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INFECTION AND IMMUNITY, Apr. 2004, p. 1843–1855 0019-9567/04/$08.00ϩ0 DOI: 10.1128/IAI.72.4.1843–1855.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.
MINIREVIEW
Chlamydial Persistence: beyond the Biphasic Paradigm Richard J. Hogan,1,2 Sarah A. Mathews,1 Sanghamitra Mukhopadhyay,3 James T. Summersgill,3 and Peter Timms1,2* Infectious Diseases Program1 and Cooperative Research Centre for Diagnostics,2 School of Life Sciences, Queensland University of Technology, Brisbane, Australia, and Division of Infectious Diseases, Department of Medicine, University of Louisville, Louisville, The chlamydiae are an evolutionarily distinct group of eu- that 146 million people have trachoma due to ocular infection bacteria sharing an obligate intracellular lifestyle and a unique by C. trachomatis serovars A to C and that 4.9 million of these developmental cycle that has been well characterized under are totally blind (121). Ascending infection by serovars D to K favorable cell culture conditions. This cycle begins when infec- of the female upper genital tract, known as pelvic inflammatory tious, metabolically inert elementary bodies (EB) attach to and disease, causes salpingitis, which in turn leads to fibrosis and stimulate uptake by the host cell. The internalized EB remains scarring of the fallopian tubes, and eventual complications of within a host-derived vacuole, termed an inclusion, and differ- ectopic pregnancy and tubal infertility (22). C. trachomatis entiates to a larger, metabolically active reticulate body (RB).
originating from the genital tract is also associated with reac- The RB multiplies by binary fission, and after 8 to 12 rounds of tive arthritis, which develops in 1 to 3% of patients after genital multiplication, the RB differentiate to EB asynchronously (78).
chlamydial infection (123). C. pneumoniae, which can also dis- At 30 to 84 h postinfection (PI), depending primarily on the seminate from the site of the initial infection (74), has been infecting species, EB progeny are released from the host cell to associated with cardiovascular disease (62, 102) and late-onset Alzheimer’s disease (4). In addition, unresolved respiratory C. Shortly after Moulder (79) definitively reported the bacterial pneumoniae infection may contribute to the pathogenesis of nature of chlamydiae in 1966, the genus Chlamydia was estab- chronic inflammatory lung diseases, such as asthma (43) and lished (86) and divided into two species, Chlamydia trachomatis chronic obstructive pulmonary disease (15). Similar Chlamy- and Chlamydia psittaci (85). Chlamydia pneumoniae (42) and dia-associated chronic diseases and their sequelae occur in Chlamydia pecorum (34), formerly known as strains of C. many animals, for example, trachoma-like blindness (25) or psittaci, were designated as distinct species in 1989 and 1992, infertility (71) in koalas and polyarthritis in sheep (117).
respectively. More recently, a new taxonomy was proposed that Recurrent chlamydial disease may result from either re- increases the number of species in the family Chlamydiaceae to peated infections or persistence of the organism after unre- nine and groups these species into two genera (31). However, solved infections. Indeed, the high incidence of chlamydial this review will not use the emended taxonomy, since much infections and transient immunity typically observed after in- debate continues on the issue (106).
fection (90) present difficulties in differentiating between per- Chlamydial species cause widespread infections in humans.
sistent infection and reinfection. Nonetheless, characterization C. trachomatis serovars D to K are considered the world’s most of the in vitro persistent phase of chlamydiae and multiple lines common sexually transmitted bacterial pathogens (40) and, of in vivo evidence suggest that chlamydiae persist in an altered following vertical transmission through an infected birth canal, form during chronic disease. This review will provide an up- cause neonatal conjunctivitis (105) and pneumonia (12). Re- date on chlamydial persistence, focusing on recent insights that spiratory infection with C. pneumoniae occurs in almost every- have been obtained into the molecular basis of this important one during his lifetime (120). C. pneumoniae is estimated to cause an average of 10% of community-acquired pneumonia cases and 5% of bronchitis and sinusitis cases (61). In addition, avian strains of C. psittaci have long been known to cause CHLAMYDIAL PERSISTENCE IN VITRO
Chlamydial persistence has been described as a viable but In addition to these acute chlamydial infections, chlamydiae noncultivable growth stage resulting in a long-term relation- are associated with a range of chronic diseases that are char- ship with the infected host cell (9). Such relationships have acterized by inflammation and scarring and result in significant been established in vitro, usually through deviations from con- damage to the host. The World Health Organization estimates ventional cell culture conditions for productive chlamydial de- velopment. The different in vitro persistence systems often share altered chlamydial growth characteristics, for example, a * Corresponding author. Mailing address: School of Life Sciences, loss of infectivity and the development of relatively small in- Queensland University of Technology, GPO Box 2434, Brisbane 4001, Australia. Phone: 61 7 38642120. Fax: 61 7 38641534. E-mail: clusions containing fewer chlamydiae. In addition, these sys- tems often produce common ultrastructural traits (Table 1).
TABLE 1. Selected in vitro ultrastructural observations of atypical persistent chlamydiae 0–12 h PI, normal EB-to-RB differentiation; enlarged RB; 20–48 h PI, membranes within released as membrane sheets); 36–48 h PI, budding and internal subdivision to normal 10% amino acids (48 h PI), RB-sized swollen containing few RB-like forms with multiple cytoplasmic nucleoid-like masses, ring forms 24 h PI, electron-dense material surrounding inclusions; 72 h PI, inclusions containing normal-sized RB with dense and wavy outer chlamydiae, which were generally enlarged 5–7 days PI, vacuolization and electron-dense 18–32 h PI, small inclusions containing few chlamydiae, including maxi-RB 2–5 times the size of normal RB; 32–44 h PI, lysis of 10–50% of host cells featured either (i) protoplast-like bodies, or (iii) dense, 100- nm-diameter oval bodies bounded by single 48 h postsplit, 10% of inclusions were either proportion of aberrant RB 4–5 times the 5 h after transfer, mosaic-like display of RB in a Similar development of abnormal forms was reported for infections (with or without cyclohexamide) with C. trachomatis serovar E or C. psittaci MN/Cal-10, 6BC, b Moulder (78) proposed that the strain had been from the lymphogranuloma venereum biovar (serovars L1 to L3).
Many studies have described enlarged, pleomorphic RB that tis serovars had differential requirements for specific amino are inhibited in binary fission and their differentiation to EB acids in McCoy cell infections that correlated with their re- but nevertheless continue to accumulate chromosomes. These spective anatomical sites of origin. Of particular note, ocular changes are generally reversible upon removal of the growth (A to C) but not oculogenital (D to I) serovars required tryp- inhibitory factor. However, despite the general similarities, tophan for normal growth (2). However, a recent study of significant differences in growth and ultrastructural character- HeLa cell infections with reference strains representing all C. istics have also been reported among different systems or trachomatis serovars failed to reproduce this finding, since the growth of all serovars was inhibited in the absence of trypto- Antibiotic-induced persistence. Many early in vitro studies
phan (33). Instead, the tissue tropisms correlated with an in- described abnormal chlamydial development after exposure to dole-rescuable phenotype, since genital (D to K and L1 to L3) antibiotics. In general, agents that target bacterial protein or but not ocular (A to C and Ba) serovars growing in tryptophan- RNA synthesis can inhibit chlamydial differentiation either deficient medium were able to generate tryptophan from ex- from EB to RB or from RB to EB, depending on when they are ogenous indole and therefore recover their infectivities (33).
added to an in vitro infection, whereas those that target DNA This finding was a crucial contribution towards establishing the or peptidoglycan synthesis specifically inhibit RB-to-EB differ- in vivo relevance of IFN-␥-mediated persistence.
entiation (78). For example, 10 ␮g of erythromycin/ml, which Depletion of nutrients other than amino acids from cell reduces ribosome activity, inhibited C. trachomatis serovar A culture medium can also induce persistence. For example, C. EB-to-RB differentiation in McCoy cells when added within trachomatis serovar L2 in McCoy cells became reversibly per- 12 h PI (24). When the erythromycin was applied at later times sistent in response to the removal of glucose from the cell PI, infections were established featuring enlarged RB that culture medium, temporarily losing infectivity and showing could not differentiate to EB (24). In contrast, early addition of abnormal morphology comparable to that seen during amino 200 U of penicillin/ml, which targets peptidoglycan cross-link- acid depletion (45). However, the major focus of recent re- ing, had no effect on C. psittaci MN/Cal-10 development in L search into deficiency-induced persistence has been iron de- cells up to 12 h PI, but from that time onwards, the RB became privation (3, 46, 95). Exposure of C. trachomatis serovar E-in- enlarged and progressively more aberrant (69) (Table 1).
fected polarized endometrial epithelial (HEC-1B) cells to 100 Upon transfer to penicillin-free medium, the productive devel- ␮M concentrations of the iron chelator deferoxamine mesylate opmental cycle resumed and normal RB were produced by (DAM) inhibited infectivity and caused significant morpholog- budding from and endosporulation-like subdivision of en- ical changes in the chlamydiae that were generally distinct from those observed in other persistence systems (95) (Table More recently, exposure of C. pneumoniae AR-39 infections 1). The persistence of C. pneumoniae TW-183 in HEp-2 cells in HeLa cells to 50 ␮g of ampicillin/ml led to the development was similarly induced by exposure to 30 ␮M DAM and main- of aberrant, giant RB (122). Interestingly, when directly com- tained for as long as 6 days, although not all of the morpho- pared to C. trachomatis infections subjected to the same con- logical features described for the C. trachomatis cultures were ditions, the persistent C. pneumoniae infections were extremely observed (3). The addition of iron-saturated transferrin led to inefficient in reactivation after the removal of ampicillin (122).
the recovery of infectivity and productive development for The explanation for this result is unclear at present, since both species, thus supporting depleted host cell iron pools as observations of C. pneumoniae exposed to gamma interferon the cause of the changes (3, 95). These studies support the (IFN-␥) or deprived of tryptophan (72) (see below) argue hypothesis that fluctuating iron levels, for example under the against slow recovery from persistence as a distinguishing fea- influence of estradiol in endometrial tissue (57), may contrib- ture of this species. These examples and many other observa- ute to the outcome of chlamydial infections in vivo.
tions of persistence induced in vitro by various antibiotics (re- Cytokine-induced persistence. Exposure of in vitro chlamyd-
viewed in reference 9) indicate that inadequate antimicrobial ial infections to cytokines, particularly IFN-␥, provides a sys- therapy may allow chlamydiae to persist in vivo.
tem of indirect deficiency-induced persistence that could plau- Deficiency-induced persistence. In contrast to persistence
sibly reflect in vivo events. In early studies, IFN-␥ was induced by antibiotics, the depletion of essential nutrients from identified as the active component in supernatant fluids from cell culture medium temporarily arrests the growth of both stimulated T cells that inhibited replication of C. psittaci 6BC chlamydiae and their host cells until the missing nutrients are in fibroblast (17) and macrophage (101) cultures that had been replaced (78). In the first ultrastructural characterization of preexposed to the supernatant. Preexposure of epithelial cells deficiency-induced persistent chlamydiae, progressive deple- for 24 h to high concentrations of IFN-␥ inhibited inclusion tion of all amino acids caused increasingly abnormal develop- formation by C. trachomatis serovar L2 (111), C. psittaci 6BC ment of C. trachomatis serovar L2 in McCoy cells (Table 1), (18), and C. pneumoniae BAL-37 (118). However, lower IFN-␥ with partial recovery of particle size after reintroduction of levels only partially restricted chlamydial development (18, amino acids (26). Induction of persistence in the same Chlamy- dia-host cell system by blood plasma concentrations of amino Persistent chlamydial infections are induced by exposing cul- acids (45) suggested that amino acid levels could directly in- tures to moderate IFN-␥ levels, usually following infection. In fluence chlamydial development in vivo.
this way, persistence was established for C. trachomatis serovar Minimum requirements for individual amino acids are likely A in HeLa cells at IFN-␥ levels as low as 0.2 ng (2.4 U)/ml (7) to be even more important than total amino acid concentra- and for C. pneumoniae A-03 in HEp-2 cells at a level of 25 tions in determining the outcome of chlamydial infections in U/ml (87). Persistence of C. trachomatis serovar A was main- vivo. Allan and Pearce (2) reported that selected C. trachoma- tained for several weeks (10). Ultrastructurally, the IFN-␥- induced persistent chlamydiae were enlarged and aberrant (7, a loss of infectivity (1, 59). These observations suggested that the 87) (Table 1). In C. trachomatis serovar A, there was also chlamydiae were surviving in a viable but culture-negative state.
evidence of budding and endopolygeny, the production of multi- The addition of neither tryptophan nor antibodies against IFN-␥ ple progeny from a single enlarged form, during resumption of (nor antibodies against tumor necrosis factor alpha and IFN-␣ for productive infection after removal of IFN-␥ from the cultures C. trachomatis) (59) counteracted the chlamydial growth inhibi- (10). These morphological observations were consistent with tion (1, 59). This finding, coupled with the unique morphological those from other persistence induction systems (26, 69). However, characteristics of this model (Table 1), led to the suggestion that a direct comparison of IFN-␥- and amino acid depletion-induced a cytokine-independent mechanism is at least partially responsi- persistent C. trachomatis serovars E and L2 in HeLa cells revealed ble for monocyte-induced persistence (59). The mechanism is also different growth characteristics between the two systems, since thought to be oxygen independent (100), but its precise nature only IFN-␥-exposed cultures showed decreases in inclusion size and the number of infected cells (52).
Phage infection of chlamydiae. Observations that lytic infec-
The most important mechanism underlying the effects of tion by naturally occurring bacteriophages can alter chlamydial IFN-␥ on chlamydial growth in cultured human cells is trypto- development in vitro suggest yet another persistence induction phan depletion through activation of the host tryptophan-de- mechanism. An early report of Chp1 infection of C. psittaci grading enzyme indoleamine 2,3-dioxygenase (IDO). Byrne et N352 described the formation of enlarged, distended RB (98).
al. (18) showed that growth restriction of C. psittaci 6BC in More recently, Hsia et al. (49) thoroughly characterized the human epithelial cells preexposed for 24 h to 20 ng (240 U) of ultrastructural effects of ⌽CPG1 infection on C. psittaci GPIC.
IFN-␥/ml could be reversed by the addition of tryptophan The maxi-RB observed had the basic characteristics of aber- following infection. IDO induction was later confirmed to be rant bodies seen in other cell culture models of persistence and the major mechanism of IFN-␥-mediated persistence for C. were of a similar size to the distended RB induced by Chp1 trachomatis serovar A in HeLa cells (6) and C. pneumoniae A-03 in aortic smooth muscle cells (88). Recently, tryptophan If phage-induced chlamydial persistence represents an in depletion provided an important link between IFN-␥ and dif- vivo reality, it will only be relevant to certain chlamydial ferential tissue tropisms among C. trachomatis serovars. Cald- strains, since infecting phage was evident in neither the major- well and colleagues (20) showed that, in agreement with the ity of complete genome sequences nor an additional analysis of previous study of direct tryptophan depletion (33) (see above), six clinical C. pneumoniae isolates (55). However, the C. pneu- C. trachomatis serovar D, I, or L2 but not serovar A in HeLa moniae AR-39 genome sequence revealed that this strain is cells displayed the indole-rescuable phenotype after exposure infected by a phage, ⌽Cpn1/⌽AR-39 (96), that is very similar (24 h preexposure for serovar L2) to 5 ng (60 U) of IFN-␥/ml.
to ⌽CPG1 at the nucleotide level (5). Interestingly, a recent Interplay between the IFN-␥ concentration and other fac- seroepidemiological study showed that seropositivity to tors may affect the outcome of a chlamydial infection in vivo.
⌽Cpn1/⌽AR-39 Vp1 antigen correlated more strongly with Such factors could include the susceptibility of the infecting the presence of aortic abdominal aneurysm than did seropos- strain to IFN-␥-mediated inhibition (76) and the activity of itivity to C. pneumoniae AR-39 EB (55). One of the authors’ other cytokines such as tumor necrosis factor alpha (112, 118) conclusions was that phage-bearing strains may become per- and interleukin-1 (21) that may synergistically enhance the sistent more readily in vivo, resulting in lower cultivability.
effects of IFN-␥ on chlamydial growth. In the case of trypto- There is the intriguing possibility that ⌽Cpn1/⌽AR-39 itself phan depletion as the mechanism of inhibition, additional fac- could be at least partially responsible for an increased ten- tors such as the availability of exogenous indole and ability of dency of C. pneumoniae AR-39 to become persistent. Studies the infecting strain to use it (20, 33) and the IDO expression are warranted to determine whether ⌽Cpn1/⌽AR-39 can af- level of the host cell type (103) may also contribute. However, fect C. pneumoniae AR-39 growth in a similar way to the despite the evident importance of tryptophan catabolism, induction of C. psittaci GPIC maxi-RB formation by ⌽CPG1 other mechanisms such as the inducible nitric oxide synthase and whether some maxi-RB and inclusions are indeed able to effector pathway and iron deprivation could also be attribut- persist (escaping phage-induced lysis).
able to IFN-␥, thus adding to the potential complexity of the in Continuous infections. In contrast to all other persistence
vivo situation (50). In addition, the relative importance of systems, continuous cultures become spontaneously persistent these different mechanisms is likely to vary among host species.
when both chlamydiae and host cells are free to multiply in the For example, inducible nitric oxide synthase induction seems absence of stresses (even spontaneous monocyte persistence to play a central role in IFN-␥-dependent inhibition of exper- must involve unidentified stresses on the chlamydiae). Contin- imental chlamydial infection in mice, whereas IDO induction uous cultures are characterized by cycles consisting of several in response to chlamydial infection is yet to be observed in days of mostly inclusion-free host cell multiplication followed by rapid chlamydial multiplication leading to partial (84) or Monocyte infections. In contrast to other persistence sys-
almost complete (65, 80) host cell destruction. These cycles tems, chlamydiae become spontaneously persistent following continue indefinitely when the cultures are maintained by pe- infection of monocytes. Cell culture infections of freshly iso- riodic washing and growth medium replacement.
lated human monocytes were morphologically characterized Some continuous cultures seem to be established by a ge- for C. trachomatis serovar K (59) and C. pneumoniae strain netic block, as opposed to blocks caused by inhibitors or defi- Kajaani 7 (1). In both studies, no normal RB (only aberrant ciencies, in the progression of a productive cell culture infec- RB) were observed at any time over the monocyte infection tion (65, 84). For example, an early report described period and chlamydial mRNA continued to be detected, despite continuous C. psittaci 6BC infections of Chang’s human liver cells that showed large fluctuations in the percentage of inclu- tence and continuous cultures. The in vitro persistent state that sion-positive host cells among successive passages (84). Isola- chlamydiae enter after experiencing this broad range of hostile tion of chlamydiae and host cells from selected passages re- conditions may, at least in part, represent a general stress vealed chlamydial variants that infected parent Chang’s human response that chlamydiae have evolved to ensure their survival liver cells with higher efficiency than wild-type C. psittaci and under harsh conditions. This hypothesis is supported by the host cell variants that were more resistant to infection by the observation that heat shock of C. trachomatis serovar L2 in chlamydial variants (84). Thus, chlamydiae, their host cells, or BGM cells at 42°C induced a morphological response that was both may undergo population shifts favoring genotypes that similar to those often observed in other persistent cell culture are more suited to a long-term persistent relationship.
systems (53) (Table 1). However, one potential inconsistency More recently, Kutlin et al. (64) established a continuous with this hypothesis is the occasional observation of aberrantly culture system in HEp-2 cells for C. pneumoniae CM-1 and enlarged RB during productive cell culture infections (60, TW-183. The authors suggested that population shifts analo- 122). Could such occurrences indicate localized areas of nutri- gous to those described for C. psittaci (84) and C. trachomatis ent depletion in a cell culture? Further studies with more biovar trachoma (65) occur in the C. pneumoniae system, al- relevant systems, for example, polarized cells and cells other though corresponding characterizations of isolated C. pneu- than epithelial cells, are required to further validate in vitro moniae and host cells have not been reported. Ultrastructur- persistence in relation to in vivo events.
ally, continuous C. pneumoniae infections at 48 h postsplit largely resembled a productive infection, except for a subpopu- EVIDENCE FOR CHLAMYDIAL PERSISTENCE IN VIVO
lation of atypical inclusions that were either partially or fully occupied by aberrantly enlarged RB resembling those de- The recognition that chlamydiae are likely to cause persis- scribed in other persistence systems (63) (Table 1). These tent infections in their hosts dates back to early descriptions of infections also showed reduced sensitivity to azithromycin or latent psittacosis in birds (73). Since these early studies of ofloxacin at levels up to four times the MIC, as indicated by a natural animal infections, the body of evidence that has accumu- significantly slower reduction of inclusion-forming units when lated for the existence of persistence in vivo has come mostly from compared over 6 days to primary infections similarly exposed experimental animal infections and clinical data from human dis- from 0 h PI onwards (64). However, this property is not re- ease. Various characteristics link these in vivo infections to the stricted to continuous cultures or even to established persistent well-studied cell culture models of persistent chlamydial infection.
cultures in general, since a similar effect was observed when Among the most convincing lines of evidence for persistence in established (48 h PI onwards) primary C. trachomatis serovar K vivo are observations of altered morphological forms in vivo, infections of HEp-2 cells were exposed to ofloxacin at up to detection of chlamydial macromolecules in diseased hosts in the four times the MIC over 18 days (30). In that study, the antibiotic absence of cultivability, recurrences that occur when reinfection is itself induced persistence, which in turn was considered to be unlikely, and clinical antibiotic resistance.
responsible for the reduced antimicrobial susceptibility (30).
Electron microscopic visualization in diseased tissues of There is a special case of continuous culture that cannot be morphologically aberrant chlamydial forms resembling those explained even by the genetic block hypothesis. Moulder et al.
observed in vitro (Table 1) indicates that such forms are un- (80) reported continuous C. psittaci 6BC infections of L cells likely to be mere laboratory artifacts. Nanagara et al. (81) from which all isolated chlamydiae and host cells were found to showed that atypical, pleomorphic RB with poorly defined be indistinguishable from their wild-type counterparts. In ad- outer membranes dominated within infected fibroblasts and dition, nine clones taken from cultures with 25% inclusion- macrophages in synovial membrane samples from patients with positive cells were initially inclusion free but subsequently all C. trachomatis-associated reactive arthritis or Reiter’s syn- gave rise to persistently infected populations (80). To explain drome, despite antibiotic therapy. More recently, C. pneu- these observations, the authors proposed that some chlamyd- moniae forms of a similar size to aberrant RB seen in vitro iae in a cryptic form survived each wipeout of host cells and were observed within macrophages in aortic valve samples that every host cell was always infected by either cryptic or from patients with degenerative aortic valve stenosis (113). In productive chlamydiae (80). Moulder (78) has since raised the addition, miniature C. trachomatis forms have been observed possibility that cryptic bodies may be related to single, inclu- in total ejaculate and expressed prostatic secretion samples sion-bound, dense oval bodies observed in C. trachomatis-L from patients with chronic chlamydial prostatitis (70) and in cell cultures (107) (Table 1) that behaved similarly to the C. the oviducts of mice experimentally infected with C. trachoma- psittaci 6BC-L cell cultures (80). However, the precise mor- tis biovar mouse pneumonitis (92). These miniature forms may phology of cryptic chlamydiae remains unknown.
correspond to budding forms seen in vitro (10, 69). However, Despite their relatively protected intracellular niche, chla- observations of ultrastructurally aberrant chlamydiae alone fall mydiae are subject to a variety of insults, particularly in intact short of proving that chlamydiae persist in vivo, since the hosts with competent immune systems. Examples of naturally viability of these particles is uncertain.
occurring insults include shortages of essential nutrients, which The presence of viable but atypical chlamydiae in vivo is may be brought about by host-elaborated factors such as cyto- suggested by the detection of chlamydial macromolecules at kines and hormones, and phage hyperinfection of certain chla- diseased sites in the absence of cultivable organisms. Chlamyd- mydial strains. In addition, antibiotics specifically target chla- ial DNA and antigen are often detected in tubal biopsy spec- mydiae in multiple ways. Apart from these known factors, imens from culture-negative women with postinfectious tubal there are other mechanisms that remain incompletely ex- infertility following antibiotic treatment (91). Even more con- plained, including those underlying monocyte-induced persis- vincingly, persistence of noncultivable chlamydiae in the va- gina, uterus, and oviduct of ewes experimentally infected with cin, and ofloxacin at concentrations above 4 ␮g/ml) (115). This a naturally occurring strain of C. psittaci was documented for indicated the presence of a more global resistance mechanism more than a year by the detection of DNA or antigen in these such as the induction of a persistent phenotype that is refrac- tissues (89). Although the detection of chlamydial DNA or tory to multiple antibiotics, for example, through membrane antigen could reflect the presence of chlamydial cell debris alterations that affect drug intake. In some cases, the explana- remaining from resolved infections, PCR data showing that tion for resistance could be more complex; certain genotypes UV-inactivated C. pneumoniae organisms were cleared far could confer antibiotic resistance by encouraging development more efficiently than live organisms from inoculated mice (74) of the persistent phenotype. Such a scenario seems to occur in argue against long-term persistence of chlamydial macromol- tetracycline-resistant porcine C. trachomatis strains, which pro- ecules. In addition, chlamydial RNA has been detected in the duced large aberrant RB in response to the antibiotic at 2 absence of cultivability in experimental trachoma of primates ␮g/ml (66). Could the gyrA mutations that developed in cell (48) as well as in synovial biopsy samples from patients with culture in response to fluoroquinolone exposure (29) also favor reactive arthritis or Reiter’s syndrome (35, 93). Since RNA is the formation of a persistent phenotype, since alterations to highly labile, its detection in vivo strongly suggests the presence of DNA gyrase could inhibit RB-to-EB differentiation? viable organisms and correlates with similar data indicating viable Taken together, the in vivo data suggest that chlamydiae but culture-negative chlamydiae in vitro (1, 30, 37, 59).
persist in their hosts. However, these studies do not conclu- Experimental and clinical data provide evidence for reacti- sively prove that the chlamydiae persist in an altered form. For vating persistent chlamydiae in vivo. Early reports described example, detection of chlamydial macromolecules in the ab- individuals who had experienced acute ocular C. trachomatis sence of cultivability could also reflect low-grade productive infection as children living in areas of trachoma endemicity infections that are not detectable by the culture methods used.
and showed no more symptoms until they developed active Similarly, although Beatty et al. (10) definitively proved that in trachoma decades after leaving those areas (119), indicating vitro aberrant forms can give rise to productive infections, in that the recurrences were more likely due to reactivations of vivo data indicating reactivation could also represent enhance- persistent infection than to reinfections. Particularly convinc- ment of an inapparent productive infection. A final line of ing evidence of periodic reactivation of persistent infections evidence for altered forms in vivo that will be discussed below comes from long-term studies that described individuals expe- comes from studies that have demonstrated similarities in chla- riencing multiple recurrent infections with chlamydial isolates mydial gene or protein expression trends between persistent cell of the same genotype. In a study of women with genital C. culture systems and tissue samples from sites of chronic disease.
trachomatis infection, Dean et al. (28) demonstrated recur- rences of the original ompA genotype over 2 to 4.5 years, MOLECULAR BASIS OF CHLAMYDIAL PERSISTENCE
despite administration of accepted treatment regimens. Al- though this study was limited by the inability to control for Although our understanding of the molecular basis of chla- reexposure to untreated partners, data from another study mydial persistence is still at an early stage, the rate of progress showed that 10% recurrence of genital C. trachomatis infection in this area has significantly increased in recent years. This has can occur even among subjects who report either abstinence or been made possible primarily by the completion of several 100% condom use following treatment with azithromycin (56).
genome sequencing projects (54, 96, 97, 116) and by recent Preliminary genotyping data from Dean et al. and Hammer- improvements in molecular methodology, in particular, quan- schlag et al. (27, 44) suggested that C. pneumoniae can also titative PCR, microarrays, and proteomics. While persistence persist for many years after initial respiratory infection. Animal is a simple term, there are several systems for chlamydial infection models provide additional evidence for reactivation persistence with a significant number of variables that make of persistent infections in vivo. In mice infected with either C. direct comparison of results difficult. Variables include the trachomatis (125) or C. pneumoniae (67), infections that had infecting chlamydial species and strain, the origin of host cells, become asymptomatic reactivated to productive infections af- the in vitro persistence induction, and times PI chosen for ter treatment with cortisone. Since the reactivation occurred analysis. Despite this complexity, some common themes are specifically after suppression of the immune system, these ob- emerging from the results of molecular studies undertaken to servations supported the hypothesis that immune factors such date (Table 2). The categories described below represent a as IFN-␥ play a significant role in persistence in vivo.
current overview of a rapidly advancing field and are therefore Several studies have reported resistance of chlamydial iso- likely to be modified as further data are generated, particularly lates to antibiotics (reviewed in reference 99). Whether these from global microarray and proteomics studies.
data reflect direct resistance or phenotypic resistance mani- MOMP and cHSP60. Until the recent availability of com-
fested by altered chlamydial forms is unclear. Chlamydiae are plete chlamydial genome sequences, most molecular studies of capable of developing true genotypic resistance to antibiotics persistence focused on relative levels of the major outer mem- in vitro. For example, C. trachomatis serovar L2 mutants iso- brane protein (MOMP) and chlamydial heat shock protein 60 lated from cell culture after several rounds of exposure to (cHSP60). Using immunoelectron microscopy, Beatty et al. (8) various fluoroquinolones consistently showed a point mutation found that exposure of C. trachomatis serovar A infections in in gyrA (encoding DNA gyrase subunit A), suggesting that HeLa cells to 0.5 ng (6 U) of IFN-␥/ml caused cHSP60 levels DNA gyrase is the primary target of these antibiotics (29).
to increase slightly (1.4-fold) and MOMP levels to decrease However, a recent study described C. trachomatis isolates as- twofold, after correcting for the decreased surface-to-volume sociated with treatment failure that were resistant to multiple ratio in enlarged persistent forms. Subsequent immunoblot drugs with diverse molecular targets (doxycycline, azithromy- analyses showed a decreased MOMP-to-cHSP60 ratio over TABLE 2. Postgenomic molecular studies of chlamydial persistence a MOMP was only down-regulated in serovar A.
b Expression of these genes and proteins was normalized in genital serovars but not in serovar A after the addition of indole.
c Only genes that were confirmed by rtRT-PCR to be differentially expressed have been included.
10-day C. trachomatis serovar K infections in either HEp-2 cell C. trachomatis serovar K-infected monocyte cultures (36–38).
cultures exposed to 0.5 ␮g of ciprofloxacin/ml (30) or unex- Finally, two-dimensional (2-D) protein gel electrophoresis posed monocyte cultures (37). In addition, semiquantitative data showed that exposure to 100 U of IFN-␥/ml caused down- reverse transcriptase PCR (sqRT-PCR) analyses demonstrated regulated MOMP expression in C. trachomatis serovar A (108) selectively down-regulated transcription of the ompA gene in but not in serovars D and L2 (108, 109) in HeLa cells. Since the latter two serovars are among the most resistant to IFN-␥ (76), tence systems; all genes studied were strongly expressed during those cultures may have required preexposure at the concen- productive infections of HEp-2 cells used as controls (19, 38).
tration used to become persistent. Indeed, preexposure of In C. trachomatis serovar K-infected monocytes, polA, dnaA, HeLa cells to 50 U of IFN-␥/ml was sufficient to induce per- mutS, and parB transcripts were produced over the whole sistence of C. trachomatis serovar D and down-regulation of 7-day infection period, whereas ftsK and ftsW were not de- ompA, as assessed by microarray and real-time RT-PCR tected after 1 day PI (38). Expression profiles in synovial tissue (rtRT-PCR) (13). Ge´rard et al. (39) recently used rtRT-PCR samples from patients with C. trachomatis-associated reactive to quantitate the relative transcript levels of the three groEL arthritis reflected the in vitro results; polA, dnaA, mutS, and genes (encoding cHSP60 homologues) in C. trachomatis sero- parB were weakly detected but neither ftsK nor ftsW was de- var K-infected monocytes over 7 days. Interestingly, transcrip- tected (38). In C. pneumoniae TW-183-infected HEp-2 cell tion of groEL-2 was markedly higher than that of groEL-1 and cultures exposed to 0.5 ng (6 U) of IFN-␥/ml, polA, dnaA, groEL-3 in monocyte cultures, a trend not seen in HEp-2 cell mutS, and minD expression was steady, whereas that of ftsK control infections (39). However, the data also seemed to sug- and ftsW was absent (19). When the C. pneumoniae infections gest that groEL-1 and groEL-3 expression was strongly down- were exposed to an IFN-␥ concentration (0.15 ng [1.8 U]/ml) regulated in monocyte persistence relative to the controls. In that caused no significant morphological alterations to the addition, many other recent studies, mostly involving C. pneu- chlamydiae compared to unexposed controls, ftsK and ftsW moniae, have reported data contrary to a decreased ompA/ expression was present but significantly attenuated (19). Taken MOMP-to-groEL/cHSP60 ratio, indicating that this may not be together, these studies demonstrated that down-regulated ftsK as universal a marker of persistence as was once thought (47, and ftsW expression occurs in different in vitro persistence models and in vivo and that these genetic alterations can pre- In general, the in vivo data support the decreased ompA/ cede evidence of morphological alterations during the estab- MOMP-to-groEL/cHSP60 ratio described in many cell culture studies of persistent C. trachomatis. Immunoelectron micro- More recent cell culture data have been inconclusive regard- scopic (81) and sqRT-PCR (35, 36, 38) analyses of synovial ing cell division and DNA replication gene expression during tissue samples from patients with C. trachomatis-associated persistence. In our own laboratory (R. J. Hogan, D. A. Good, chronic arthritis demonstrated diminished levels of MOMP S. A. Mathews, S. Mukhopadhyay, J. T. Summersgill, and P.
and ompA transcripts, respectively. In apparent contrast, re- Timms, unpublished data), rtRT-PCR analysis of C. pneu- cent data from similar samples indicate down-regulated ex- moniae A-03-infected HEp-2 cells exposed to 50 U of IFN- pression of all three groEL genes, including the virtual absence ␥/ml revealed strong down-regulation of ftsK but insignificant of groEL-3 transcripts (39). Nonetheless, many studies have differential expression of ftsW. Microarray studies of persistent reported enhanced production of cHSP60-specific antibodies C. trachomatis serovar D in HeLa cell cultures have also pro- in various chronic chlamydial infections (reviewed in reference vided mixed data; exposure of such infections to 100 U of penicillin/ml was associated with up-regulated ftsK and un- Whether an altered MOMP-to-cHSP60 ratio has any signif- changed ftsW expression (82), whereas exposure to IFN-␥ lead icance to chlamydial pathogenesis beyond serving as a marker to unchanged ftsK and down-regulated ftsW expression (13).
of persistence in vivo is a subject of much debate. Beatty et al.
These discrepancies are not surprising, since the precise func- (7) proposed that reduced levels of MOMP, an immunopro- tions of chlamydial FtsK and FtsW are unlikely to be directly tective antigen, could enable chlamydiae to avoid the develop- related. FtsW is a predicted septum-peptidoglycan biosynthetic ment of protective immunity. At the same time, according to protein involved in cell wall formation, whereas FtsK is re- the traditional immunological paradigm of chlamydial patho- quired for chromosome segregation (116). Similarly, DNA rep- genesis, steady or increased cHSP60 levels would promote lication gene expression profiles were varied in the microarray immunopathology through delayed-type hypersensitivity or study of IFN-␥-exposed C. trachomatis, with some genes up- cross-reactivity with HSP60 from either the human host or regulated (dnaB, topA, and xerC) and others down-regulated other pathogens. Another potential explanation for the rela- (dnaA-2, dnlJ, and ihfA) (13).
tive abundance of cHSP60 during C. trachomatis persistence is Energy metabolism. Using a similar experimental design to
its function as a stress response chaperone (77), which is likely that of their previous study (38) (see above), Ge´rard and col- to be of particular importance under the conditions that induce leagues (36) studied genes encoding enzymes predicted to be persistence. In support of this view, the production of five involved in energy metabolism in both cultured C. trachomatis- proteins thought to be major heat shock proteins, including infected monocytes and synovial tissue samples from patients cHSP60, was strongly increased as assessed by one-dimen- with C. trachomatis-associated reactive arthritis. Since genes sional gel electrophoresis after heat shock of C. trachomatis encoding enzymes belonging to glycolysis (pyk, gap, and pgk) and the pentose phosphate pathway (gnd and tal) were found Cell division and chromosome replication and partitioning.
to be selectively down-regulated in vitro and in vivo relative to Recent sqRT-PCR investigations have provided molecular in- genes encoding enzymes in the tricarboxylic acid cycle (mdhC sights into the common observation that persistent chlamydiae and fumC), the authors concluded that persistence may be are inhibited in cell division and yet continue to accumulate characterized by a shift from a partial to a full reliance on the chromosomes (Table 1). These studies analyzed expression of host cell for ATP (36). The microarray expression data for genes encoding products predicted to function in DNA repli- genes encoding tricarboxylic acid cycle enzymes in IFN-␥-in- cation (polA, dnaA, and mutS), chromosome partitioning (parB duced persistence of C. trachomatis were mixed. Genes encod- and minD), and cell division (ftsK and ftsW) in various persis- ing 2-oxoglutarate dehydrogenase (sucA, sucB-1, and sucB-2) and succinate thiokinase (sucC and sucD) were down-regu- CPAF. Another immunoavoidance strategy that has been
lated, whereas genes encoding other enzymes in the cycle were identified in both C. trachomatis (126) and C. pneumoniae (32) either up-regulated (fumC and sdhB) or unchanged (mdhC, is the secretion of a chlamydial protease-like activity factor sdhA, and sdhC). There was little evidence of down-regulation (CPAF) into the host cell cytoplasm that cleaves eukaryotic in glycolysis or the pentose phosphate pathway (13). Also in transcription factors required for both major histocompatibil- apparent contrast to the results of Ge´rard et al. (36), 2-D gel ity complex class I and II antigen expression. Recent rtRT- analysis of C. pneumoniae A-03 exposed to 50 U of IFN-␥/ml PCR (R. J. Hogan, S. A. Mathews, S. Mukhopadhyay, J. T.
showed up-regulated Pgk and unchanged Pyk levels (75).
Summersgill, and P. Timms, unpublished data), 2-D gel (110), Tryptophan metabolism. Tryptophan metabolism in relation
and immunoblot (46) analyses of IFN-␥-exposed chlamydial to persistence has recently become an area of intensive re- infections, in addition to immunoblotting of iron-depleted C. search. Using 2-D gel analysis, Shaw and colleagues (108, 109) pneumoniae cultures (46), revealed little or no differential ex- showed that exposure of C. trachomatis serovars A, D, and L2 pression of CPAF between persistent and productive infec- to 100 U of IFN-␥/ml caused significant tryptophan-reversible tions. These data indicated that CPAF production is important up-regulation of the ␣ and ␤ chains of chlamydial tryptophan in both persistent and normal infections. Interestingly, Heuer synthase. Induction of this enzyme is a plausible mechanism by et al. (46) demonstrated inhibited CPAF translocation to the which chlamydiae would counteract tryptophan deficiency in- host (HEp-2) cell cytoplasm during both IFN-␥- and iron de- duced by exposure to IFN-␥. However, another important find- ficiency-induced persistence of C. pneumoniae CWL-029.
ing was the presence of frameshift mutations in the gene en- These authors reasoned that if CPAF is translocated by the coding the ␣ chain, trpA, for serovars A and C and the resultant same mechanism as other chlamydial proteins, and if this synthesis of a truncated ␣ chain by these serovars (109).
mechanism is inhibited during persistence, then the resultant More recent studies (20, 33, 124) have developed the initial general decrease in chlamydial antigen processing and presen- observations of Shaw and colleagues (108, 109) into a para- tation would reduce the requirement for CPAF activity during digm that links the tissue tropisms of C. trachomatis serovars to this phase (46). The absence of CPAF protease activity on host their relative abilities to synthesize tryptophan. Fehlner- cell proteins could also reduce the availability of readily trans- Gardiner et al. (33) cloned and sequenced a section of the portable amino acids (including tryptophan) to chlamydiae and plasticity zone, a highly variable region of the chlamydial ge- therefore contribute to the maintenance of persistence.
nome that contains the trpBA operon encoding both chains of Late genes. Down-regulated expression during persistence
tryptophan synthase, from 15 reference strains representing all of genes and proteins that are specifically expressed late in the human-infecting serovars of C. trachomatis. With the exception productive developmental cycle is a common observation most of serovar B, which was missing the trpBA operon, these au- likely reflecting the inhibited RB-to-EB differentiation that thors concluded that ocular (A to C and Ba) but not genital (D characterizes persistence. A well-known example is the 60-kDa to K and L1 to L3) serovars have the trpA mutation (33).
cysteine-rich protein (CRP), which is abundant in EB enve- Further investigation with several experimental approaches re- lopes. The 60-kDa CRP was either markedly diminished or vealed that this mutation was responsible for the inability of completely absent in early studies of C. trachomatis strains ocular serovars to synthesize tryptophan from indole (33) (see exposed to ␤-lactam antibiotics (23, 104), IFN-␥ (7, 10), or above). Caldwell et al. (20) continued the sequencing approach heat shock (53). Another study reported the absence of the to confirm that this paradigm also applies to clinical isolates of Hc-1 and Hc-2 DNA-binding proteins (involved in chromo- C. trachomatis and used rtRT-PCR to analyze trpBA expres- somal condensation) in IFN-␥-exposed C. trachomatis, al- sion for selected ocular (A) and genital (E) serovars in infec- though interestingly, the 60-kDa CRP was not down-regulated tions of HeLa cells. While both serovars strongly up-regulated (52) (Table 2). Similarly, both persistence microarray studies their trpBA expression in response to 5 ng (60 U) of IFN-␥/ml, carried out to date reported down-regulated hctB (encoding only serovar E trpBA expression returned to original levels Hc-2) expression in C. trachomatis, whereas omcB (encoding following the addition of 100 ␮M indole, providing further the 60-kDa CRP) down-regulation was associated with expo- support for the paradigm that ocular serovars are not indole sure to IFN-␥ (13) but not to penicillin (82). As was the case rescuable (20). The authors concluded that the evolution of for ompA/MOMP, many other studies have also failed to con- genital strains to utilize indole for tryptophan synthesis repre- firm omcB/60-kDa CRP down-regulation in persistent cultures sents an immunoavoidance strategy, since these serovars are (47, 52, 68, 114). Therefore, hctB/Hc-2 down-regulation cur- more likely to survive under IFN-␥ pressure in vivo (20).
rently appears to be a more reliable marker of persistence.
C. pneumoniae does not contain a trpBA operon in its ge- Nonetheless, the penicillin microarray study (82) strongly sup- nome (54) and may therefore have an alternative strategy to ported the late gene shut-down hypothesis, since 14 of the 15 counteract the effects of IDO activity, which this species no most down-regulated genes in that study were subsequently doubt encounters in vivo (103). A recent study revealed that confirmed to be late genes (14, 83) (Table 2). Finally, this respiratory strains of C. pneumoniae possess multiple copies of hypothesis was used to explain selective down-regulation of the tyrP gene, encoding a tyrosine-tryptophan permease, while late-stage-specific type III secretion genes in an sqRT-PCR vascular strains encode only one copy (41). Since the presence study of C. pneumoniae CM-1-infected HEp-2 cell cultures of extra tyrP copies correlated with increased mRNA levels and exposed to 40 ng (480 U) of IFN-␥/ml (114).
higher uptake of the substrate tyrosine in respiratory strains, Microarray analysis: new persistence gene categories iden-
the authors hypothesized that a reduced capacity for amino tified. The recent microarray study of IFN-␥-exposed C. tra-
acid transport may contribute to a greater tendency of vascular chomatis by Belland et al. (13) provided the first published strains to become persistent in vivo (41).
transcriptome-wide data set for chlamydiae in a persistence system. While many of the differentially expressed genes fall tryptophan residues in C. trachomatis serovar L2 than are con- into categories discussed above, other groups were identified tained in C. psittaci GPIC (16).
that have not previously been considered in the context of chlamydial persistence. Many rl and rs genes (encoding ribo- somal proteins) were markedly up-regulated, and none were CONCLUDING REMARKS
significantly down-regulated (13). Early genes that were up- regulated in persistence included euo and the incD-G operon, While the exact molecular mechanism by which chlamydiae although another early gene (oppA-4) was reported to be enter and exit the persistent phase is not yet understood, there down-regulated (13). The euo result was consistent with data is little doubt that this stage plays an important role in chla- from C. trachomatis serovar L2-infected HeLa cells cultured in mydial development. The well-accepted biphasic paradigm of the absence of glucose (51), conditions known to induce per- chlamydial development warrants the addition of a persistent sistence in this species (45). In that study, transcripts from euo phase that represents a critical survival mechanism of this but not from eight other genes of interest were detectable by well-adapted intracellular pathogen. Down-regulated expres- sqRT-PCR (51) (Table 2). Other novel observations from the sion of the major structural protein MOMP was one of the first microarray data included up-regulation of three members of a markers of persistence but has not been confirmed in all sub- gene family encoding phospholipase D-like enzymes and sequent studies on persistence. On closer examination of these down-regulation of four clp and three opp genes, encoding data, most C. trachomatis studies reported down-regulation of proteins involved in proteolysis and peptide transport, respec- ompA/MOMP, whereas the C. pneumoniae studies observed up-regulation of ompA/MOMP. Perhaps this could be ex- Belland et al. (13) used rtRT-PCR to clarify the magnitude plained by different roles played by MOMP in the two species.
of relative changes for 15 genes (Table 2). For example, three In C. pneumoniae, MOMP is apparently less important struc- genes, CT228, euo, and trpB, with up-regulated microarray turally, whereas its role as a porin might be more critical.
changes of a similar magnitude (approximately 6-fold) showed Another example of a species-specific persistence marker rtRT-PCR changes of 12.6-, 28.7-, and 458-fold, respectively relates to the elegant work of Caldwell et al. and Fehlner- (13). The dramatic variation in these figures emphasizes the Gardiner et al. (20, 33), who clearly showed the key role of a importance of quantitative PCR in verifying microarray data, functional tryptophan synthase in the development of C. tra- and perhaps this should include data for genes that show no chomatis experiencing tryptophan depletion. This is also an significant relative change by microarray analysis.
example of an expression pattern that relates to the key met- Regulatory mechanisms in persistence. The gene expression
abolic limitation in a particular system of persistence. Such data described in the above categories imply that many persis- patterns are presumably not a direct effect of persistence but tence gene profiles are transcriptionally regulated. Based on rather the chlamydiae trying to compensate for limitations in their microarray data, Belland et al. (13) proposed the exis- other parts of the same or related pathways.
tence of a chlamydial persistence stimulon that is more com- While most persistence patterns appear complex at this plex than a general stress response. The strongly up-regulated stage of our understanding of chlamydial cell biology, it is euo expression observed during persistence in that study may hoped they can be unraveled by the multiple molecular ap- provide the first insight into the molecular mechanisms of proaches that are currently being pursued, for example RT- regulation, since these authors proposed that Euo may con- PCR, proteomics, and microarrays. Since these methods com- tribute towards silencing late gene expression in persistence plement each other, their combined use will be critical for (13). The precise mechanisms of regulation for other differen- answering biological questions about persistence. The estab- tially expressed gene categories remain unknown. Another un- lishment of gene and protein expression patterns representing answered question is to what degree a persistence stimulon persistence in vitro will allow the development of persistence may vary among persistence systems resulting from different assays to be applied to in vivo samples, resulting in the corre- lation of model systems to clinical reality.
In addition, translational control may contribute to some An important question that remains is whether the persis- observed profiles, at least in IFN-␥-mediated persistence. Be- tence phenotype is simply a survival response by chlamydiae to atty and colleagues (6) proposed that direct control at the a range of unfavorable environmental conditions, with the sub- translational level may explain their observations of steady sequent complex gene expression profiles being the uncon- cHSP60 levels and reduced membrane antigen levels in IFN- trolled outcome. Alternatively, have chlamydiae evolved over ␥-mediated persistence. This proposal is based on gene se- millions of years to deliberately enter the persistent state to quence data revealing that both MOMP and the 60-kDa CRP, maximize evasion of host cell immune mechanisms and in- but not cHSP60, contain significant numbers of tryptophan crease growth and survival opportunities in different host residues and hence require adequate tryptophan levels for niches? Indeed, can we further document the overall preva- their synthesis (6). Brown and Rockey (16) also suggested this lence of this persistent state in vivo? Have different ecotypes of mechanism to potentially explain their observation that expres- Chlamydia (serovars and strains) evolved subtly different ways sion of an unidentified antigen (termed SEP) that localizes to of responding to environmental cues so as to enter this quies- the septum of dividing chlamydiae was severely attenuated in cent phase to enhance survival or transmission? As we con- IFN-␥-exposed C. trachomatis serovar L2 but not in IFN-␥- tinue to better understand the molecular and cellular mecha- exposed C. psittaci GPIC and not after exposure of either nisms of chlamydiae, we will also begin to understand the role strain to ampicillin. If SEP is a peptidoglycan, the inhibition of persistence in this uniquely adapted obligate intracellular may have been due to a biosynthetic enzyme containing more ACKNOWLEDGMENTS
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and Cooperative Research Centre for Diagnostics Postgraduate Re- synthesis of membrane proteins of Chlamydia trachomatis LGV2 serotype.
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laboratory has been supported by NIH grants HL68874 and AI51255.
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