection argues against artificial cleavage.

Assuming autotransporter-like translocation and processing for other members of the Pmp superfamily, earlier results of others appear in new light: for PmpG8, -G11 and -G8 in C. pneumoniae, smaller polypeptides were observed (Grimwood, Olinger, and Stephens, 2001). For other Pmps, positive reacting bands at different sizes could be observed depending on the probe being denatured by boiling or not, which is in agreement with the known temperature-dependent running behavior of proteins containing porelike beta-barrel structures (McCafferty et al., 1996; Knudsen et al., 1999; Christiansen et al., 1999). In C. psittaci, only the N-terminus of Pmps was found to be surface exposed forming a beta helix immunogenic in ovine infection, consistent with the model of a C-terminal translocator unit incorporated in the OM (Everett and Hatch, 1996; Longbottom et al., 1998; Vretou ef a/., 2003).

Our EM and immunofluorescence experiments complemented by limited proteolysis show unequivocally that the N-terminal part of PmpD is present on the surface of chlamydial cells. The ring-like pattern in Fig. 6 sometimes appearing in cubical or spherical configuration indicates presence of N- pmpD on RB. Surface localization is in agreement with the differential fixation method used for Pmp6, -20 and -21 (Vandahl et al, 2002). However, unlike the above authors, we focused majority of our work on the N-terminal, passenger portion of PmpD.

In the limited trypsin digestion, we used Hsp70 as a negative control, though it has been shown by other groups to be surface-exposed in C. trachomatis. However, accessibility of the substrate-binding domain occurred only after incubation with reducing agents disturbing disulfide bridges in the membrane (Raulston et al., 2002). Similarly, it might be protected or less accessible than N-pmpD at least at the trypsin concentrations used. In addition, there may be structural differences between C. trachomatis and C. pneumoniae. Binding of N-pmpD to the membrane was strong and could not be disrupted by heat treatment, high ionic strength, chelation of divalent cations or moderate pH-shifts (Fig. 5A). In isolated OM-fractions (insoluble in 2% Sarkosyl) as well as in Chlamydial Outer Membrane Complexes (COMC) (insoluble in 2% SDS) a proportion of N-pmpD could be recovered in the absence of reducing agents (Fig. 5B). The COMCs are a cluster of cysteine- rich proteins highly cross-linked by disulfide bridges forming a stabilizing lattice below the OM. Partial release of N-pmpD from the OM (solubilized by 2% Sarkosyl) and from COMC (solubilized by 2% SDS) under non-reducing conditions indicates an interaction with one or more proteins involved in the COMC-cluster only for the small percentage of PmpD molecules (N- terminus) that co-isolate with OM and COMCs, while the majority has other interaction partners. Alternatively, the non-covalent binding to the OM is weakened by detergent treatment in general and sensitive only to Sarkosyl and SDS to a similar extend. However, incubation of EB with several classes of detergents had different effects on recovery of N-pmpD in the insoluble fraction with 2% zwittergent or NP-40, bringing more N-pmpD into solution than Triton X-100, Tween, CHAPS or the surfactant saponin, independent from reducing or non-reducing conditions (data not shown).

Although the passenger domain of autotransporters are often secreted, the mature N-pmpD could not be observed on the outside of the chlamydial inclusion membrane nor in the cytoplasm. Therefore, we assume that N- pmpD must act at the surface of EB where it stays strongly attached to the components of the OM. The staining pattern visualized by electron microscopy showed additionally a strong association of N-pmpD with vesicle-like structures (Fig. 7) that could possibly be derived by fission or simply by shedding from the chlamydial OM. This type of chlamydial vesicles was observed before but so far no physiological role was ascribed (Heuer et al., 2003).

To date, no function was assigned to PmpD or to other members of Pmp family. PmpD shares homology with known adhesins [OmpA (Rickettsia spp.), TibA (E. coli ETEC), FN0291 (F. nucleatum), HMWA (Y. pestis)]. Presence of the highly repetitive tetra-aminoacid motifs in Pmps suggests their possible role in adhesion to membranes of different host cell types (Everett and Hatch, 1995). Our experiments demonstrated reduction in the chlamydial infectivity by anti-N-pmpD antibodies for up to 95% in a concentration-dependent manner. In contrast, monoclonal antibodies against C. pneumoniae MOMP and genus-specific antibodies against chlamydial LPS showed no reduction in infectivity but increased uptake of EB instead, possibly because of a Fc-receptor mediated uptake of opsonized chlamydial EB (Su, Spangrude, and Caldwell, 1991 ; Scidmore et al., 1996). This makes a simply sterical effect of the antibodies blocking the interaction of a second chlamydial adhesin with the receptor of the host cell unlikely, but it cannot be fully excluded. Neutralizing capacity of antibodies and antiserum have been demonstrated for other OM-components of Chlamydiaceae with indications for a role in adherence as for PorB (C. trachomatis), Hsp70 (C. trachomatis), cell wall-associated glycosaminoglycan (C. trachomatis and C. pneumoniae), and MOMP (C. trachomatis) (Kawa and Stephens, 2002; Mamelak et al., 2001; Rasmussen-Lathrop et al., 2000; Caldwell and Perry, 1982). However, in contrast to C. trachomatis, MOMP of C. pneumoniae is not immunodominant and neutralizing antibodies were only reported against a conformation-dependent epitope. Antiserum against peptides representing variable domains of MOMP failed to neutralize infection and a monoclonal antibody against C. pneumoniae LPS was effective only for the strain it was raised against (Wolf et al., 2001; Peterson et al., 1996; Peterson et al., 1998). Furthermore, immunoreactive surface exposed structures like a glycolipid exoantigen (GLXA, C. psittaci and C. trachomatis), a 76-kDa and a 54-kDa protein (C. pneumoniae) as well as two unknown antigens (C. pneumoniae) served as targets for protective antibodies (Girjes et al., 1993; An et al., 1997; Gran, Hjetland, and Andreassen, 1993; Perez, Kuo, and Campbell, 1994; Wiedmann-AI-Ahmad, Schuessler, and Freidank, 1997; Puolakkainen et al., 1995). However, we could not differentiate between an inhibition of binding and uptake/invasion and a negative effect of bound antibodies on the course of the chlamydial development after entry into the eukaryotic cell.

Unlike in specimens analyzed 2 days p.i., we were unable to detect full length PmpD in purified EB (Fig. 4A). This suggested that the 170 kDa protein serves as a precursor and the N-terminal passenger part localized on the surface of EB is in fact the functional domain of PmpD. Cleavage after passing the outer membrane could be a trigger to fold into the right conformation. However, a possible contribution of M-pmpD that also contains multiple tetra-aminoacid-motifs and other components of the EB OM mediating adhesion cannot be excluded. Interestingly, recombinant N- pmpD stimulated metabolic activity and IL-8 secretion in THP-1 cells in a concentration dependent manner (Fig. 9). Induction of a proinflammatory cytokine response (IL-1β, TNF-α, IL-6) after infection with C. pneumoniae in vitro has been demonstrated for both, the monocytic cell line Mono Mac 6 and freshly isolated human peripheral blood mononuclear cells (PBMC) and IL-8 production was induced in alveolar macrophages (Heinemann et al., 1996; Kaukoranta-Tolvanen et al., 1996; Redecke et al., 1998). Cytokine release including IL-8 from human PBMC and mouse macrophages could be attributed to acellular components without the need of viable Chlamydiae and Hsp60, respectively (Netea et al., 2000; Bulut et al., 2002). At present, it is unclear whether the observed stimulating property of recombinant N- pmpD could be modulated by additional surface components of EB or by factors secreted into the target host cell during early infection. NF-κB- dependent induction of the inflammatory mediators IL-6, IL-8 and MCP-1 in human endothelial cells with recombinant Pmp20 or Pmp21 was recently demonstrated (Niessner et al., 2003).

Given that PmpD interacts directly with the host cells and mediates early immunostimulatory events during an ongoing infection and inflammation, passive administration of antibodies or stimulation of the humoral response against PmpD could possibly be used as therapeutic strategies. Furthermore, the demonstrated neutralizing effect of antibodies against N- pmpD supports a protective role of the humoral immune system during the early infection and makes PmpD an important target for anti-chlamydial vaccination.

ABBREVIATIONS

CHAPS 3-([3-cholamidopropyl]dimethylammonio)-1 - propanesulfonate COMC chlamydial outer membrane complex

C-pmpD C-terminal part of PmpD

DTT dithiothreitol

EB elementary body / elementary bodies

2-ME beta-mercaptoethanol FBS fetal bovine serum

MCP-1 monocyte chemoattractant protein-1

MOI multiplicity of infection

M-pmpD middle part of PmpD

N-pmpD N-terminal part of PmpD NF-κB nuclear factor-κB

OM outer membrane p.i. post infection

PFA paraformaldehyde

PMA phorbol 12-myristate 13-acetate Pmp polymorphic membrane protein

PMSF phenylmethylsulfonylfluoride

RB reticulate body / reticulate bodies

SDS sodium dodecyl sulfate

STF^® STRECK TISSUE FIXATIVE TNF-α tumor necrosis factor-α

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Claims

Claims

1. Use of (i) a chlamydial polymorphic membrane protein or/and an immunogenic fragment thereof, or/and (ii) antibodies against a chlamydial polymorphic membrane protein or/and against an immunogenic fragment thereof, for the manufacture of a medicament for the treatment or/and prevention of a chlamydial infection.

2. Use according to claim 1, wherein the medicament causes stimulation of a humoral response against the chlamydial polymorphic membrane protein or/and the immunogenic fragment thereof.

3. Use according to claim 1 or 2, wherein the polymorphic membrane protein is selected from the group consisting of PmpG, PmpA/l, PmpH, PmpE, PmpE/F, PmpA, PmpB, PmpD, CPJ0015, CPJ0017 and CPJ0019.

4. Use according to any of the claims 1 to 3, wherein the polymorphic membrane protein is PmpD.

5. Use according to any of the preceding claims, wherein the polymorphic membrane protein or the immunogenic fragment thereof is encoded by a nucleic acid comprising (a) the nucleotide sequence of SEQ.ID.NO:1, (b) a nucleotide sequence corresponding to the sequence of (a) within the scope of the degeneracy of the genetic code, (c) a nucleotide sequence which is at least 70 % homologous to the sequence of (a) or (b), or (d) a fragment of the sequences of (a), (b) or (c).

6. Use according to any of the preceding claims, wherein the polymorphic membrane protein or the immunogenic fragment thereof comprises (a) the amino acid sequence of SEQ.ID.NO:2, (b) an amino acid sequence which is at least 70 % homologous to the 5 sequence of (a), or (c) a fragment of the sequences of (a) or (b).

7. Use according to any of the preceding claims, wherein the immunogenic fragment has a length of a least 6 amino acids, preferably at least 10o amino acids, more preferably at least 50 amino acids, most preferably at least 100 amino acids.

8. Use according to any of the preceding claims wherein the immunogenic fragment of the polymorphic membrane protein is a polypeptides comprising N-pmpD (amino acid 122-655 in SEQ.ID.NO:2), a polypeptide comprising M-pmpD (amino acid 670-1114 in SEQ.ID.NO:2), a polypeptide comprising the sequence of amino acids 16 to 670 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence ofo amino acids 670 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1114 in SEQ.ID.NO:2, or/and a polypeptide comprising a sequence starting at amino acid 31 in SEQ.ID.NO:2, amino acid 16 in SEQ.ID.NO:2, or amino acid 122 in SEQ.ID.NO:2, and terminating at amino acid 660 in SEQ.ID.NO:2, at5 amino acid 655 in SEQ.ID.NO:2, or at amino acid 647 in SEQ.ID.NO:2.

9. Use according to any of the preceding claims, wherein the antibodies are antibodies against PmpD, a polypeptide comprising N-pmpD, a polypeptide comprising M-pmpD, a polypeptide comprising the sequenceo of amino acids 16 to 670 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 670 to 1145 in SEQ.ID.NO:2, a polypeptide comprising the sequence of amino acids 671 to 1114 in SEQ.ID.NO:2, or/and a polypeptide comprising a sequence starting at amino acid 31 in SEQ.ID.NO:2, amino acid 16 in SEQ.ID.NO:2, or amino acid 122 in SEQ.ID.NO:2, and terminating at amino acid 660 in SEQ.ID.NO:2, at amino acid 655 in SEQ.ID.NO:2, or at amino acid 647 in SEQ.ID.NO:2.

10. Use according to any of the claims 1 to 9, wherein the chlamydial infection is an infection with microorganisms from the genus Chlamydia, e.g. Chlamydia trachomatis.0

11. Use according to any of the claims 1 to 9, wherein the chlamydial infection is an infection with microorganisms from the genus Chlamydophila, e.g. Chlamydophila pneumoniae. s

12. Antibodies against a polymorphic membrane protein or an immunogenic fragment thereof as defined in any of the claims 1 and 3 to 8 for use as a vaccine for treatment or/and prevention of a chlamydial infection or/and for diagnosis of a chlamydial infection. o

13. Antibodies of claim 12, which are anti-N-pmpD antibodies or/and anti-M- pmpD antibodies.

14. A pharmaceutical composition comprising as an active agent for the prevention or/and treatment of infections with chlamydiae5 (i) a polymorphic membrane protein or/and an immunogenic fragment thereof as defined in any of the claims 1 and 3 to 8, or/and (ii) an antibody as defined in any of the claims 1 and 3 to 9, optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives.0

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is for use in human medicine.

16. The pharmaceutical composition of claims 14 or 15 comprising at least one further active ingredient for the prevention or/and treatment of chlamydial infections.

5 17. The pharmaceutical composition of claim 16, wherein the at least one further active ingredient is selected from antibiotics or/and amino acids.

18. The pharmaceutical composition of claims 16 or 17, wherein the at least one further active ingredient is selected from macrolides, quinolones ando combinations thereof.

19. The pharmaceutical composition of any of the claim 16 to 18, wherein the at least one further active ingredient is selected from the group consisting of L-leucine, L-isoleucine, L-methionine, and L-phenylalanine.5

20. The pharmaceutical composition of claims 16 to 19 for the treatment of patients with chronic chlamydial infections.

21. A screening method for identification of a compound suitable foro treatment, prevention or/and diagnosis of chlamydial infections, comprising the steps (a) providing a cell capable of secreting cytokines, (b) contacting a compound with chlamydiae or/and the cell, (c) determining the infectivity of the chlamydiae by cytokine secretion of5 the cell, and (d) selecting a compound which reduce the infectivity of the chlamydiae.

22. A screening method for identification of a compound suitable for0 treatment, prevention or/and diagnosis of chlamydial infections, comprising the steps (i) incubating a eukaryotic cell, e.g. a cell of the immune system, in the presence of a chlamydial polymorphic membrane protein or/and an immunogenic fragment thereof with a compound, (ii) measuring the interaction of the polymorphic membrane protein or/and the immunogenic fragment thereof with the cell, and (iii) selecting a compound which is able to suppress or reduce the interaction of the polymorphic membrane protein or/and the immunogenic fragment thereof with the cell.

23. The screening method according to claim 22, wherein the polymorphic membrane protein or/and the immunogenic fragment thereof is PmpD, a polypeptide comprising N-pmpD or/and a polypeptide comprising M- pmpD.

24. The screening method according to claim 22 or 23, wherein the interaction in step (ii) is measured by the metabolic activity of the cell, and wherein in step (iii) compounds are selected which suppress metabolism-enhancing effect of PmpD, N-pmpD, or/and M-pmpD by competition with the cell for binding with PmpD, N-pmpD, or/and M- pmpD.

25. Inhibitor of PmpD, N-pmpD, or/and M-pmpD for treatment, prevention or/and diagnosis of chlamydial infections.

26. A method for treating or/and prevention of a chlamydial infection, the method comprising the administration of (i) a chlamydial polymorphic membrane protein or an immunogenic fragment thereof, or/and (ii) antibodies against a polymorphic membrane protein or against an immunogenic fragment thereof, in a amount effective in therapy or/and prevention to a subject in need thereof.

27. The method of claim 26 for stimulation of a humoral response against the chlamydial polymorphic membrane protein or/and the immunogenic fragment thereof in an amount effective to elicit a humoral response.