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"So, Nat’ralists observe, a Flea

"Hath smaller Fleas that on him prey.

"And these have smaller Fleas to bite ‘em,

And so proceed ad infinitum."

Like Dean Swift's fleas, parasitic chlamydiae are themselves victims of even smaller life forms, viruses. Viruses differ from bacteria in consisting of only a single kind of nucleic acid (genetic material), DNA or RNA, and in using host cell ribosomes for their protein synthesis. Viruses which infect bacteria are called bacteriophages . Four different chlamydial phages have been identified, infecting the Chlamydophila only.

The first phage to be characterised was found  in an isolate of Chlamydophila psittaci  recovered from a severe outbreak of respiratory infection that occurred in England in a duck-processing plant in East Anglia [Richmond et al., 1982]. The phage, named Chp1, was purified and found to be a member of the Microviridae, a family of bacteriophages), the best-known member of which is PhiX174. The new phage, which apparently infected only C. psittaci, was placed in a unique subfamily, the Chlamydiavirinae [Storey et al., 1989]. Subsequently it has been found that Chp1-like infection of C. psittaci is common in duck chlamydiosis and may lead to some of the features of the disease. Growth of Chp1-infected C. psittaci in laboratory cell culture can lead to a spontaneous loss of the phage infection [Storey et al., 1989].

Phage_Fig 1. Electron micrograph of chlamydial reticulate bodies infected with crystalline arrays of Chp1 phage particles. [With kind permission of Charles Ashley and modified from Richmond, R. J., Stirling, P. & Ashley, C. R. (1982). FEMS Microbiology Letters 14, 33 - 35.]

A distinct but related phage, Chp2 [Liu et al., 2000] was subsequently identified from a Macedonian isolate of C. abortus, an organism which causes abortion in sheep [See: C. abortus infections in sheep]. Chp2 does not infect members of the genus Chlamydia and infects only selected species of the genus Chlamydophila, including C. abortus, C. felis, and C. pecorum but not C. caviae or C. pneumoniae, despite the fact that these species support the replication of very closely related bacteriophages [Everson et al., 2002].

Phage_Fig 2. Electron micrograph of a negatively stained suspension of Chp2. Some of the negative stain has penetrated the capsid shell of the 22 nm diameter particles. [Electron micrograph with kind permission of Dr Ian Clarke, University of Southampton. See: Liu, B. L., Everson, J. S., Fane, B., Giannikopoulou, P., Vretou, E., Lambden, P. R. & Clarke, I. N. (2000). Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. Journal of Virology 74, 3464 - 3469].

Phage_Fig 3. Electron micrograph of a thin section of a cell infected with C. abortus which is itself infected with phage Chp2. Note that the phage has ruptured many of the reticulate bodies. [Electron micrograph by kind permission of Dr Ian Clarke. See: Liu, B.L. et al., (2000). J. Virol 74, 3464 - 3469].

A third phage, phiCPG1, [Hsia et al., 2000a & b] was identified from an isolate of the guinea pig inclusion conjunctivitis (GPIC) agent,  C. caviae. All of these Chlamydophila species were formerly known as C. psittaci. 

Phage_Fig 4. Electron micrograph of phage phiCPG1 attached to ruptured multilamellar membranes of C. caviae. Each of these chlamydial microviridae has quite distinct effects on the host cell. [Electron micrograph courtesy of Dr Patrik Bavoil: Hsia, R-C., Ohayon, H., Gounon, P., Dautry-Varsat, A & Bavoil, P. M. (2000). Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis. Microbes and Infection 2, 761 - 772].

Finally the genome of phiCPAR39 was found during the sequencing of the whole genome of Chlamydophila pneumoniae [Bavoil et al., 2000]. The sequence of the genome of this phage shows 96.8% homology (i.e. virtual identity) with the C. caviae phage phiCPG1. Immunofluorescent staining with specific monoclonal antibody demonstrated the presence of phage coat protein in C. pneumoniae while electron microsopy showed the presence of phage particles [Everson et al., 2000].

All these phages are small isometric particles of approximately 25 nm in diameter [1 nm is the width of10 hydrogen atoms].

The genomes of Chp1, Chp2 and phiCPG1  and phiCPAR39 have been fully characterized [Storey et al., 1989; Liu et al., 2000; Hsia et al., 2000b; Bavoil et al., 2000], consisting of circular, single-stranded DNA with genes (open reading frames) encoding 3 viral structural proteins, VP 1-3 and 2 further postulated genes ORF 4 & 5 (in Chp1 and 2; ORF means open reading frame), which correspond to VG 4 and 5 in phiCPG1. Additional smaller open reading frames are also present and there are some minor differences in the structural organisation of the genome [Liu et al., 2000]. Comparison of the nucleotide sequences showed substantial similarities with each other (see table below) and with genes of a similar virus called SpV4, infecting Spiroplasma melliferum, a helical bacterium which is pathogenic for honey bees [Hsia et al., 2000b]. Recently a related bacteriophage, MH2K, which infects Bdellovibrio bacteriovorus has been described [Brentlinger et al., 2002]. Surprisingly the coat protein of MH2K showed closer similarity to Chp2 than was shared between Chp1 and Chp2.

The function of some of the chlamydial virus genes has been tentatively identified. VP1 is a major structural protein similar to the coat (capsid) F protein of phiX174. When the genomic sequences of  phiCPG1, phiAR39, Chp2, and Chp1 were compared it was found that Chp1 had diverged significantly in its nucleotide sequence from the other three phages, which formed a closely related group. The VP1 major viral capsid proteins of phiCPG1 and phiAR39 were almost identical. VP2 and VP3 are structural proteins and it has been suggested that VP3 performs a scaffolding - like function but has evolved into a structural protein [Lie et al., 2000].

Table: Percentage sequence similarity of selected genes of Chp1, Chp2, phiCPG1, phiCPAR39 & SpV4. (Source: References 3, 5 & 6).

	Chp1 v Chp2	Chp1 v phiCPG1	Chp1 v phiCPAR39	phiCPG1 v phiCPAR39	SpV4 v phiCPG1
Genome		50.1%	50.1%	96.8%	n/a
VP1	48.3%	54.5%	54.3%	99.5%	46%
VP2	27.3%	38.2%	37.6%	99.5%	34.8%
VP3	26.0%	28.8%	28.8%	98.7%	-
ORF4	19.9%	36.8%	n/a	n/a	42.9%
ORF5	27.1%	34.1%	34.1%	98.8%	-

Morphologically, there are differences in the replication of these phages. Chp1 unlike Chp2 and phiCPG1 forms crystalline arrays inside the C. psittaci reticulate body [Richmond et al., 1982], [Phage_Fig 1]. Particles of phiCPG1 tend to associate strongly with chlamydial membrane [Hsia et al., 2000a]; [Phage_Fig 4]. Chp1 was considered to only attach to (and infect) chlamydial reticulate bodies. In contrast,  phiCPG1 (Figure below) attaches to fresh (not aged) elementary bodies, infection taking place as soon as the elementary bodies differentiate into metabolically active reticulate bodies [Hsia et al., 2000a].

Phage_Fig 5. Phage particles (arrowed) of phiCPG1 attached to an elementary body of C. caviae. [Electron micrograph courtesy of Dr Patrik Bavoil modified from:  Hsia, R-C. et al., (2000).  Microbes and Infection 2, 761 - 772].

In phiCPG1 phage-infected reticulate bodies, cell division is inhibited, producing abnormally large "maxi" reticulate bodies which do not mature into elementary bodies. These maxi reticulate bodies bear a superficial resemblance to the similar structures observed in the persistent chlamydial developmental cycle, when chlamydial development is temporarily halted by gamma interferon or penicillin [See: incomplete development].

Release of chlamydial phages is probably by lysis (rupture) of the infected reticulate body [Phage_Fig 3] and, subsequently, the surrounding inclusion membrane. The related phiX174 phage, which infects the common gut bacterium Escherichia coli, has a protein which interacts with a bacterial protein (slyD) to stimulate enzymatic activity that leads to rupture of the bacterium [researchers: it’s an FK506 binding protein related to the FKBP family of peptidyl-prolyl cis-trans-isomerases]. Interestingly, a similar group of proteins is found in the Chlamydiaceae [Hsia et al., 2000b] suggesting that the same kind of mechanism may occur.

The apparent similarity of "maxi", phiCPG1-phage infected reticulate bodies with the structures observed in gamma interferon-treated, persistently chlamydial-infected cells, may be coincidental. Chp1 and Chp2 seem to produce rather different effects. However, it has been suggested that phage infection may be another factor leading to the tendency of some chlamydial strains to cause delayed, persistent or severe chlamydial infection; at the present time we just don’t know. 

In the laboratory, phage infection generally reduces chlamydial inclusion formation and viability. However phage infection did not appear to reduce the clinical severity of duck-derived ornithosis in the East Anglia outbreak from which Chp1 was isolated. The scenario of chlamydiae with an associated chlamydial virus infecting an animal or human is a complex three-way relationship, where the outcome is presently hard to predict. As phage phiCPG1 is a pathogen of the C. caviae guinea pig agent, this latter scenario might be explored experimentally in guinea pig infections. The observation that a fragment of a close relative of phiCPG1 has become incorporated into the C. pneumoniae genome, together with the presence of the whole phage, phiCPAR39, is unusual. It indicates that similar phages may be a suitable vehicle for the experimental manipulation of the genome of chlamydiae. Moreover it is possible that phages that we now know can infect different Chlamydophila species [Everson et al., 2002],  may have played a role in the evolution of the Chlamydiaceae or the Chlamydiales].

[INC & MEW] May, 2002

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References

Bavoil, P.M., Hsia, R-C., Brunham, R., Fraser, C. M. & Read, T. D. (2000) Chlamydia psittaci GPIC and Chlamydia pneumoniae are infected by virtually identical bacteriophages. Page 23: In: Proceedings of the Fourth Meeting of the European Society for Chlamydial Research (Saikku, P. ed)., pub Editrice Esculapio, Bologna, Italy. ISBN 88-86524-41-2.

Brentlinger, K. L., Hafenstein, S., Novak, C. R., Fane, B. A., Borgon, R., McKenna, R., Agbandje-McKenna, M. (2002). Microviridae, a family divided: isolation, characterization, and genome sequence of phiMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus. Journal of Bacteriology 184, 1089 - 1094.

Everson J. S., Garner, S. A., Fane, B., Liu, B. L., Lambden, P. R. & Clarke, I. N. (2002). Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular Bacterium Chlamydophila abortus. Journal of Bacteriology 184, 2748 - 2754.

Hsia, R-C., Ohayon, H., Gounon, P., Dautry-Varsat, A & Bavoil, P. M. (2000a). Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis. Microbes and Infection 2, 761 - 772.

Hsia, R-C., Ting, L-M. & Bavoil, P. M. (2000b). Microvirus of Chlamydia psittaci strain guinea pig inclusion conjunctivitis: isolation and molecular characterization. Microbiology UK, 146, 1651 - 1660. Full article 

Kalman, S., Mitchell, W., Marathe, R., et al., (1999). Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nature Genetics 21, 385-389. Full article Chromosome map C. pneumoniae Functional assignments table of genes in C. pneumoniae and C. trachomatis 

Liu, B. L., Everson, J. S., Fane, B., Giannikopoulou, P., Vretou, E., Lambden, P. R. & Clarke, I. N. (2000). Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. Journal of Virology 74, 3464 - 3469. Full article  

Read, T. D., Fraser, C. M., Hsia, R. C. & Bavoil, P. M. (2000). Comparative analysis of Chlamydia bacteriophages reveals variation localized to a putative receptor binding domain. Microbial Comparative Genomics 5, 223 - 231.

Richmond, S. J., Stirling, P. & Ashley, C. R. (1982). Virus infecting the reticulate bodies of an avian strain of Chlamydia psittaci. FEMS Microbiology Letters 14, 31 - 36. [The initial discovery]

Storey, C.C., Lusher, M. & Richmond, S. J. (1989). Analysis of the complete nucleotide sequence of Chp1, a phage which infects avian Chlamydia psittaci. Journal of General Virology 70, 1321 - 1327; also 3381 - 3390. [First sequence]

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