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Unraveling the basic biology and clinical significance of the chlamydial plasmid

Daniel D. Rockey

Chlamydial plasmids are small, highly conserved, nonconjugative, and non­ syndromes caused by otherwise related integrative DNA molecules that are nearly ubiquitous in many chlamydial strains and species. species, including Chlamydia trachomatis. There has been significant recent Such variability is not found in progress in understanding chlamydial plasmid participation in host–microbe chlamydia. Several chlamydial species interactions, disease, and immune responses. Work in mouse model systems contain one of a homologous set of and, very recently, in nonhuman primates demonstrates that plasmid- 7,500 base pair plasmids with a copy deficient chlamydial strains function as live attenuated vaccines against number that is approximately four­ genital and ocular infections. Collectively, these studies open new avenues fold greater than that of the chromo­ of research into developing vaccines against trachoma and sexually transmitted some (Thomas et al., 1997; Pickett et al., chlamydial infections. 2005; Fig. 1). Within C. trachomatis clinical isolates, the plasmid is virtually Human pathogenic chlamydiae are These obligate intracellular bacteria ubiquitous. There are occasional stud­ members of a successful and unique develop within a membrane-bound ies showing plasmid-negative clinical lineage of bacteria (Collingro et al., vacuole termed the inclusion (Fig. 1), strains, but little is known about the 2011), which infect and cause disease in and existence within the inclusion de­ epidemiology and significance of these a wide variety of animals (Longbottom fines much about the biology of the relatively rare isolates (Peterson et al., and Coulter, 2003). Although anti­ lineage. The challenges to understanding 1990; An et al., 1992; Farencena et al., biotic chemotherapy is quite effective in and preventing chlamydial disease are 1997). Chlamydial plasmids are non­ treating diagnosed primary uncompli­ amplified by the difficulties of work­ conjugative and nonintegrative. They cated ocular and urogenital chlamydial ing with an obligate intracellular bacte­ do not encode antibiotic resistance infections, prevention of the serious rial species, in which the development genes and do not show signs of genetic pathological sequelae, which can de­ of a practical system of genetic manipu­ flexibility. This plasmid and the equally velop after infection, and prevention lation is only in its infancy. (Binet and curious chlamydiaphage found in some of reinfection are much more difficult Maurelli, 2009; Kari et al., 2011). strains (Storey et al., 1989) represent tasks (Byrne, 2010). Most chlamydial the entire complement of extrachro­ mosomal elements carried by Chla- infections are asymptomatic and thus The chlamydial plasmid mydia spp. There is a single example of go undiagnosed and untreated, which In most microbial systems, autono­ an integrated plasmid in Chlamydia suis can lead to chronic inflammation and mously replicating plasmids function as (Dugan et al., 2004), but this is clearly irreversible tissue and organ pathology. tools of genetic variation and are built the exception and not the rule. The Journal of Experimental Medicine In some countries and communities, with replicative backbones associated Nucleotide sequence identity among limited access to medical care and in­ with a variety of functional cassettes plasmids from Chlamydia spp. ranges fection screening technologies further encoding niche-specific genes or gene from 69 to 99%, with very high identity hampers timely diagnosis and treat­ sets. Often these plasmids can be hori­ within C. trachomatis (Thomas et al., ment (Centers for Disease Control zontally transferred, which leads to 1997; Seth-Smith et al., 2009). Sequence and Prevention [CDC], 2011). Repeat rapid distribution or reassociation of comparisons suggest that the plasmids infections are common, and the risk phenotypes among strains (Koonin et al., evolved in parallel with the different of developing the serious sequelae of 2001). The spread of antibiotic resis­ chlamydial species. The C. trachomatis ocular (e.g., blindness) or urogenital tance is often a function of sharing plasmid carries a set of five open reading infection (e.g., infertility) increases of plasmids (or other laterally trans­ frames (ORFs), which share identity with multiple infections (Darville and ferred genetic elements) among other­ with episomal maintenance genes com­ Hiltke, 2010). wise susceptible strains. Such plasmid mon to other plasmids, and a set of transfer is a major and evolving prob­ lem in infectious disease worldwide. D.D. Rockey is at the Department of Biomedical © 2011 Rockey This article is distributed under the terms of an Sciences, Oregon State University College of Many serious diseases are directly the Attribution–Noncommercial–Share Alike–No Mirror Sites license Veterinary Medicine, Corvallis, OR 97331 for the first six months after the publication date (see http://www result of virulence factors carried by plas­ .rupress.org/terms). After six months it is available under a Crea- mids, and variation in plasmid structure tive Commons License (Attribution–Noncommercial–Share Alike CORRESPONDENCE 3.0 Unported license, as described at http://creativecommons.org/ D.D.R.: [email protected] is often associated with different disease licenses/by-nc-sa/3.0/).

The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 11 2159-2162 2159 www.jem.org/cgi/doi/10.1084/jem.20112088 However, clinical isolates with a dele­ tion in the target sequence used in some diagnostic kits (the Swedish Vari­ ant) have been identified. This can lead to false negative test results in patients (Unemo and Clarke, 2011). Studies have reported an apparent clonal expan­ sion of C. trachomatis isolates carrying this deletion. It is possible that the in­ ability to detect infections caused by strains carrying the deleted plasmid has contributed to the rapid expansion of the variant strains in patient populations. As mentioned, the lack of routine genetic tools has been a significant bar­ rier to rapid progress in chlamydial re­ search. Manipulation of the chlamydial plasmid is a logical approach to intro­ duce DNA into these organisms, but such efforts have been frustrating and generally unproductive. However, a very recent study describes successful trans­ formation experiments using hybrid shut­ tle vectors built with pBR325 and the entire chlamydial plasmid (Wang et al., 2011). The vectors are propagated in both E. coli and C. trachomatis, and the system was used to generate green fluor­ escent C. trachomatis. This is an exciting and highly significant development in the study of chlamydiae. The chlamydial plasmid appears to be critical for growth in vivo (Russell et al., 2011), but it is not required for growth in vitro. Multiple laboratories have described phenotypic changes as­ sociated with plasmid-deficient strains as compared with closely related wild type strains. Creation of plasmid-deficient isogenic strains began with the careful Figure 1. Chlamydial growth and plasmid structure. The obligately intracellular chlamydiae work of Akira Matsumoto, who devel­ develop within a host vacuole termed the inclusion. The two developmental forms have different functions in growth. Elementary bodies (EB) are infectious but minimally metabolically active, oped a plaque-purification technique for whereas reticulate bodies (RB) grow and divide, but cannot infect. As shown in a magnified reticulate chlamydial strains (Matsumoto et al., body, the chlamydial plasmid has eight ORFs, which encode plasmid maintenance and chlamydia- 1998) and noted that unlike wild-type specific functions. ORF5 encodes Pgp3, which is secreted from the bacterium and the inclusion and C. trachomatis strains, three of his plaque- accumulates in the cytosol of the host cell. Pgp3 is immunogenic in many host species. The chla- purified clones of C. trachomatis did not mydial plasmid is also associated with the accumulation of glycogen granules (purple) in the lumen accumulate glycogen within the inclu­ of the inclusion and, as discussed in the text, functions in virulence in animal model systems. sion. Each glycogen-negative clone was then shown to lack the chlamydial three genes encoding proteins of The chlamydial plasmid also has a plasmid. Subsequent investigations of unknown function, each of which is practical side. It is a common target for plasmid-negative strains (O’Connell chlamydia specific. Human strains of nucleic acid amplification test (NAAT)– and Nicks, 2006; Carlson et al., 2008; Chlamydophila pneumoniae lack plasmid, based diagnostics of human infections O’Connell et al., 2011) confirmed the but equine C. pneumoniae strains and (Fredlund et al., 2004). NAAT-based absence of glycogen and examined other veterinary chlamydial species are tests have been, and remain, a valuable transcription across the chlamydial plasmid positive. aspect of chlamydial disease control. genome relative to wild-type strains.

2160 Emerging role for conserved chlamydial plasmids | Rockey Minireview

Although the precise scope and nature of and it is hypothesized that Pgp3 serves question remains unaddressed, it appears plasmid-chromosome cross-regulation is a similar function. that some aspect of chlamydial plasmid not fully understood, transcription of a Chlamydial vaccine studies are com­ biology is strongly selected for during set of chlamydial chromosomal genes is plicated by the possibility that the de­ successful infection or transmission of negatively affected by the absence of bilitating pathology associated with the pathogen within a host population plasmid. One C. trachomatis gene that is chlamydial ocular and urogenital infec­ (Russell et al., 2011). It is likely that this down-regulated in the plasmid-negative tions represents collateral damage of competitive advantage tips the scale to­ strains is glgA, a glycogen biosynthetic innate and/or adaptive immune re­ ward pathogenesis in human infections. gene. Thus, it is hypothesized that ele­ sponses (Rockey et al., 2009; Farris and In addition, as repeated and/or persistent ments encoded by the plasmid regulate, Morrison, 2011). Protection, in contrast, chlamydial infections are strongly asso­ and perhaps directly participate in, is thought to be serovar or genovar ciated with severe pathology, it is likely the process of glycogen accumulation specific, but neither the mechanisms of that inflammatory responses elicited by within chlamydial inclusions. pathogenesis nor the mechanisms of the bacterium are associated with serious protection have been clearly elucidated. disease sequelae. Thus, the plasmid may Linking disease and vaccines Several candidate protective antigens have encode or control expression of proteins to the chlamydial plasmid been explored (Rockey et al., 2009), but that are responsible for successful bac­ Subsequent in vivo studies using plasmid- no molecule has emerged as a clear and terial growth and/or of the stimulation deficient strains demonstrated that the practical choice as a single protective of a deleterious immune response. The plasmid is a virulence factor that signifi­ antigen. Work in the mouse system sug­ search for deleterious chlamydial antigens cantly impacts clinical disease in mouse gests that delivery of antigens, or a com­ has a long history, and genus-common model systems. For example, although bination of antigens, is required to antigens are thought to be major players plasmid-negative strains grew similarly generate a protective response. Indeed, in this pathogenetic response. Perhaps a to isogenic wild-type strains, they failed to a live attenuated veterinary chlamydial major immunopathogenetic determinant activate Toll-like receptor 2–dependent vaccine, generated via chemical muta­ is encoded by the plasmid, or the gene immune responses either in vivo or genesis, is commercially available and products encoded/controlled by the in vitro (O’Connell et al., 2011). Other routinely used to reduce the incidence of plasmid allow persistence to a level that work indicated that plasmid-negative abortion in sheep (Burall et al., 2009). facilitates deleterious responses to other C. trachomatis strains had significantly In this issue of the Journal of Experi- chlamydial macromolecules. Under the higher 50% infective dose values relative mental Medicine, Kari et al. describe ex­ latter model, absence of the plasmid to a wild-type matched isolate (Carlson periments demonstrating that a plasmid- would allow the bacterium to be cleared et al., 2008). The plasmid-deficient strains deficient C. trachomatis strain is both before an immunopathogenic response also functioned as successful attenuated avirulent in ocular infections of ma­ can be generated or amplified. Alterna­ live vaccines in mice, wherein prior caques and functions as a live attenuated tively, glycogen accumulation might be infection with plasmid-negative strains vaccine in that system. These fascinat­ a metabolic equivalent of a virulence limited the pathology associated with a ing studies conclusively demonstrate factor, providing energy stores as chla­ subsequent infection by wild-type strains that the plasmid functions as a virulence mydiae fight to grow and survive in a (O’Connell et al., 2007). factor in C. trachomatis ocular infections, hostile host environment. The near universal carriage of this consistent with the work conducted in Although the mechanisms of atten­ single plasmid by C. trachomatis has led the genital tract in the murine model uated virulence and/or of immune pro­ to speculation about its importance in system. In addition to the colonization tection are not clearly defined by Kari human chlamydial infections. Early studies, Kari et al. demonstrated that et al., this study is a seminal contribu­ work demonstrated that chlamydia- three of the six animals infected with tion to the field of chlamydia patho­ infected individuals produce antibodies the plasmid-negative strain were com­ genesis and forms the basis for continued specific for at least one plasmid-encoded pletely protected against challenge by an exploration of the role of the plasmid protein, Pgp3, which is encoded by isogenic and virulent plasmid-positive in the chlamydial infectious process. ORF5 (Comanducci et al., 1994). Fur­ strain. Analysis of the major histocom­ Future studies, perhaps encouraged by ther work explored the possible utility patibility complex genotypes of each this work, will need to address the of Pgp3 as a vaccine candidate (Donati animal demonstrated that all three of challenging issues of antigenic variation et al., 2003). Recent work has shown the solidly protected animals shared a and host genetic variation in designing that native Pgp3 is a trimeric molecule common M1 haplotype, suggesting a vaccine strategies. that is transported to the host cell cytosol potential role for CD4+ T cells in the during the infectious process (Li et al., observed differential protection. Richard Morrison of the University of Arkansas for 2008; Chen et al., 2010; Fig. 1). Other What role does the plasmid play in Medical Sciences is acknowledged for critical reading of this manuscript. Editorial comments were also chlamydial proteins are delivered to the chlamydial infections and why is it main­ provided by members of my laboratory. cytosol, where they function to turn tained as an extrachromosomal element The Rockey laboratory is supported by Public the host cell into a “chlamydia factory,” in this pathogen? Although the latter Health Service awards AI069214 and AI086469.

JEM Vol. 208, No. 11 2161 REFERENCES the spread of chlamydial infection from the induce immune pathology and protect against An, Q., G. Radcliffe, R. Vassallo, D. Buxton, W.J. lower to the upper genital tract in C3H/HeN oviduct disease. J. Immunol. 179:4027–4034. O’Brien, D.A. Pelletier, W.G. Weisburg, J.D. mice. Vaccine. 21:1089–1093. http://dx.doi O’Connell, C.M., Y.M. AbdelRahman, E. Green, Klinger, and D.M. Olive. 1992. Infection with .org/10.1016/S0264-410X(02)00631-X H.K. Darville, K. Saira, B. Smith, T. Darville, a plasmid-free variant Chlamydia related to Dugan, J., D.D. Rockey, L. Jones, and A.A. Andersen. A.M. Scurlock, C.R. Meyer, and R.J. Belland. Chlamydia trachomatis identified by using mul­ 2004. Tetracycline resistance in Chlamydia suis 2011. Toll-like receptor 2 activation by tiple assays for nucleic acid detection. J. Clin. mediated by genomic islands inserted into Chlamydia trachomatis is plasmid dependent, Microbiol. 30:2814–2821. the chlamydial inv-like gene. Antimicrob. Agents and plasmid-responsive chromosomal loci are Binet, R., and A.T. Maurelli. 2009. Transformation Chemother. 48:3989–3995. http://dx.doi.org/10 coordinately regulated in response to glucose and isolation of allelic exchange mutants of .1128/AAC.48.10.3989-3995.2004 limitation by C. trachomatis but not by C. mu- Chlamydia psittaci using recombinant DNA Farencena, A., M. Comanducci, M. Donati, G. Ratti, ridarum. Infect. Immun. 79:1044–1056. http:// introduced by electroporation. Proc. Natl. and R. Cevenini. 1997. Characterization of a dx.doi.org/10.1128/IAI.01118-10 Acad. Sci. USA. 106:292–297. http://dx.doi new isolate of Chlamydia trachomatis which lacks Peterson, E.M., B.A. Markoff, J. Schachter, and .org/10.1073/pnas.0806768106 the common plasmid and has properties of L.M. de la Maza. 1990. The 7.5-kb plasmid Burall, L.S., A. Rodolakis, A. Rekiki, G.S. biovar trachoma. Infect. Immun. 65:2965–2969. present in Chlamydia trachomatis is not es­ Myers, and P.M. Bavoil. 2009. Genomic Farris, C.M., and R.P. Morrison. 2011. sential for the growth of this microorgan­ analysis of an attenuated Chlamydia abortus Vaccination against Chlamydia genital in­ ism. Plasmid. 23:144–148. http://dx.doi.org/ live vaccine strain reveals defects in cen­ fection utilizing the murine C. muridarum 10.1016/0147-619X(90)90033-9 tral metabolism and surface proteins. Infect. model. Infect. Immun. 79:986–996. http://dx Pickett, M.A., J.S. Everson, P.J. Pead, and I.N. Immun. 77:4161–4167. http://dx.doi.org/10 .doi.org/10.1128/IAI.00881-10 Clarke. 2005. The plasmids of Chlamydia tra- .1128/IAI.00189-09 Fredlund, H., L. Falk, M. Jurstrand, and M. Unemo. chomatis and Chlamydophila pneumoniae (N16): Byrne, G.I. 2010. Chlamydia trachomatis strains and 2004. Molecular genetic methods for di­ accurate determination of copy number and the virulence: rethinking links to infection preva­ agnosis and characterisation of Chlamydia paradoxical effect of plasmid-curing agents. lence and disease severity. J. Infect. Dis. 201:S126– trachomatis and Neisseria gonorrhoeae: impact Microbiology. 151:893–903. http://dx.doi.org/ S133. http://dx.doi.org/10.1086/652398 on epidemiological surveillance and interven­ 10.1099/mic.0.27625-0 Carlson, J.H., W.M. Whitmire, D.D. Crane, tions. APMIS. 112:771–784. http://dx.doi.org/ Rockey, D.D., J. Wang, L. Lei, and G. Zhong. L. Wicke, K. Virtaneva, D.E. Sturdevant, 10.1111/j.1600-0463.2004.apm11211-1205.x 2009. Chlamydia vaccine candidates and tools J.J. Kupko III, S.F. Porcella, N. Martinez- Kari, L., M.M. Goheen, L.B. Randall, L.D. for chlamydial antigen discovery. Expert Rev. Orengo, R.A. Heinzen, et al. 2008. The Taylor, J.H. Carlson, W.M. Whitmire, D. Vaccines. 8:1365–1377. http://dx.doi.org/ Chlamydia trachomatis plasmid is a transcrip­ Virok, K. Rajaram, V. Endresz, G. McClarty, 10.1586/erv.09.98 tional regulator of chromosomal genes and a et al. 2011. Generation of targeted Chlamydia Russell, M., T. Darville, K. Chandra-Kuntal, virulence factor. Infect. Immun. 76:2273–2283. trachomatis null mutants. Proc. Natl. Acad. Sci. B. Smith, C.W. Andrews Jr., and C.M. http://dx.doi.org/10.1128/IAI.00102-08 USA. 108:7189–7193. http://dx.doi.org/10 O’Connell. 2011. Infectivity acts as in vivo Centers for Disease Control and Prevention (CDC). .1073/pnas.1102229108 selection for maintenance of the chlamydial 2011. CDC Grand Rounds: Chlamydia pre­ Koonin, E.V., K.S. Makarova, and L. Aravind. cryptic plasmid. Infect. Immun. 79:98–107. vention: challenges and strategies for reducing 2001. Horizontal gene transfer in prokaryotes: http://dx.doi.org/10.1128/IAI.01105-10 disease burden and sequelae. MMWR Morb. quantification and classification. Annu. Rev. Seth-Smith, H.M., S.R. Harris, K. Persson, P. Mortal. Wkly. Rep. 60:370–373. Microbiol. 55:709–742. http://dx.doi.org/ Marsh, A. Barron, A. Bignell, C. Bjartling, Chen, D., L. Lei, C. Lu, A. Galaleldeen, P.J. Hart, 10.1146/annurev.micro.55.1.709 L. Clark, L.T. Cutcliffe, P.R. Lambden, and G. Zhong. 2010. Characterization of Li, Z., D. Chen, Y. Zhong, S. Wang, and G. et al. 2009. Co-evolution of genomes and Pgp3, a Chlamydia trachomatis plasmid- Zhong. 2008. The chlamydial plasmid- plasmids within Chlamydia trachomatis and encoded immunodominant antigen. J. Bacteriol. encoded protein pgp3 is secreted into the the emergence in Sweden of a new variant 192:6017–6024. http://dx.doi.org/10.1128/JB cytosol of Chlamydia-infected cells. Infect. strain. BMC Genomics. 10:239. http://dx.doi. .00847-10 Immun. 76:3415–3428. http://dx.doi.org/ org/10.1186/1471-2164-10-239 Collingro, A., P. Tischler, T. Weinmaier, T. 10.1128/IAI.01377-07 Storey, C.C., M. Lusher, and S.J. Richmond. 1989. Penz, E. Heinz, R.C. Brunham, T.D. Read, Longbottom, D., and L.J. Coulter. 2003. Animal Analysis of the complete nucleotide sequence P.M. Bavoil, K. Sachse, S. Kahane, et al. chlamydioses and zoonotic implications. J. of Chp1, a phage which infects avian Chlamydia 2011. Unity in variety - the pan-genome of Comp. Pathol. 128:217–244. http://dx.doi psittaci. J. Gen. Virol. 70:3381–3390. http:// the Chlamydiae. Mol. Biol. Evol. In press. .org/10.1053/jcpa.2002.0629 dx.doi.org/10.1099/0022-1317-70-12-3381 Comanducci, M., R. Manetti, L. Bini, A. Matsumoto, A., H. Izutsu, N. Miyashita, and Thomas, N.S., M. Lusher, C.C. Storey, and I.N. Santucci, V. Pallini, R. Cevenini, J.M. Sueur, M. Ohuchi. 1998. Plaque formation by Clarke. 1997. Plasmid diversity in Chlamydia. J. Orfila, and G. Ratti. 1994. Humoral im­ and plaque cloning of Chlamydia tracho- Microbiology. 143:1847–1854. http://dx.doi mune response to plasmid protein pgp3 in matis biovar trachoma. J. Clin. Microbiol. .org/10.1099/00221287-143-6-1847 patients with Chlamydia trachomatis infection. 36:3013–3019. Unemo, M., and I.N. Clarke. 2011. The Swedish Infect. Immun. 62:5491–5497. O’Connell, C.M., and K.M. Nicks. 2006. A plas­ new variant of Chlamydia trachomatis. Curr. Darville, T., and T.J. Hiltke. 2010. Pathogenesis mid-cured Chlamydia muridarum strain dis­ Opin. Infect. Dis. 24:62–69. http://dx.doi.org/ of genital tract disease due to Chlamydia tracho- plays altered plaque morphology and reduced 10.1097/QCO.0b013e32834204d5 matis. J. Infect. Dis. 201:S114–S125. http://dx infectivity in cell culture. Microbiology. Wang, Y., S. Kahane, L. Cutcliffe, R. Skilton, P. .doi.org/10.1086/652397 152:1601–1607. http://dx.doi.org/10.1099/ Lambden, and I. Clarke. 2011. Development of a Donati, M., V. Sambri, M. Comanducci, K. Di mic.0.28658-0 transformation system for Chlamydia trachomatis: Leo, E. Storni, L. Giacani, G. Ratti, and R. O’Connell, C.M., R.R. Ingalls, C.W. Andrews restoration of glycogen biosynthesis by acquisi­ Cevenini. 2003. DNA immunization with Jr., A.M. Scurlock, and T. Darville. 2007. tion of a plasmid shuttle vector. PLoS Pathog. 7: pgp3 gene of Chlamydia trachomatis inhibits Plasmid-deficientChlamydia muridarum fail to e1002258. 10.1371/journal.ppat.1002258.

2162 Emerging role for conserved chlamydial plasmids | Rockey