Transcriptional response patterns of Chlamydophila psittaci in different in vitro models of persistent infection

Abstract

The obligatory intracellular bacterium Chlamydophila psittaci is the causative agent of psittacosis in birds and humans. The capability of this zoonotic pathogen to develop a persistent phase is likely to play a role in chronicity of infections, as well as in failure of antibiotic therapy and immunoprophylaxis. To elucidate three different in vitro models for transition of C. psittaci to persistence (iron depletion, penicillin G treatment, and gamma interferon [IFN-gamma] exposure), a set of 27 genes was examined by mRNA expression analysis using quantitative real-time PCR. While the phenotypical characteristics were the same as in other chlamydiae, i.e., aberrant morphology of reticulate bodies, loss of cultivability, and rescue of infectivity upon removal of inducers, the transcriptional response of C. psittaci to persistence-inducing factors included several new and distinctive features. Consistent downregulation of membrane proteins, chlamydial sigma factors, cell division protein, and reticulate body-elementary body differentiation proteins from 24 h postinfection onward proved to be a general feature of C. psittaci persistence. However, other genes displayed considerable variations in response patterns from one model to another, which suggests that there is no persistence model per se. In contrast to results for Chlamydia trachomatis, late shutdown of essential genes in C. psittaci was more comprehensive with IFN-gamma-induced persistence, which is probably due to the absence of a functional tryptophan synthesis operon.

FIG. 1.

Decrease of infectivity of C. psittaci after exposure of infected HEp-2 cell cultures to different amounts of persistence-inducing reagents. The number of inclusion-forming units was determined from methanol-fixed immunostained cell cultures at 48 h p.i. (A) Iron depletion by addition of DAM. (B) Penicillin G treatment. (C) Exposure to IFN-γ.

FIG. 2.

Immunohistological staining of chlamydial cells in acute and persistent infection. Infected HEp-2 monolayers were incubated at 37°C and 5% CO₂ for 48 h, fixed with methanol, stained as described in Materials and Methods, and visualized using fluorescence microscopy at a magnification of ×400. (A) HEp-2 monolayers infected with C. psittaci DC15 without persistence inducer. (B) Persistent infection with iron depletion (150 μM DAM). (C) Persistent infection after treatment with 200 U/ml penicillin G. (D) Persistent infection upon exposure to IFN-γ (240 U/ml). To illustrate inclusion morphology, magnifications of typical chlamydial inclusions are shown in the lower left corner of each image. Please note that the natural size of the inclusion shown in panel A is four to five times those of the persistent inclusions shown in panels B to D. Bars, 20 μm.

FIG. 3.

Transmission electron microscopy of chlamydial inclusions in acute and persistent infection at 48 h p.i. (A) HEp-2 monolayers infected with C. psittaci DC15 without persistence inducer. A large inclusion, with numerous EBs, numerous intermediate condensing bodies, and few RBs is shown. (B) Persistent infection at iron depletion (150 μM DAM). A small inclusion containing only a few RBs is shown. (C) Persistent infection after treatment with 200 U/ml penicillin G. An inclusion densely packed with RBs of variable size and shape is shown. Two RBs are severely enlarged. Note the electron-dense depositions at the outer membranes of RBs. (D) Persistent infection upon exposure to IFN-γ (240 U/ml). A small inclusion with large RBs at the periphery and amorphous material in the center is shown. RBs are highly variable in size, shape, and appearance. Bars, 2 μm.

FIG. 4.

Reactivation of persistent chlamydial organisms. The numbers of recoverable infectious chlamydial cells (in IFU) from acute infection (Acute inf.) and persistence trials based on iron depletion by addition of DAM, penicillin G (PenG) treatment, or exposure to IFN-γ (filled columns) are compared to those from the reactivation assay, which included removal of persistence inducers at 24 h p.i. (open columns), and 48 h p.i. (shaded columns) and subsequent culture in complete growth medium for 24 h.