Persistence:
 

- Persistent Chlamydial Development:

 Persistence describes a long-term association between Chlamydiae and their host cell in which these organisms remain in a viable but culture-negative state.
 Persistence may represent a deviation from the typical development of Chlamydiae, resulting in delayed intracellular growth under the influence of exogenous factors that may not be as "typical" as cell culture growth conditions. These conditions generally delay reticulate body (RB) maturation, inhibit differentiation to infectious elemental body (EB), and are associated with gross morphological alterations of RBs typified by markedly enlarged, atypical chlamydial forms. Delays in chlamydial development in response to exogenous factors suggest an innate ability of these organisms to persist intracellulary in a unique developmental form.
  Chlamydial persistence has been established in a variety of cell culture systems; however, conclusive documentation of persistent Chlamydiae in natural infections remains to be demostrated.
 

       Altered Chlamydial Development.
 

*Persistent infections in cell culture:
 

Nutrient deficiency-induced persistence:

  Chlamydiae are able to invade the deficient host cells and upon entry remain in a noninfectious but viable state. Addition of complete medium stimulates growth and recovery of infectious Chlmaydiae.
  Depletion of cysteine interrupts chlamydial RB-to-EB differentiation in ten different serovars of C.  trachomatis. This effect is reversible, with resumed differentiation to infectious forms, upon the addition of cysteine.  Deficiency of other amino acid have a little or no effect on chlamydial development. These observations suggest that an alteration in growth and differentiation arises via the requirement for this amino acid for the biosynthesis of three cysteine-rich proteins (MOMP, 12 and 60 kda.) needed for RB-to-EB differentiation.
  Some studies show that intracellular development of C. trachomatis serovars E and L2 in a medium lacking thirteen amino acids result on reduced infectious yield, with the development od enlarged abnormal chlamidial forms. This effect is reversible, with a substantial recovery of infectivity upon the reintroduction of amino acids.
  In conclusion, under conditions in which the host´s soluble pools for metabolic needs become limited, Chlamydiae may fail to successfully compete for macromolecular precursors and hence may enter a state of arrested growth.

Antimicrobial agents and persistence:

  Treatment with penicillin has no effect on initial differentiation of the infecting EB to the RB but prevents the process of binary fission inducing the development of enlarged, morphologically abnormal chlamydial forms with a block in the production of progeny EBs. These aberrant forms become progressively larger with continued culture in the presence of the antibiotic. Because Chlamydiae are deficient in peptidoglycan the mechanism of chlamydial growth inhibition by penicillin is unknown. The penicillin have no effect on the synthesis of the MOMP but the synthesis of the cysteine-rich 60 kda protein is completely inhibited. Removal of penicillin from the extracellular culture medium results in extensive budding and internal subdivision of the aberrant chlamydial forms, producing typical RBs with maturation to infectious EBs.
  Ampicilin also blocks the conversion of RBs to EBs. The effect of ampicillin on Chlamydia-specific protein synthesis reveals that arrest of chlamydial development is accompanied by a marked inhibition in the expression of both 60 kda and 12 kda cysteine-rich proteins, while the production of MOMP is only minimally affected.
  Chloranfenicol and chlortetracycline have also been shown to interrupt the intracellular development of Chlamydiae. The effect of these inhibitors of prokaryotic protein synthesis is dependent on the stage during infection at which the Chlamydia-infected cells are exposed to these antibiotics. Addition of these inhibitors early in infection prevents primary differentiation of EB to RB ( persistence is not induced ), whereas exposure later in infection interrupts RB division and secondary differentiation.
  A nearly identical situation occurs when the effect of Erythromycin on chlamydial development is analyzed. This antibiotic not only inhibits the RB-to-EB differentiation but also induces smaller inclusions containing RBs of approximately twice the diameter of typical RBs. Erythromycin binds to the 50s subunit of the ribosome and may reduce RB ribosomal activity and subsequent protein synthesis.
  Antibiotics that inhibit nucleic acid synthesis also have been shown to inhibit chlamydial development. 5-fluorouracil affects late stages of the C. trachomatis LGV growth cycle. Treatment of 48 hours shows smaller inclusions containing only a few noninfectious larger particles present. Hydroxyurea, also blocks secondary differentiation, as before, accompanied by the inhibition of the synthesis of cysteine-rich proteins.
  Sulfonamides as trimethoprim and sulfomethazole at levels slightly below the MIC inhibit RB-to-EB differentiation characterized by the presence of gross changes in RB formation.

Immunologically induced persistence:

  The most of the studies about persistence are focus in the T-cell product gamma interferon (IFN-g) using HeLa 229 cells infected with C. trachomatis serovar A. Chlamydiae were among the first nonviral pathogens reported to induced IFNs and were shown to exhibit growth inhibition in their presence. IFN-g affects human host cells in vitro by inducing indoleamine 2,3-dioxygenase (IDO), a nonconstitutive enzyme that catalyzes the initial step in the degradation of tryptophan to N-formylkynurenine and kynurenine.
   Persistence of C. trachomatis occurs when very low levels of IFN-g are added to culture systems following infection of the host cells. This effect is reversible upon IFN-g removal from the culture medium. High levels of it inhibit completely chlamydial development.
   IFN-g-induced persistent organisms not only exhibit a highly unusual intracellular morphology but also display differential expression of key chlamydial antigens with continued synthesis of hsp-60, an immunopathological antigen, and reduced synthesis of MOMP, aprotective antigen. Besides, these forms exhibit a reduction in the levels of other structural constituents of Chlamydiae, including the 60 kda envelope protein and lipopolysaccharide.
  The effect of exogenous tryptophan on IFN-g-induced persistent development revealed that the presence of superphysiological concentrations of tryptophan interrupted IFN-g-mediated persistence, resulting in the development of typical chlamydial inclusions. Cycloheximide, an inhibitor of host protein synthesis, have the same effect of exogenous tryptophan on  IFN-g-mediated persistence.
 
 

Other potential mediators of persistence:

  Treatment of C. trachomatis L2-infected cells with AMPc reversibly interferes with the correct progression of Chlamydiae through the developmental cycle. Inclusions are small and immature and remain noninfectious in the presence of AMPc.
  Verapamil, a Ca2+ antagonist, interferes with chlamydial development inhibiting RB-to-EB differentiation.
 
 

- Persistence of Chlamydiae in vivo:

  There are evidence of it in:

Ocular infections:

  Although a clear correlation between trachoma and the infection with C. trachomatis has been established, Chlamydiae cannot be identified by tissue culture or immunocytological methods in over 20% of cases, even in the presence of severe abnormalities.
  Older individuals who have been exposed to repeated infections display complications of ocular scarring and fibrosis, corneal trauma, and blindness, even though C. trachomatis is rarely isolated from the disease tissue.
  The presence of a cryptic form of Chlamydiae in the infected tissues is suggest in studies of individuals who left  areas where trachoma was endemic and had no experienced active disease since childhood but developed acute trachoma several decades later. Therefore, viable Chlamydiae may be present in a latent , nonreplicating form, contributing to the progression of disease toward blinding trachoma.

Genital infections:

 The identification of C. trachomatis as an etiologically agent of infertility in women is well recognized. Infection of the cervix by C. trachomatis may be chronic, persisting unrecognized for months to years unless a symptomatic infection of a male partner occurs.
  Approximately 70% of women with C. trachomatis cervicitis have only mild symptoms or remain asymptomatic. Unrecognized infection can progress, ascending to the salpinges and fallopian tubes and resulting in pelvic inflammatory disease.
  Chlamydial antigens have been detected in endometrial and tubal specimens of culture-negative infertile women.
  Chlamydia-specific DNA has been identified in endocervical cells and fallopian tube tissue of women with tubal occlusion.
  In conclusion, affected individuals may harbor a persistent form of Chlamidiae that possesses the characteristics of noncultivatable RBs but retains viability and the capacity to stimulate immunophatologic changes. However, evidence of chlamydial antigen and nucleic acids in the absence of cultivable organisms only suggests the possibility of persisting Chlamydiae.
 
 

References:

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8. Oriel, J. D. 1986. The carrier state: C. trachomatis. J. Antimicrob. Chemother. 18SA:67-71.

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10. Shepard, M. K., and R. B. Jones. 1989. Recovery of Chlamydia trachomatis from endometrial and fallopian tube biopsies in women with infertility of tubal origin. Fertil. Steril. 52:232-238.

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