In this context, one of the most rap-idly growing areas of research in vacci-nology is maternal immunization,aimed at transplacental protection viamaternal antibodies. The report byMarchant (1) suggests that underappropriate conditions of prenatalstimulation, this protective net canpotentially be broadened to includethe generation of protective cellularimmunity in the neonate. Furtherresearch into the underlying mecha-nism(s) of prenatal T cell priming inthis and other congenital infectionsmay provide important new clues toguide the development of safe vaccinesthat aim to achieve a comparable end.

1. Marchant, A., et al. 2003. Mature CD8+ T lympho-cyte response to viral infection during fetal life. J. Clin.Invest. 111:1747–1755. doi:10.1172/JCI200317470.

2. Wegmann, T.G., Lin, H., Guilbert, L., and Mos-mann, T.R. 1993. Bidirectional cytokine interac-tions in the maternal-fetal relationship: is suc-cessful pregnancy a Th2 phenomenon? Immunol.Today. 14:353–356.

3. Krishnan, L., Guilbert, L.J., Wegmann, T.G., Belo-sevic, M., and Mosmann, T.R. 1996. T helper 1response against Leishmania major in pregnantC57BL/6 mice increases implantation failure andfetal resorptions. J. Immunol. 156:653–662.

4. Munn, D.H., et al. 1998. Prevention of allogeneicfetal rejection by tryptophan catabolism. Science.281:1191–1193.

5. Roth, I., et al. 1996. Human placental cytotro-phoblasts produce the immunosuppressivecytokine interleukin 10. J. Exp. Med. 184:539–548.

6. Guller, S., and LaChapelle, L. 1999. The role ofplacental Fas ligand in maintaining immuneprivilege at maternal-fetal interface. Semin.Reprod. Endocrinol. 17:39–44.

7. Hilkens, C.M., Kalinski, P., de Boer, M., andKapsenberg, M.L. 1997. Human dendritic cellsrequire exogenous interleukin-12-inducing fac-tors to direct the development of naive T-helpercells toward the Th1 phenotype. Blood.90:1920–1926.

8. Szekeres-Bartho, J., Faust, Z., Varga, P., Szereday,L., and Kelemen, K. 1996. The immunologicalpregnancy protective effect of progesterone ismanifested via controlling cytokine production.Am. J. Reprod. Immunol. 35:348–351.

9. Ulevitch, R.J. 1999. Endotoxin opens the Tollgatesto innate immunity. Nat. Med. 5:144–145.

10. Holt, P.G., and Sly, P.D. 2002. Interactionsbetween RSV infection, asthma, and atopy: unrav-eling the complexities. J. Exp. Med. 196:1271–1275.

11. Rowe, J., et al. 2001. Heterogeneity in diphtheria-tetanus-acellular pertussis vaccine-specific cellu-

lar immunity during infancy: relationship to vari-ations in the kinetics of postnatal maturation ofsystemic Th1 function. J. Infect. Dis. 184:80–88.

12. Goriely, S., et al. 2001. Deficient IL-12(p35) geneexpression by dendritic cells derived from neona-tal monocytes. J. Immunol. 166:2141–2146.

13. Yap, G., Pesin, M., and Sher, A. 2000. IL-12 isrequired for the maintenance of IFN-γ produc-tion in T cells mediating chronic resistance to theintracellular pathogen, Toxoplasma gondii. J. Immunol. 165:628–631.

14. White, G.P., Watt, P.M., Holt, B.J., and Holt, P.G.2002. Differential patterns of methylation of theIFNγpromoter at CpG and non-CpG sites under-lie differences in IFNγ gene expression betweenhuman neonatal and adult CD45RO– T-cells. J. Immunol. 168:2820–2827.

15. Hassan, J., and Reen, D.J. 2001 Human recentthymic emigrants-identification, expansion andsurvival characteristics. J. Immunol. 167:1970–1976.

16. Deacock, S.J., et al. 1992. Evidence that umbilicalcord blood contains a higher frequency of HLAclass II-specific alloreactive T cells than adultperipheral blood. A limiting dilution analysis.Transplantation. 53:1128–1134.

17. Mbawuike, I.N., et al. 2001. HLA-restricted CD8+

cytotoxic T lymphocyte, interferon-γ, and inter-leukin-4 responses to respiratory syncytial virusinfection in infants and children. J. Infect. Dis.183:687–696.

18. Hermann, E., et al. 2002. Human fetuses are ableto mount an adultlike CD8 T-cell response. Blood.100:2153–2158.

The Journal of Clinical Investigation | June 2003 | Volume 111 | Number 11 1647

New insights into a persistent problem —chlamydial infections

Richard P. Morrison

Department of Microbiology, Montana State University, Bozeman, Montana, USA

Tissue tropism of clinical ocular and genital Chlamydia trachomatis strainsis shown to be linked to the tryptophan synthase genotype (see related arti-cle beginning on page 1757). It is suggested that, in the presence of IFN-γ,which depletes available tryptophan, there exist unique host-parasite inter-actions that may contribute to persistent chlamydial infection.

J. Clin. Invest. 111:1647–1649 (2003). doi:10.1172/JCI200318770.

Chlamydial infections are noted for thebroad array of clinically distinct mani-festations that they produce, rangingfrom acute self-limiting ocular andgenital infections to chronic inflam-matory diseases that result in blindnessor infertility. Trachoma, an ocular

infection, is caused by Chlamydia tra-chomatis serovars A–C and is a primarycause of preventable blindness. Themajority of C. trachomatis serovars(D–K) cause urogenital infection,which can progress to serious genitaltract disease in men and women. Agenetic basis for the remarkable tissuetropism of the various chlamydialserovars (i.e., ocular vs. urogenitalstrains) was first proposed by Fehlner-Gardiner et al., based upon their analy-sis of the tryptophan synthase genecluster (trpRBA) of laboratory strainsof chlamydiae (1). They found that thetryptophan synthase genotype is close-ly linked to the tissue tropism of ocular

and genital chlamydial strains; uro-genital serovars possess a functionaltryptophan synthase and are capable ofutilizing indole as a substrate for tryp-tophan synthesis, whereas ocular iso-lates possess a nonfunctional trypto-phan synthase. In this issue of the JCI,Caldwell et al. have extended that earli-er work to include the characterizationof tryptophan synthase genes fromhundreds of clinical isolates, therebyconfirming the correlation betweenchlamydial tryptophan synthase geno-type and tissue tropism (2).

Between the extremes of acute self-limiting infection and chronic inflam-matory disease lies the notion of persistent and/or chronic states ofchlamydial infection. Cell culture sys-tems have been used for a number ofyears to demonstrate and studychlamydial persistence in vitro (3).However, the experimental documen-tation of persistent infection in thehuman host and of the mechanismsof in vivo persistence has been a muchgreater challenge. A particularlyintriguing hypothesis put forth by theauthors is that tryptophan synthasegenes function as chlamydial viru-lence factors and may be involved inthe maintenance of persistent/chron-ic chlamydial infection. That hypoth-

Address correspondence to: Richard P.Morrison, Department of Microbiology,Lewis Hall, Room 109, Montana StateUniversity, Bozeman, Montana 59717, USA.Phone: (406) 994-7959; Fax: (406) 994-4926;E-mail: morrison@montana.edu.Conflict of interest: The author has declaredthat no conflict of interest exists.Nonstandard abbreviations used: elementarybody (EB); reticulate body (RB); indoleamine2,3-dioxygenase (IDO).

1648 The Journal of Clinical Investigation | June 2003 | Volume 111 | Number 11

esis is particularly relevant when oneconsiders the key role played by IFN-γin host defense against chlamydialinfection. Caldwell et al. propose amechanism by which genital chlamy-dial strains may counter the growthinhibitory effects of IFN-γby utilizingindole produced by vaginal microbialflora as a substrate for tryptophansynthesis, thus allowing for the con-tinued survival of chlamydiae withina growth-inhibiting environment (2).

The intracellular developmentalcycle of ChlamydiaKey to understanding the pathophysi-ology of chlamydial disease is anappreciation of the chlamydial devel-opmental cycle (Figure 1) (4). Chlamy-dial infection is initiated by the attach-ment to a susceptible host cell of theinfectious, metabolically inert elemen-tary body (EB), which then enters thecell within a membrane-bound vesicle,

termed an inclusion. The EB rapidlydifferentiates into a reticulate body(RB), which replicates by binary fissionwithin the confines of the inclusion.Following several rounds of replica-tion, the RBs reorganize to form infec-tious EBs, which are released by cytol-ysis from the cell to initiate newinfections. Deviations from this typi-cal developmental cycle have beenexperimentally induced by a variety ofstimuli, including IFN-γ, antibiotics,and nutrient deprivation (3). Pertinentto the current discussion is the abilityof these stimuli, particularly IFN-γ, toalter chlamydial growth and facilitatepersistent or chronic infection.

Experimental persistence in cell cultureIt has long been known that IFN-γalters the growth of C. trachomatis incell culture systems (5). Chlamydialserovars and species display differen-

tial susceptibilities to the inhibitoryeffects of IFN-γ, which range fromminimal to marked inhibition ofchlamydial growth (6). In addition,IFN-γ induces persistent chlamydialinfections in cell culture systems thatare characterized by viable, but non-cultivatable, chlamydiae, which dis-play aberrant morphology (7, 8). Theseaberrant forms of chlamydiae remainviable for over one month in cell cul-ture and differentiate into infectiousEBs when IFN-γ is removed from thecell culture system (9, 10).

In theory, the mechanisms by whichIFN-γcould inhibit chlamydial growthare many, due to the pleomorphiceffects of IFN-γ on host cell function.However, one mechanism, which isparticularly germane to the currentdiscussion, is the induction by IFN-γof the tryptophan-decyclizing enzymeindoleamine 2,3-dioxygenase (IDO).Depletion of intracellular pools of

Figure 1Chlamydial infection is initiated by the attachment of the infectious EB to the host cell, followed by entry of the EB into a membrane-bound vesi-cle, termed an inclusion. The inclusion evades fusion with host lysosomes, and the EB rapidly differentiates into an RB that replicates by binaryfission within the inclusion. Following several rounds of replication, the RBs reorganize and form infectious EBs, which are released from the cell.Under certain conditions, such as in an inflammatory environment where IFN-γ is produced, the intracellular growth of ocular and urogenitalchlamydial strains may be altered. IFN-γ induces cellular IDO, which results in a marked decrease in available tryptophan. Depletion of trypto-phan either results in chlamydiae cell death or causes chlamydiae to adopt a noninfectious, nonreplicating form that retains viability (persistence).The outcome is dependent on the level and duration of tryptophan depletion and the tryptophan synthase genotype of the infecting chlamydialstrain. Persistent forms of chlamydiae can redifferentiate into infectious EBs upon removal of IFN-γand subsequent replenishing of intracellulartryptophan pools. Alternatively, even in an IFN-γ–rich environment, strains of chlamydiae that possess a functional tryptophan synthase (i.e., gen-ital strains) may use indole (perhaps produced by local microbial flora) as a substrate for tryptophan synthesis to counter the growth inhibitoryeffects of IFN-γ. This model is far from complete, and the biological processes involved are likely much more complex and interrelated than depict-ed here. However rudimentary, though, the proposed model provides a reasonable representation of our current understanding.

tryptophan by IDO essentially starveschlamydiae of this essential aminoacid, rendering them incapable of dif-ferentiating into infectious EBs (Fig-ure 1) (10, 11). As noted by Caldwell etal., the resistance pattern of the vari-ous chlamydial strains to the inhibito-ry effects of IFN-γ correlates to poly-morphisms in tryptophan synthasegenes (2). Thus, ocular serovars, whichhave a nonfunctional tryptophan syn-thase and are unable to synthesize tryp-tophan, are more sensitive to IFN-γ–mediated inhibition than genitalserovars, which have a functional tryp-tophan synthase and are capable ofutilizing indole as a substrate for tryp-tophan synthesis.

Clinical persistencePersistence of chlamydial infection inthe human host is at best incompletelydefined (12, 13). Chlamydial infectionsare known to be particularly insidious,and chronic inflammatory conditionsare a frequent consequence of untreatedchlamydial infection. Because chlamy-diae respond to a variety of environ-mental stimuli that alter their growthcharacteristics (e.g., IFN-γ), there existsthe potential for chlamydiae to estab-lish a chronic or persistent relationshipwith the host. If persistent or chronicinfections are established, then thoseinfections may serve as a reservoir fornew infections, contribute to the im-munopathological consequences ofinfection, or require alternative thera-peutic approaches. Thus, understand-ing the consequences of persistentchlamydial infection could be impor-tant to the control and prevention ofchlamydial disease.

IFN-γ is an important mediator ofprotective immunity to chlamydialinfection (14). Consequently, the pos-sibility that chlamydiae may grow in anenvironment rich in IFN-γ and thepotential for chlamydiae to establishpersistent or chronic infections in thepresence of IFN-γ are two infectionoutcomes that could have importantclinical implications. Caldwell et al. dis-cuss quite eloquently how genital

strains of chlamydiae could utilizeindole produced by vaginal microbesto survive in an IFN-γ–rich, trypto-phan-limiting environment (Figure 1)and how survival under such condi-tions might facilitate the developmentof chronic infections (2). Moreover, ifthe in vitro observation of IFN-γ–mediated persistent infection holds forhuman infection, then perhaps in anIFN-γ–rich environment, chlamydiaecould persist as morphologically aber-rant, nonculturable forms. If so, wouldthose persistent chlamydiae be suscep-tible to therapeutic doses of antimicro-bials typically prescribed to treat infec-tion? Although apparent treatmentfailures have been reported, there is nodirect in vivo evidence to support thenotion that those failures resultedfrom persistent chlamydiae being morerefractory to antimicrobial chemother-apy. However, recent studies using anin vitro cell culture system suggest thatpersistent forms of chlamydiae aremore refractory to antimicrobial ther-apy than chlamydiae grown under nor-mal cell culture conditions (P. Wyrick,personal communication).

Much remains to be learned aboutthe pathogenesis of chlamydial dis-ease, but the study by Caldwell et al.represents a seminal contribution toour understanding of chlamydiae-hostinteractions (2). Further characteriza-tion of the polymicrobial environmentof the female genital tract duringchlamydial infection and the develop-ment of experimental animal modelsof defined polymicrobial infectionswill be needed to assess the full impactof tryptophan synthase gene polymor-phisms on the pathogenesis ofchlamydial disease. Additional studieswill also be needed to more fullyunderstand why ocular strains havelost functional tryptophan synthaseactivity even though they face thesame IFN-γ selection pressure as geni-tal strains and to define what otherbiological differences may existbetween ocular and genital strains thatmight contribute to tissue tropismand disease pathogenesis. Investigat-

ing the interplay between chlamydiaeand its environment will impart amore thorough understanding ofhost-microbial interactions and mayprovide important insight into novelapproaches for the treatment and pre-vention of chlamydial disease.

AcknowledgmentsThe author acknowledges grant sup-port from the National Institutes ofHealth (AI-038991) and the AmericanHeart Association (0150034N).

1. Fehlner-Gardiner, C., et al. 2002. Molecular basisdefining human Chlamydia trachomatis tissue tro-pism: a possible role for tryptophan synthase. J. Biol. Chem. 277:26893–26903.

2. Caldwell, H.D., et al. 2003. Polymorphisms in Chla-mydia trachomatis tryptophan synthase genes differ-entiate between genital and ocular isolates. J. Clin.Invest. 111:1757–1769. doi:10.1172/JCI200317793.

3. Beatty, W.L., Morrison, R.P., and Byrne, G.I. 1994.Persistent chlamydiae: from cell culture to a para-digm for chlamydial pathogenesis. Microbiol. Rev.58:686–699.

4. Hackstadt, T. 1999. Cell biology. In Chlamydia:intracellular biology, pathogenesis, and immunity. R.S.Stephens, editor. American Society for Microbiol-ogy Press. Washington, D.C., USA. 101–138.

5. Kazar, J., Gillmore, J.D., and Gordon, F.B. 1971.Effect of interferon and interferon inducers oninfections with a nonviral intracellular microorgan-ism, Chlamydia trachomatis. Infect. Immun. 3:825–832.

6. Morrison, R.P. 2000. Differential sensitivities ofChlamydia trachomatis strains to inhibitory effectsof gamma interferon. Infect. Immun. 68:6038–6040.

7. Beatty, W.L., Byrne, G.I., and Morrison, R.P. 1993.Morphologic and antigenic characterization ofinterferon-gamma mediated persistent Chlamydiatrachomatis infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 90:3998–4002.

8. Beatty, W.L., Morrison, R.P., and Byrne, G.I. 1994.Immunoelectron-microscopic quantitation of dif-ferential levels of chlamydial proteins in a cell cul-ture model of persistent Chlamydia trachomatisinfection. Infect. Immun. 62:4059–4062.

9. Beatty, W.L., Morrison, R.P., and Byrne, G.I. 1995.Reactivation of persistent Chlamydia trachomatisinfection in cell culture. Infect. Immun. 63:199–205.

10. Beatty, W.L., Belanger, T.A., Desai, A.A., Morrison,R.P., and Byrne, G.I. 1994. Tryptophan depletion asa mechanism of gamma interferon-mediatedchlamydial persistent. Infect. Immun. 62:3705–3711.

11. Byrne, G.I., Lehmann, L.K., and Landry, G.J. 1986.Induction of tryptophan catabolism is the mecha-nism for gamma-interferon-mediated inhibitionof intracellular Chlamydia psittaci replication in T24cells. Infect. Immun. 53:347–351.

12. Dean, D., Suchland, R.J., and Stamm, W.E. 2000.Evidence for long-term cervical persistence ofChlamydia trachomatis by opm1 genotyping. J. Infect.Dis. 182:909–916.

13. Campbell, L.A., et al. 1993. Detection of Chlamydiatrachomatis deoxyribonucleic acid in women withtubal infertility. Fertil. Steril. 59:45–50.

14. Morrison, R.P., and Caldwell, H.D. 2002. Immu-nity to murine chlamydial genital infection. Infect.Immun. 70:2741–2751.

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