Arch Microbiol DOI 10.1007/s00203-013-0912-8

Mini-Review

Chlamydia bacteriophages Joanna S´liwa‑Dominiak · Ewa Suszyn´ska · Małgorzata Pawlikowska · Wiesław Deptuła 

Received: 18 June 2013 / Revised: 8 July 2013 / Accepted: 9 July 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract  Phages are called “good viruses” due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G−) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, ϕCPG1 ϕCPAR39 (ϕCpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better. Keywords  Bacterial viruses · Chlamydia · Infection · Genome

Communicated by Erko Stackebrandt. J. S´liwa‑Dominiak (*) · E. Suszyn´ska · M. Pawlikowska · W. Deptuła  Department of Microbiology, Faculty of Biology, University of Szczecin, 71‑412 Szczecin, Poland e-mail: [email protected]

Introduction Bacteriophages are viruses that infect bacteria. Their morphological structure usually differs from that of other viruses. Most of them, contrary to other viruses, have permanent tails but, similarly to other viruses, most phages are composed of nucleic acid carrying genetic information and a protein coat (Da˛browska et al. 2005). Bacteriophages need to have a sensitive host with a specific receptor in order to replicate in the host cells (Da˛browska et al. 2005). There are 18 families and 1 genus (not assigned to a family) of bacteriophages, of which 16 families, as well as a genus not classified into any family, are bacteriophages-containing genetic material in the form of DNA, whereas 2 families contain RNA (Ackermann and Prangishvili 2012; King et al. 2012). Bacteriophages of free living hosts are the most highly characterised on a molecular level, but little is known about phages that infect intracellular bacterial hosts, which have complex developmental cycles, especially if the host bacterium has an obligate intracellular replication phase (Salim et al. 2008). An example of such phages are bacteriophages belonging to the family Microviridae that infect the genus Chlamydia. Members of the family Microviridae are polyhedral, non-enveloped, and prokaryotic viruses with T  = 1 icosahedral symmetry, about 30 nm in diameter and have a single-stranded DNA (ssDNA) (Ackermann and Prangishvili 2012; King et al. 2012; Krupovic and Forterre 2011; Roux et al. 2012). The capsid consists of 12 pentagonal trumpet-shaped pentomers (King et al. 2012). Bacteriophages belonging to Microviridae are divided into the subfamily Gokushovirinae and the genus Microvirus. Recently, a new tentative subfamily Alpavirinae was found through bacterial genome analysis and confirmed by metagenomic analysis (Kim et al. 2011; Krupovic and Forterre 2011; Roux et al. 2012). Description of Alpavirinae is restricted to prophages residing in the genomes of bacteria

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Arch Microbiol

belonging to two genera of the phylum Bacteroidetes: Prevotella and Bacteroides (Krupovic and Forterre 2011; Roux et al. 2012). The subfamily Gokushovirinae is represented by three genera, namely Chlamydiomicrovirus, Bdellomicrovirus, and Spiromicrovirus (King et al. 2012). Members of the genus Microvirus infect enterobacteria and share a morphology and genome organisation typified by Enterobacteria phage phiX174 (King et al. 2012). Members of the Gokushovirinae infect obligate intercellular parasitic bacteria (Bdellovibrio spp. and Chlamydia spp.) and Mollicutes (Spiroplasma spp.), and share the morphology, typified by Spiroplasma phage 4 (SpV4) (King et al. 2012). Genomes are circular positive sense ssDNA molecules. As with morphological, biochemical, and biophysical properties, genome sizes appear to fall into two size ranges. Microvirus genomes are 5.3–6.1 kb, while gokushovirus genomes are considerably smaller, 4.4–4.9 kb (King et al. 2012). The smaller genomes reflect the absence of genes encoding major spike and external scaffolding proteins (King et al. 2012). Bacteria of Chlamydia are infected by phages of the genus Chlamydiomicrovirus. The first-known representative of the genus Chlamydiamicrovirus was bacteriophage Chp1 (Chlamydiaphage 1). It was discovered in 1982, when phage infection was described in two Chlamydia psittaci isolates (Storey et al. 1989a; Storey et al. 1989b). Crystalline arrays of viruses within distended reticulate bodies (RB) and free viruses within mature chlamydial inclusions were observed using electron microscopy in isolates grown in coculture on a McCoy cell monolayer (Liu et al. 2000; Storey et al. 1989a; Storey et al. 1989b). Since then, four more species of chlamydiaphages have been identified, namely: Chp2 (Chlamydiaphage 2) (Everson et al. 2002; Liu et al. 2000; Salim et al. 2008; Skilton et al. 2007), Chp3 (Chlamydiaphage 3) (Garner et al. 2004), ϕCPG1 (Hsia et al. 2000a; Hsia et al. 2000b; Rank et al. 2009), and ϕCPAR39 (ϕCpn1) (Chlamydia pneumoniae AR39, bacteriophage) (Everson et al. 2003; Hoestgaard-Jensen et al. 2011; Karunakaran et al. 2002; Read et al. 2000a; Rupp et al. 2007) (Table 1). The latest isolated bacteriophage is Chp4 (Chlamydiaphage 4) originating from C. abortus (Sait et al. 2011). The isolated bacteriophages (Table 1) were shown to infect Chlamydia species such as C. psittaci, C. abortus, Table 1  Chlamydiaphages, species of chlamydia from which they were isolated, as well as species of chlamydia that can be infected in laboratory conditions

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C. felis, C. caviae, C. pecorum, and C. pneumoniae. All chlamydiaphages isolated to date are members of the Microviridae and are genetically and structurally related to phages of Spiroplasma melliferum and Bdellovibrio bacteriovorus and more distantly to the coliphage phiX174. The phage host range is specified by a peptidic loop of the major capsid protein, VP1 (Read et al. 2000b), and sequence variation within the loop is consistent with the observed overlapping host ranges of the phages. For instance, ϕCPG1 and ϕCPAR39, which have identical VP1 sequences, are both able to infect Chlamydia caviae and C. pneumoniae. However, Chp2, whose VP1 sequences differs, may also infect C. caviae and other Chlamydia species (Everson et al. 2002), suggesting that distinct receptors to multiple phage exist at the surface of chlamydia. Receptors for these phages have not been identified. The chlamydial host range for the various chlamydiaphages is varied, but all are lytic for their respective hosts (Rank et al. 2009). Chlamydias are small, Gram-negative (G−) microbes showing some structural similarities with viruses (Mårdh et al. 1989; Pawlikowska and Deptuła 2012). Similarly to viruses, they are very small (0.2–1.5 μm in diameter). They form inclusion bodies, have a complicated developmental cycle which involves intracellular multiplication in live cells of the host, and show ATP dependence on the host (Pawlikowska and Deptuła 2012). Chlamydias are, however, classified as bacteria due to the structure of the cellular wall, presence of both nucleic acids, DNA and RNA, synthesis of cellular elements using their own enzymatic apparatus, and presence of cellular organelles and cytoplasm, as well as sensitivity to antibiotics and various compounds (iodine, phenol, acids and alkalis) (Bergey et al. 2011; Pawlikowska and Deptuła 2007). Chlamydia is not to be confused with Porochlamydia that is a member of the family Coxiellaceae (phylum XIV, Proteobacteria, class Gammaproteobacteria, order Legionellales) (Bergey et al. 2011). Chlamydias cause dangerous diseases in mammals, termed chlamydiosis (Deptuła et al. 2002; Pawlikowska and Deptuła 2012). In humans, these bacteria are etiological agents of diseases, such as psittacosis caused by C. psittaci, or respiratory tract diseases caused by C. pneumoniae biotype TWAR, while in animals, the infection may result in enteritis in cattle caused by C. psittaci serotype WC,

Chlamydiaphage

Host

Host range

Chp1

C. psittaci

No data

Chp2

C. abortus, C. psittaci

C. felis, C. pecorum

Chp3

C. pecorum

C. abortus, C. caviae, C. pecorum, C. felis

ϕCPG1

C. caviae, C. psittaci

C. caviae, C. pneumoniae

ϕCPAR39 (ϕCpn1)

C. pneumoniae (C. pneumoniae AR39)

C. abortus, C. caviae, C. pecorum, C. pneumoniae

Chp4

C. abortus

No data

Arch Microbiol

or chronic bowel diseases caused by C. pecorum, as well as miscarriages in sheep caused by C. abortus (Deptuła et al. 2002; Pawlikowska and Deptuła 2012). The susceptibility of chlamydias to antibiotics has been tested in yolk sac of chicken embryos or cell culture (Bergey et al. 2011). The growth of chlamydias in culture is inhibited by the tetracyclines, macrolides, azalides, chloramphenicol, rifampin, and fluoroquinolones (Bergey et al. 2011). Chlamydial multiplication is not blocked by aminoglycosides, bacitracin, or vancomycin (Bergey et al. 2011). Chlamydia suis bacteria are sensitive to sulphonamides, whereas other species are resistant (Bergey et al. 2011). Beta-lactam antibiotics (penicillin), which inhibit the synthesis of peptidoglycan, interrupt the developmental cycle by preventing the maturation of reticulate bodies into elementary bodies. If these antibiotics are removed, development proceeds normally (Bergey et al. 2011). The drugs of choice for treatment of chlamydial infections are tetracyclines, macrolide, and azalides. Longacting antibiotics (doxycycline) and those antibiotics that achieve high intracellular concentrations (azithromycin) are preferred. Chlamydial strains resistant to sulphonamides, penicillin, chlorotetracycline, and rifampin have been produced by in vitro passage in the presence of these drugs. However, stable drug-resistant mutants of C. trachomatis or C. pneumoniae have not been isolated (Bergey et al. 2011). The developmental cycle of chlamydias comprise several phases and is characterised by the presence of two coexisting morphological forms: pathogenic form termed “elementary body” (EB) and “reticulate body” (RB) with metabolic activity (Deptuła et al. 2002; Mårdh et al. 1989; Pawlikowska and Deptuła 2012). The cycle begins when EBs bind to receptors on the target cell of the host. EBs enters the cell through endocytosis or phagocytosis. Within the host cell, a phagosome (early inclusion) is formed, which moves to the area of the nucleus, where EBs transform into a non-infectious form of reticulate bodies. RBs multiply by cross-division inside the inclusion which increases its dimension. Near the middle of the lifecycle, RBs condensate forming EBs. This process can be observed in the form of intermediate bodies within the host cell. When the number of EBs reaches the level of 100–1,000 per cell, they are released from the inclusion to the cytoplasm of the host cell. The infected cell ruptures releasing EBs outside the cell, which can lead to infection of new cells (Pawlikowska and Deptuła 2012).

Characteristics of chlamydiaphages (Table 2) Chp1 (Chlamydiaphage 1) The phage Chp1 has been isolated from cells infected with avian C. psittaci (Liu et al. 2000; Storey et al. 1989a; Storey et al. 1989b). Chp1 can infect other chlamydial strains, but

Table 2  Characteristics of chlamydiaphages Chlamydiaphage

Genome size (kb)

GC content (%)

Number of ORFs

Chp1 Chp2 Chp3 ϕCPG1 ϕCPAR39 (ϕCpn1)

4.88 4.56 4.54 4.53 4.52

36.6 40.9 41.0 41.0 No data

8 8 8 9 6

Chp4

4.53

42.2

8

its host range is restricted to avian C. psittaci (Bevan and Labram 1983; Liu et al. 2000). Molecular analyses of Chp1 showed that it has a single-stranded circular DNA genome of 4,877 bp (Bevan and Labram 1983; Liu et al. 2000; Storey et al. 1989a; Storey et al. 1989b). GC pair content at the level of 36.6 % is extremely low. Chp1 has 5 open reading frames (ORFs) 1–5 with ATG “start” codon, which in each case is preceded by a ribosome-binding site, meaning that each of the reading frames can potentially encode protein (Liu et al. 2000; Storey et al. 1989a; Storey et al. 1989b;). ORF6 and 2, as well as ORF1 and 7, overlap. ORF8 can potentially encode protein comprising 36 amino acids, but because in 50 % it is composed of arginine, it is unlikely to encode a functional protein. Moreover, there are additional sites of transcription initiation, namely ORF2a, 2b, 4a, 5a, which potentially allow Chp1 to encode four additional proteins (Storey et al. 1989a; Storey et al. 1989b). Based on N-terminal ends of amino acid sequence, it was proposed that three structural proteins VP1, VP2, and VP3 are encoded, by ORF1–3. VP1 shows partial similarity to the F capsid main structural protein of bacteriophage phiX174, whereas protein encoded by ORF4 reveals homology with protein A (nicking/closing protein) (Storey et al. 1989b). Chp2 (Chlamydiaphage 2) The phage Chp2 was isolated from C. abortus and C. psittaci (Everson et al. 2002; Liu et al. 2000; Salim et al. 2008; Skilton et al. 2007). Its genome is 4,567 bp in size and shows the presence of 8 open reading frames (ORFs) which potentially encode 8–10 proteins, depending on the usage of second start codons within ORF2 (Liu et al. 2000). It is similar in overall organisation to the Chp1 genome, except for the location of ORF6, which is found in a different relative position within the genome (Liu et al. 2000). Seven of the ORFs (1–5, 7, and 8) have sequence homologies with Chp1. The difference between two such genomes lies in the different spatial organisation and lack of congeneric ORF6 template, as well as lack of ORF4a and 5a. ORF1–3 encode viral structural proteins VP1–VP3. Similarly as in Chp1, proteins of the viral capsid of Chp2 show the greatest

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affinity to the proteins of capsid ϕSpV4, which, however, only features one such protein, while chlamydiaphages feature at least three (VP1–3). Furthermore, similarity was noted between small protein encoded by ORF8 and binding protein J of coliphage phiX174 which is a structural component of its virion (Liu et al. 2000). It was shown that there are no proteins in Chp1, Chp2, and SpV4 that share any homologies to the coliphage external scaffolding protein (protein D), which is the most conserved protein of the coliphages in the Microviridae (Liu et al. 2000). The protein D was suggested to mediate the placement of spike protein pentamers on the coat protein and the organisation of the coat protein at the threefold axes symmetry (Dokland et al. 1997; Dokland et al. 1999; Liu et al. 2000). None of these functions may be required in the chlamydiaphage (Liu et al. 2000). Chp3 (Chlamydiaphage 3) Chp3 was the first bacteriophage isolated from elementary bodies of C. pecorum from sheep (Garner et al. 2004). The host range is limited to C. abortus, C. caviae, and C. pecorum, and can additionally infect C. felis (Everson et al. 2003). Its genome comprises of 4,544 bp and encodes eight ORFs organised similarly as in the other chlamydiaphages. Protein encoded by ORF8 is similar to protein J of phiX174 and is characterised by a high degree of sequence conservation in Chp2, Chp3, CPG1, and CPAR39. It was evidenced that VP1 is the main and the largest protein of the capsid and is characteristic for all described chlamydiaphages. The analysis of its amino acid sequence has revealed the presence of two areas of significant divergence between amino acids 216 and 299 and 462–467 (Read et al. 2000b). It has been hypothesised that the IN5 loop, which comprises the larger part of these two regions, is exposed in the surface of the virion. The other area (462–467 bp), also referred to as Ins, is also exposed on the virion surface and most probably interacts with IN5 (Garner et al. 2004). Chlamydiaphage ϕCPG1 ϕCPG1 is a bacteriophage isolated from C. psittaci (Hsia et al. 2000a; Hsia et al. 2000b; Rank et al. 2009). Its genome comprises of 4,529 bp and shows the presence of five main, non-overlapping open reading frames: ORF1–3 encode viral proteins VP1–3, while ORF4 and 5 encode proteins VG4 and 5 (Hsia et al. 2000b). As compared to other chlamydiaphages, it shows the greatest similarity in genome organisation and homology of sequence with Chp1 that infects avian C. psittaci (Hsia et al. 2000b). VP1 sequence reveals high similarity between Chp1, ϕCPG1, and Spv4, showing an important role of the protein as the main structural component of the capsid (Hsia et al.

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2000b). Furthermore, it is reported that the genome segment comprising VG4 and the above-mentioned sequences can encode signals important for replication initiation and/ or regulation of the phage’s gene expression (Hsia et al. 2000b). ϕCPAR39 (Chlamydia pneumoniae AR 39 strain bacteriophage) The ϕCPAR39 bacteriophage isolated from C. pneumoniae strain AR39 is also referred to as ϕCpn1 (Everson et al. 2003; Hoestgaard-Jensen et al. 2011; Karunakaran et al. 2002; Read et al. 2000a; Rupp et al. 2007). The host range of ϕCPAR39 is limited to four chlamydia species: C. abortus, C. caviae, C. pecorum, C. pneumoniae (Everson et al. 2003). It is the first chlamydiaphage related to chlamydia species that causes diseases in humans. Its genome comprises of 4,524 bp, which encodes six ORFs—ORF8, ORF4, ORF5, and VP1–VP3 of the Microviridae family (Hoestgaard-Jensen et al. 2011). The ϕCPAR39 is unique among chlamydiaphages because it does not lyse the membranes of the phage-infected, abnormally large C. pneumoniae AR39 RB, which was demonstrated by the distinct staining of individual RB (Everson et al. 2003; HoestgaardJensen et al. 2011). Electron micrographs revealed intact RB membranes within which the phage particles formed beaded patterns (Hoestgaard-Jensen et al. 2011). Chp4 (Chlamydiaphage 4) Chp4 has been isolated from C. abortus. It has a circular ssDNA molecule of 4,530 bp (Sait et al. 2011). Chp4 is predicted to contain eight ORFs organised in a manner characteristic of chlamydiaphages. ORFs1–3 usually encode: VP1—the putative major coat protein, VP2—the putative minor spike protein, and VP3—the putative scaffolding protein. ORFs4 and 5 are predicted to encode non-structural proteins homologous to the ϕX174 A and C proteins involved in synthesis of viral DNA and packaging of DNA into the viral capsid (Sait et al. 2011). In turn, ORF8 is homologous to the protein J of the ϕX174, that is, involved in DNA packaging and probably in viral attachment to host’s cell (Sait et al. 2011). No homologues have been predicted for ORFs6 and 7, which overlap ORFs2 and 1, respectively, other than in Chlamydiamicroviridae (Sait et al. 2011). Chlamydiaphage affinity The assessment of chlamydia phage affinity is usually based on the analysis of two genome regions: VP1 and ORF4, which are present in all phages of this genus. Differences in VP1 sequence principally refer to regions:

Arch Microbiol

IN5 and INS. In silico analyses have shown that these regions are located next to each other, which indicates their major role in determination of tissue tropism in various chlamydiaphages. Phylogenetic analysis of the VP1 protein sequence was performed to determine the degree of relatedness between Chp4 and other members of the family Microviridae and the genus Chlamydiamicrovirus (Sait et al. 2011). Chp4 was found to be more closely related to ϕCPAR39 and ϕCPG1 than to Chp2 or Chp3 (Sait et al. 2011). Differences between the chlamydiaphage VP1 sequences were found to occur mainly in the IN5 and INS regions of the VP1 outer coat protein (Sait et al. 2011). It is suggested that regions IN5 and INS participate in receptor recognition. It was observed that attachment of Chp2 or Chp3 to a susceptible host of C. abortus does not prevent ϕCPAR39 binding, which suggests the presence of various receptors specific for the chlamydiaphage on host cell surface (Sait et al. 2011). It was evidenced that a fragment of ORF4 occurs in all chlamydiaphages. Phylogenetic analysis of chlamydiaphages ϕCPG1, ϕCPAR39, Chp2, and Chp1 suggests that the replication-initiation protein encoded by ORF4 region evolved quicker than other proteins. The function of this protein is not proven, but the central part of the sequence is characteristic of many prokaryotic Rep proteins, e.g.: RepA of E. coli or in phage ϕLf (Read et al. 2000b). Chlamydiaphages and chlamydia developmental cycle Many studies on chlamydiaphages point at their impact on the development of chlamydias and their pathogenicity. Studies have shown that the phage DNA does not integrate into the chlamydial chromosome, nor it is related to the cryptic plasmid found in many chlamydial strains (Liu et al. 2000). Salim et al. (2008) noted that the infection with chlamydiaphage Chp2 has a strong impact on the development of C. abortus by inhibiting bacterial cell division of the bacteria. In this case, the transformation of RB to EB is blocked, with a simultaneous enlargement of reticulate bodies without cellular divisions (Salim et al. 2008). It was shown that between hour 36 and 48 post-infection, rapid replication of Chp2 genome occurs, which is coincident with the expression of viral proteins and the replication of the host chromosome (Salim et al. 2008). As a result, the development of this bacteriophage leads to chlamydia cell lysis, accompanied by the appearance of paracrystalline structures (Salim et al. 2008). These data indicate that bacteriophage life cycle is coordinated with the developmental cycle of its bacterial host (Salim et al. 2008). Hsia et al. (2000a) studied the lifecycle of ϕCPG1 using transmission electron microscopy. The study demonstrated that the phage infection takes place upon EBs differentiation into metabolically active RBs. Bacteria infected with the phage

follow an altered developmental path (Hsia et al. 2000a). Similarly to Chp2, cell division is inhibited and RBs grow in an unnatural manner (maxiRB) without maturing to EB (Hsia et al. 2000a). These forms finally undergo lysis at a later phase of chlamydia development cycle, releasing abundant phage progeny in the inclusion, and after lysis of the inclusion membrane, the changed bacteria are released into the cytosol of host cell (Hsia et al. 2000a). On the basis of ultrastructural analyses, the authors of the cited study (Hsia et al. 2000a) proposed a model of the mechanism of infection and transmission of ϕCPG1 chlamydiaphage, which is as follows: (a) ϕCPG1 gains access to the replicating chlamydia by attaching to the EBs, which occurs without interference with the early phases of chlamydia infection; (b) ϕCPG1 s infect chlamydia directly after the transformation of EBs into RBs; (c) inhibition of the division of the infected RBs and their unnatural growth (maxiRB-mRB) occurs; (d) mRB is formed at the place of phage replication, composing its elements; (e) ϕCPG1 directs infected mRB lysis, leading to release of abundant progeny; (f) ϕCPG1 released from mRB binds to receptors on the surface of the external chlamydia membranes; (g) the release of ϕCPG1 induces lysis of the parasitic inclusion membrane, causing the mixing of inclusion contents with cytosol; (h) loss of cellular structural integrity occurs, leading to host cell lysis caused by ϕCPG1 bacteriophage activity or mechanisms induced by its presence. Further analyses of interactions of the phage with chlamydia in their natural animal host were performed on guinea pigs, which were inoculated in the conjunctiva with suspension containing C. caviae infected with ϕCPG1 (Rank et al. 2009). The effects were surprising, as it turned out that the presence of the phage inhibited the development of chlamydia and decreased the pathological response that mostly depended on the volume of chlamydia in the affected tissue (Rank et al. 2009). Similar effect has been shown in the case of chlamydiaphage ϕCPAR39 infecting C. pneumoniae, which did not cause lysis of the membrane of abnormally large RBs of C. pneumoniae strain AR39 (Everson et al. 2002). However, this property was limited to one species (C. pneumoniae), and the infection of other chlamydia (C. caviae, C. abortus, C. pecorum) with ϕCPAR39 led to lysis of RBs (Everson et al. 2003). It was also observed (Hoestgaard-Jensen et al. 2011) that the infection with phage ϕCPAR39 variously affects synthesis of membrane proteins Pmp 10, Cpn0796, Cpaf, and Inc, which are secreted by C. pneumoniae during the

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infection of the host cell cytoplasm or are released to the inclusion membrane. It was noted that protein Pmp10 could not be detected in phage-infected C. pneumoniae inclusions (Hoestgaard-Jensen et al. 2011). This could be explained either by lower production of that protein or by a faster degradation, as its incorporation into the membrane could be compromised by the phage infection (HoestgaardJensen et al. 2011). The Cpn0796 was also nearly absent in phage—infected inclusions, but secreted from phage-infection-free inclusions (Hoestgaard-Jensen et al. 2011). So far, there has been no answer to the question why secretion and expression of this protein were reduced (HoestgaardJensen et al. 2011). Cpaf is produced and secreted late in the lifecycle of C. pneumoniae (Hoestgaard-Jensen et al. 2011). During phage infection, Cpaf is synthesised, but not secreted into the cytoplasm of the host cell (HoestgaardJensen et al. 2011). It was shown that it was accumulating in the chlamydial envelope (Hoestgaard-Jensen et al. 2011) Furthermore, the presence of inclusion membrane proteins IncA was detected, which surround the infected RBs, and presumably can be involved in a specific defence against the development of C. pneumoniae infected with the phage (Hoestgaard-Jensen et al. 2011).

Conclusion The relations between chlamydiaphages, their prokaryotic host (chlamydia) and indirectly their eukaryotic host are very complex. Each of the three partners possesses specific defence mechanisms against potential harm inflicted by the other. It is unknown how the presence of phage affects the biology of the Chlamydia. It was suggested that phages effect on chlamydial pathogenicity because there were found significant correlation between phage antibodies and abdominal aortic aneurysm in 61 C. pneumoniae seropositive individuals (Karunakaran et al. 2002). Moreover, the well-known phenomenon often discussed as related to phage infection are different growth characteristics of chlamydial isolates in cell culture. Also, there are no data about coevolution of these bacteria and their phages. It is unclear whether these phages have coevolved with their host from a single ancestor or whether they, or specific phage genes, have been independently “acquired” at later stages of evolution (Hsia et al. 2000b). Although other members of the Microviridae have been described as lytic (Read et al. 2000b), the high homology of the chlamydial gene Cpn 0222 to the phage ORF4 gives strong evidence that phage integration occurred at least once in C. pneumoniae history (Rupp et al. 2007). The complicated developmental cycle of chlamydia makes it difficult to investigate the gene sequences of chlamydiaphages, their regulation, and biological function of the gene products. This, in turn,

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