Chlamydial plasmids and bacteriophages

Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.


INTRODUCTION
Chlamydia are absolute intracellular pathogens in humans, as well as vertebrates and invertebrates, all present in the environment, including aquatic environments (Pawlikowska & Deptuła, 2012).Their virulence is principally due to components of the outer membrane (Inc and Pmp proteins, related to the third type secretion system) and various secretory proteins (e.g.glycosyltransferase), chromosomally encoded (Rockey et al., 2000;Nunes et al., 2013), and their virulence is supplemented with extrachromosomal factors, specifically plasmids (Song et al., 2013(Song et al., , 2014)).Chlamydia are distinct prokaryotes because they carry features that are intermediate between bacteria and viruses.The most characteristic feature that differentiates them from bacteria is their unique development cycle, lasting 48-72 hours, with three forms that include an infectious elementary body (EB), a noninfectious reticulate body (RB), as well as intermediate body (IB) (Horn, 2012;Pawlikowska & Deptuła, 2012).Their genetic information, as in all prokaryotes, is written in a rather small bacterial chromosome (approx.1 Mbp) (Nunes et al., 2013), but some of their genotypic and phenotypic features are supplemented with extrachromosomal carriers, that is plasmids (Song et al., 2013(Song et al., , 2014) ) and bacteriophages (Hsia et al., 2000).
Plasmids are small, usually circular DNA particles which coexist in bacterial cells, carrying genetic information apart from the bacterial chromosome, and they are capable of independent replication (Petersen, 2011).They can transfer genes that are not usually present in the bacterial chromosome, and despite not being essential for bacterial cell functioning, they can condition features necessary for their survival (Petersen, 2011).Among others, plasmids carry genes responsible for resistance to antibiotics (resistance plasmids), genes encoding toxins, e.g.bacteriocins, genes conditioning degradation of chemical compounds (catabolic plasmids), genes conditioning virulence, and conditioning one of the variation mechanisms in prokaryotes -conjugation (conjugative plasmids).An important feature of plasmids present among prokaryotes, including chlamydia, is the fact that they can be transferred among bacterial cells, including horizontal gene transfer (Petersen, 2011).This feature of plasmids is a useful biotechnological tool used in genetic engineering (Petersen, 2011).Due to the range, plasmids have been divided into plasmids with narrow host range (NHR), such as those that replicate and exist exclusively in the native host and closely related species, and plasmids with broad host range (BHR), characterised with capacity of colonizing many unrelated species (Petersen, 2011).Chlamydial plasmids actually colonise just some species of chlamydia that are pathogenic to humans and animals (Pickett et al., 2007).
Bacteriophages (phages) -bacterial viruses, including chlamydia, are all-present in the environment, as they have been isolated from water, soil, but also desert sand, hot springs, and even from humans or animals (Clockie et al., 2011).They are characterised by narrow host range, although there are also phages infecting various bacterial species.After infecting bacteria, the phages can undergo two basic lifecycles -lytic or lysogenic, which begin in the same way, that is by adsorption on a bacterial cell via specific receptors.Further phases of the lifecycle involve permeation into the bacterial cell, release of the genetic material and passage into an eclipse phase, namely taking control over the host's metabolism.The next stages are replication of the genetic material, synthesis of viral proteins, formation of new bacteriophage particles and their release (Clockie et al., 2011).Phage genetic material can be either DNA or RNA, in a single-or double-stranded form, in circular or linear form (Clockie et al., 2011;King et al., 2012).The current systematic of bacteriophages differentiates 10 families and 1 genus (not assignated to a family), among which 8 families and as well as the genus not classified into any family are bacteriophages containing genetic material in the form of DNA, whereas just 2 families contain RNA (Krupovic et al., 2011;King et al., 2012).There is also a proposal of a new family of bacteriophages -Sphaerolipoviridae, containing dsDNA as genetic material (Pawlowski et al., 2014).Chlamydial bacteriophages belonging to the Gokushovirinae group in the Microviridae family, contain a single-stranded circular DNA particle (ssDNA) and their virion is in the cubic form and does not have an envelope (King et al., 2012;Roux et al., 2012).

CHARACTERISTICS OF CHLAMYDIAL PLASMIDS
Chlamydial plasmids are present in seven species of chlamydia (C. trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum, C. caviae) and their length is about 7.5 kbp, present in the quantity of 4 to 10 copies (Pickett et al., 2007;Rockey, 2011).Their replication is strictly related to the replication cycle of those bacteria, especially with divisions of RB (Ferreira et al., 2013).Chlamydial plasmids are not conjugative plasmids, they mostly do not code antibiotic-resistance and do not have capacity for integration, except for integrative plasmid revealed in C. suis (Dugan et al., 2004).The main role of plasmids in chlamydia is their participation in accumulation of glycogen, which is an additional material necessary for divisions inside chlamydial inclusion, which is related to the correlation of plasmid genes with the gene of glycogen synthase glgA, located at the chlamydial chromosome (Carlson et al., 2008;Wang et al., 2011).
Chlamydial plasmids contain non-coding RNA (Abdelrahman et al., 2011) and eight open reading frames (ORF1-8) (Table 1), whereas the functions of five of them have been thoroughly recognised (Li et al., 2008;Ferreira et al., 2013;Song et al., 2013).While comparing the sequences of plasmids isolated from various species of Chlamydia, it seems that their genetic similarity would range from 69 to 99% (Thomas et al., 1997).Whereas in the case of plasmids isolated from C. trachomatis, from the strains representing 15 serotypes, high homology of their sequence was found (only 1% of differences was recorded), and is considered to be the most conserved among chlamydial plasmids (Thomas et al., 1997;Ferreira et al., 2013), similarly as C. felis plasmids (Di Francesco et al., 2010;Harley et al., 2010).Such high homology and conservative nature of plasmids of C. trachomatis and C. felis may suggest that they have evolved in parallel with C. trachomatis and C. felis (Seth-Smith et al., 2009).Among others, their homology and conservative nature is a result, of the fact that there is a 22 bp region located between ORF8 and ORF1, which occurs in four copies in the plasmid sequence (Thomas et al., 1997).Similar repetitive sequences have been revealed in plasmids originating from avian strain C. psittaci (Hugall et al., 1989;Lusher et al., 1989), from C. felis and C. cavie (Lusher et al., 1989), and they have also been identified in plasmids from C. pneumoniae strain N16 (equine biotype) and C. muridarum strain E58 (Hugall et al., 1989;Everett et al., 1999).In the case of plasmids from C. trachomatis strains, their conservative sequences differ as compared to plasmids of C. psittaci by substitution of two nucleotides (Thomas et al., 1997).When analysing nucleotide sequences of open reading frames, it was evidenced that proteins encoded by ORF1 (pgp7) and ORF2 (pgp8) are homologues of, integrase and recombinase, respectively, and are in charge of regulation of plasmid replication, while ORF3 (pgp1) is a homologue of DnaB helicase and participates in unwinding of the double DNA strands during their replication.In turn, ORF4 encodes Pgp2 protein which, depending on chlamydial species, contains from 345 to 245 amino-acids, but its function has not yet been determined.One of the major frames is ORF5, which encodes 28kDa protein Pgp3, which can be a marker of chlamydial infections (Comanducci et al., 1994;Li et al., 2008), although its function for other chlamydial features remains unknown.ORF6 encodes Pgp4 protein that contains 101-102 amino-acids, while ORF7 and OEF8 encode proteins Pgp5 and Pgp6, respectively, which may be involved in plasmid replication (Thomas et al., 1997).Gong et al. (2013) have shown by using a C. trachomatis plasmid with pORF deletion, that Pgp3, Pgp4, Pgp5 and Pgp7 are not required for maintenance, and also minimum 30 nucleotides were found in pgp3 which encode a region required for pgp4 expression.In C. muridarum plasmid with deleted pORFs a new function of Pgp5 was discovered, which negatively regulates some plasmid-dependent genes (Liu et al., 2014).Despite the occurrence of plasmids in many chlamydial species (C. trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum, C. cavie), their role in the development cycle of these bacteria has not yet been fully understood.It was evidenced that plasmids encode various proteins involved in the replication of the plasmid itself, but it was not determined whether they affect the interaction with the host's cell; it was only demonstrated that Pgp3 protein is secreted to the cytosol of cells infected by chlamydia (Li et al., 2008).The spreading mechanism of this protein is similar, but not identical, to the distribution of chlamydial CPAF factor (Chlamydia Protease-like Activity Factor), genome-encoded protease secreted to cytosol of a cell infected with chlamydia (Li et al., 2008).It was demonstrated that Pgp3 protein stimulates murine macrophages in vitro to release pro-inflammatory cytokines: MIP-2 (murine IL-2 homologue) and TNFα, and can act as a receptor for TLR4 (Li et al., 2008).This protein can also be used in anti-chlamydial vaccines (Donati et al., 2003).The role of plasmids in pathogenicity of Chlamydia is not yet fully understood, although it has been shown that the most important for plasmids are genes pgp1 (ORF3), pgp2 (ORF4), pgp6 (ORF8) and pgp8 (ORF2), while of slightly less importance are pgp3 (ORF5), pgp5 (ORF7) and pgp7 (ORF1) (Song et al., 2013).However, pgp7 and pgp8 genes are very significant for chlamydial plasmids, as they show homology with the family of phage proteins (integrase/recombinase) (Song et al., 2014).It was also shown that pgp4 gene affects the ability to accumulate glycogen in chlamydial inclusions, and the Pgp4 protein is a transcriptional regulator of plasmid and chromosomal genes of chlamydia, responsible for their virulence (Song et al., 2013(Song et al., , 2014)).Also, no correlation was found between the quantity of plasmid copy in chlamydia and its pathogenicity (Last et al., 2014).An experiment with plasmid-free C. trachomaits -serovar L2, transformed with a plasmid carrying a deletion of a plasmid CDS5 gene, used in mice with a urogenital infection, showed that inflammatory response in mice was enhanced (Ramsey et al., 2014).What this means is that the CDS5 plasmid plays a critical role in virulence and infectivity of C. trachomatis (Ramsey et al., 2014).This was confirmed in mice with a urogenital infection which were treated with plasmid-less serovar E of C. trachomatis, which showed that infectivity of this strain was reduced when compared to the wild strain of C. trachomatis carrying plasmid (Sigar et al., 2014).
Chlamydial plasmids, due to their low variability, have found practical application in diagnostics, principally as a target in DNA amplification in NAAT (nucleic acid amplification test) (Fredlund et al., 2004;Schachter et al., 2006).With the use of this method, a new strain of C. trachomatis -Sweden2 (serotype E), was described in 2006 (Ripa & Nilsson, 2006).The difference in this strain resulted from the deletion of a 377 bp long fragment from the plasmid DNA, and the impossibility of detecting this strain using standard NAAT (Ripa & Nilsson, 2006).Studies of the evolution of both, C. trachomatis strains and their plasmids indicated that both the chlamydial chromosome and the plasmid DNA evolved in parallel, and both depend on the biotype or serotype of chlamydia (Seth-Smith et al., 2009).These results suggest that the presence of plasmids with deletion within urogenital strains may cause difficulty in diagnosis of those chlamydial infections, and consequently leads to latent infections, effects of which can be severe, such as infertility.Another application of chlamydial plasmids is an attempt to use them in anti-chlamydial vaccines.It is known that chlamydial strains which do not carry plasmids are less pathogenic, and sometimes do not activate an immunological response (O'Connell et al., 2007).It was demonstrated that C. muridarum, deprived of plasmids both in vivo and in vitro, does not activate a TLR2-dependent immunological response (O'Connell et al., 2007); this was also confirmed in the study with C. cavie on Guinea pigs (Frazer et al., 2012).In turn, Miyairi et al. (2011) did not observe differences in TLR2 activation in the case of the C. psittaci strain 6BC with and without plasmids.In addition, this study has shown that infection with both forms of the C. psittaci strain 6BC renders the infection (Miyairi et al., 2011).Those studies (Miyairi et al., 2011), as well as another (Kari et al., 2011), suggest that chlamydial strains deprived of plasmids can be applied for immunisation, which may provide protection against infection with chlamydial strains carrying plasmids or weaken the effects of such infections.

CHARACTERISTICS OF CHLAMYDIAL BACTERIOPHAGES (CHLAMYDIAPHAGES)
Chlamydial bacteriophages belong to the Chlamydiamicrovirus genus, which together with Bdellomicrovirus and Spiromicrovirus form the Gokushovirinae group, which belong with Microvirus to the aforementioned Microviridae family (King et al., 2012;Roux et al., 2012).The first chlamydial phage, denoted as Chp1 bacteriophage (Chlamydiaphage 1), was described in 1982 in reticulate bodies (RB) and inclusions of C. psittaci isolated from ducks (Liu et al., 2000).At present, five more chlamydiaphages are known, namely Chp2 (Chlamydiaphage 2), Chp3 (Chlamydiaphage 3), φCPG1 (Guinea pig chlamydiaphage), φCPAR39 (φCpn1; Chlamydia pneumoniae AR39 bacteriophage) and Chp4 (Chlamydiaphage 4), which primarily infect C. psittaci, C. abortus, C. felis, C. cavie, C. pecorum and C. pneumoniae.Viral genomes similar to chlamydiaphages were also found in the human gut, lung and feces (Roux et al., 2012).The mutual feature of these chlamydiaphages is the similar size of their genomes (4.5-4.8 kbp) and encoding of eight open reading frames (ORF1-8) (Table 2), arranged in the following order: VP1-VP2-VP3-ORF8-ORF4-ORF5, wherein ORF6 and ORF7 overlap ORF1 and ORF2, respectively (Sait et al., 2011;Roux et al., 2012).The most important are frames ORF1-3 and ORF8, which encode the major capsid proteins VP1a major capsid protein (ORF1), VP2 -a minor spike protein (ORF2), and VP3 -a scaffold protein (ORF3).The VP1 protein comprises two major areas that are exposed on the virion surface that likely interact with each other (Garner et al., 2004).The first region is located at position 216-299 bp, and reminds IN5 loop, while another area, referred to as INS, is located between 462 and 467 bp.Protein encoded by ORF8 is similar to protein J of φX174 phage and is characterised with high degree of sequence conservation in Chp2, Chp3, CPG1 and CPAR39.Another common feature of chlamydiaphages is that their lifecycle is a lytic cycle, although the release of new particles from the bacterial cell is still mysterious.In the case of φX174 phage, a gene encoding protein E, a specific inhibitor of peptydoglycan synthesis (acting as penicillin) was detected in its genome, which results is cell lysis (Young, 1992;Bernhardt et al., 2000;Zheng et al., 2008).A homolog of this gene is also present in the chlamydial genome (Kalman et al., 1999).The function of this gene does not only refer to the inclusion membrane where replication of chlamydia and phage occur, but also to the cellular membrane of the eukaryotic cell where chlamydia multiply (Hsia et al., 2000).
Chlamydiaphage Chp1 was isolated, as mentioned above, from C. psittaci infected ducks.Its genome is comprised of 4877 bp, encodes 11 proteins, and the content of GC pairs is relatively low, as amounting to 36.6%.Open reading frames have an ATG start codon preceded with the region which could be an RBS (ribosome-binding site), which suggests that each of them may encode protein.Moreover, there are additional places for starting transcription, namely ORF2a, 2b, 4a, and 5a, which allow for encoding four additional proteins (Storey et al., 1989a(Storey et al., , 1989b)).The organisation of ORFs is such that reading frames ORF 6 and 2, as well as ORF1 and 7 overlap, while ORF8 probably encodes a non-functional protein.The analysis of N-terminal amino acid sequences showed that structural proteins VP1, VP2 and VP3 are encoded by ORF1-3, while VP1 protein shows partial similarity with major structural protein of the F capsid of φX174 bacteriophage (a typical species of Microvirus), and the protein encoded by ORF4 reveals homology with protein A (Storey et al., 1989b).Chlamydiaphage Chp2 was identified in C. abortus and C. psittaci (Liu et al., 2000;Salim et al., 2008).The length of its genome amounts to 4567 bp and encodes eight open reading frames, of which ORF1-5, ORF7 and ORF8 are homologous to sequences of Chp1.Similarly as Chp1, the Chp2 phage has three structural proteins VP1, VP2 and VP3.However, capsid proteins show the greatest similarity to proteins of phage φSpV4 (a typical phage of Spiromicrovirus).Similarity was also noticed between the small J protein encoded by ORF8 of the Chp2 phage with a J binding protein of phage φX174 (Liu et al., 2000).Chlamydiaphage Chp3 was isolated from elementary bodies of C. pecorum and, similarly as in previously described chlamydial phages, its genome has also 4544 bp and is organised into eight ORFs (Garner et al., 2004).Chlamydiaphage Chp4 is a phage infecting C. abortus, and its genome is comprised of 4530 bp, and also features 8 ORFs.ORF 4 and ORF5 encode non-structural proteins similar to proteins A and C isolated from φX174 phage, involved in synthesis of phage DNA and packaged into capsids.Chlamydiaphage φCPG1 (Guinea pig chlamydiaphage) isolated from C. psittaci (currently C. caviae), with the genome sized 4529 bp, features eight open reading frames, where ORF1-3 encode structural proteins (VP1-3), while ORF4 and ORF5 encode proteins VP4 and VP5.The segment of the genome comprising protein VP4 and sequences above the region may contain signals important for replication and/or regulation of gene expression of the phage (Hsia et al., 2000).The analysis of VP1 protein sequences showed high similarity between homologous sequences of Chp1, φCPG1 and φSpV4 (Hsia et al., 2000).The described chlamydiaphage φCPAR39, also known as φCpn1, was isolated from C. pneumoniae -strain AR39.This is the first and only chlamydial phage isolated from bacteria causing diseases in humans.Its genome is comprised of 4524 bp, and divided into eight reading frames (Karunkaran et al., 2002;Rupp et al., 2007;Hoestgaard-Jensen et al., 2011).In case of bacteriophages of C. caviae and C. pneumonia, three chlamydiaphage genes were found in genomes of their host (Read et al., 2003).This is similar to the Microviridae-related prophages integrated in the genome of Bacteroidetes and integration into the host's genome can establish lysogeny (Krupovic & Forteree, 2011).
The description presented indicates that all five chlamydiaphages (Chp1, Chp2, Chp3, Chp4, φCPG1, φCPAR39) have similar genome structure, although for the assessment of similarity or differences between them two regions present in all of them are used, namely the region encoding protein VP1 and ORF4 (Read et al., 2000;Everson et al., 2002;Sait et al., 2011).In the VP1 region, two characteristic regions are considered, that is IN5 and INS, closely located, and play a role in recognising the host's receptors (tissue tropism determination) (Everson et al., 2002).Sait et al. (2011) observed that adsorption of Chp2 or Chp3 phages on C. abortus, does not prevent adsorption of φCPAR39, which indicates and proves the presence of various receptors on the surface of chlamydia.It was also evidenced that replication initiation protein, encoded in the ORF4 region, is the fastest evolving protein as compared to other chlamydial proteins, and despite the fact that its function has not been entirely recognised, it was documented that its central sequence is characteristic of many prokaryotic Rep proteins (controlling replication) (Read et al., 2000).The impact of bacteriophages on chlamydial development is still questioned, and thus the course and pace of infecting the macro-organism with them.In the studies on infection with Chp2 of C. abortus, inhibition of cellular division was observed in the form of blocking the transformation of RB into EB bodies, with simultaneous increase in the volume Chlamydial plasmids and bacteriophages of RB bodies (Salim et al., 2008).It was also determined during the C. abortus cycle that between hour 36 and 48, increased replication of Chp2 phage genome occurs, coinciding with expression of phage proteins (VP3 -procapsid proteins) and replication of bacterial chromosome (Salim et al., 2008).In the case of φCPG1 phage, it was evidenced that infection of C. psitacii -Guinea pig strain (presently C. caviae) occurs upon transformation of infectious EB bodies into metabolically active forms, namely RB.At that time, reticulate bodies RB grow (formation of maxiRB), which become the place of phage replication and maturation (Hsia et al., 2000).Both, in the case of phage Chp2 infecting C. abortus, and φCPG1 infecting C. caviae, when new bacteriophage particles are formed, lysis of chlamydia as host's cell occurred.Chlamydial phages can occur in the cell's cytosol, but most frequently bind to the cellular membrane of EB bodies, and are transferred in such a form together with chlamydia to other eukaryotic cells.In case of mixed intranasal infection with C. caviae and stock of phage φCPG1, a positive reaction in serum of Guinea pigs -high level of antibody to VP1 was observed (Rank et al., 2009).

CONCLUSION
Plasmids and bacteriophages occurring in chlamydia show highly conservative nucleotide sequence, similarly as their hosts.Chlamydial plasmids generally do not carry for e.g. the genes of antibiotic resistance, as in other bacteria, except for plasmid C. suis, which is additionally an integrative plasmid, but carry the genes "cooperating" with chromosomal genes in accumulation of glycogen and encode proteins that may take part in virulence by interaction with chromosomal genes (Pgp4), and immunogenic proteins (Pgp3).They are used in diagnostics, DNA amplification tests, or as markers of chlamydial infection in the form of proteins encoded by them.They can be used as elements of vaccines preventing chlamydioses in animals.However, bacteriophages infecting chlamydia decrease their pathogenicity and distort their cycle by their replication.The easy transfer of infectious EB bodies into other cells can be used in the treatment for chlamydioses, particularly in the case of concealed and latent infections, principally at the time of chlamydial resistance to antibiotics used by choice of treatment (tetracyclines).Considering the presented and the still undiscovered properties of plasmids and chlamydiaphages, additional elements of genetic information present in chlamydia, it seems they will allow creating new tools for fighting chlamydioses in the future.