This study was undertaken to elucidate three different in vitro models for changes in mRNA expression during the transition of C. psittaci to the persistent state and to work out common and distinctive features of chlamydial persistence by comparisons with published data on Chlamydia trachomatis and C. pneumoniae. While the phenotypical criteria were in line with observations from the literature, 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 was found to display several new and distinctive features.
The first persistence model, DAM-induced iron depletion, resembles the local microenvironment of inflamed infected tissues (1
). Under our experimental conditions, it appeared to be the most resilient model, having the highest recovery rate from persistence at 24 h p.i. At the same time, we observed the lowest total number of upregulated genes among the models we tested, which indicates a relatively straight transition to the persistent state.
Exposure to 240 U/ml IFN-γ caused the most intensive transcriptional response of chlamydial cells at the early stage, where 8 of the 27 genes were significantly upregulated. At the late stage, the most complete shutdown of mRNA expression among the three models occurred, which is in line with the observation that the highest loss of infectivity occurred in the IFN-γ trials (Fig. ). Many studies in the literature were based on the IFN-γ model (3
), which is thought to reflect locally elevated levels of this cytokine during chlamydial infection in vivo. In this context, persistence can be regarded as an alternative branch of the chlamydial developmental cycle designed to evade the host's immune response (5
). Susceptibility of C. psittaci
to IFN-γ may be expected, as this species has no functional tryptophan synthesis operon (G. S. A. Myers, personal communication). Early studies of avian Chlamydia psittaci
had already shown inhibition of intracellular replication and inclusion development as a result of IFN-γ treatment (7
). Current data on stress response factors and surface proteins are showing good agreement with recent findings on C. pneumoniae
(which also lacks the trp
operon) in IFN-γ and iron depletion models (39
). In contrast, Chlamydophila caviae
(formerly Chlamydia psittaci
GPIC strain) harbors a complete tryptophan biosynthesis operon consisting of six genes, trpRDCFBA
, whose expression is regulated by the amino acid. This set of genes enables C. caviae
to recycle tryptophan and thus accounts for the IFN-γ-resistant phenotype displayed in indolamine dioxygenase-expressing host cells (41
). Similarly, genital strains of Chlamydia trachomatis
were found to possess functional trpBA
genes, which enable them to utilize exogenous indole for tryptophan biosynthesis (12
). This characteristic difference provides an explanation for our observation that the late shutdown of essential genes in C. psittaci
with IFN-γ-induced persistence was more comprehensive than that in Chlamydia trachomatis
D strains (5
Penicillin-induced persistence imitates in vivo conditions of inadequate antibiotic treatment. The fact that the response pattern of this model was markedly different from the other two should be due to the distinct mechanism of action, i.e., targeting cell wall synthesis rather than chlamydial metabolic pathways. Furthermore, Timms (39
) pointed to an overlap between IFN-γ and iron depletion models, as IFN-γ also downregulates transferrin receptors, which results in lower host cell iron levels.
One of the most interesting findings of the present study is the conspicuous upregulation at 12 h p.i. of late genes coding for membrane proteins (omcA, pomp91B, omcB) and for proteins involved in DNA condensation (hctA), RB-EB differentiation (ctcB), type III secretion system (incA), signal transduction (pkn1), and transcriptional regulation (rpoN), particularly in the IFN-γ model. This seems to illustrate that, in response to challenge by the host immune system, the chlamydial cell tries to accelerate its developmental cycle by prematurely initiating processes that would normally occur at a later stage and/or enhance already ongoing expression of genes that are essential for long-term survival. Nonetheless, this transient increase in mRNA abundance does not result in measurable EB formation, as one would expect from the massive upregulation of hctA and some membrane proteins.
Another characteristic feature of the present persistence models is downregulation of the vast majority of genes at 48 h p.i. Notably, the group of the most intensely downregulated genes includes omcA, omcB, pomp91B, hctA, and ctcC, all of which were transiently upregulated at 12 h p.i. This mRNA expression pattern underlines the hypothesis that the processes prematurely initiated and probably not completed can no longer be maintained by the chlamydial cell at the late stage, and it further illustrates the idea that mRNA expression rates at 48 h p.i. do not merely reflect a general shutdown but show differential regulation.
It is also important that the persistent state of C. psittaci can be extended considerably. Our observation that a sizeable proportion of chlamydial cells remained visibly persistent and viable at least until 96 h p.i. with all three models (data not shown) additionally illustrates the chronic aspect of this phenomenon.
Summarizing the qrt-PCR data, we postulate that downregulation of genes coding for membrane proteins, transcription regulators, cell division factors, and EB-to-RB differentiation factors is associated with establishment and maintenance of the persistent state in vitro, independent of the inducing reagent. The downregulation of genes and proteins that are specifically expressed late in the productive developmental cycle most likely reflects the inhibited RB-to-EB differentiation. This is a common observation with persistence models, and it led to the late gene shut-down hypothesis (16
Regarding regulation of individual genes during transition to persistence, the present data do not support the suggestion that a decrease in the ompA
ratio is a universal persistence marker. No steady decline of the ratio was observed for any of the C. psittaci
models as mRNA expression rates of both genes were oscillating along the time axis, and clear downregulation at the late stage was observed only with IFN-γ-induced persistence. Recent studies on C. pneumoniae
also reported data showing that this ratio was not suitable as a marker for all chlamydial persistence systems (16
Cell division protein FtsW is assumed to be involved in cell wall formation through septum peptidoglycan synthesis (37
). The present study demonstrates the association of low mRNA expression levels of ftsW
with persistence of C. psittaci
, which is in agreement with reports of downregulation in several in vitro persistence models of Chlamydia trachomatis
) and C. pneumoniae
). Consistent downregulation of this gene from 24 h p.i. onward is in accordance with the observation that, in persistent infection, chromosome partitioning still takes place, whereas cell division is inhibited (8
Interestingly, the CADD gene was found to be downregulated at the last stage of the present IFN-γ experiments after having been transiently upregulated at 24 h p.i. This is in contrast to data from IFN-γ-induced persistence of Chlamydia trachomatis
, where it was downregulated at 24 h p.i. and upregulated at 48 h (5
). CADD shares homology with the death domains of tumor necrosis factor family receptors and induces apoptosis when transiently transfected to noninfected cells (36
). Although it is not known at present whether CADD is the dominant protein governing apoptosis, a recent study reporting the crystal structure of this protein (35
) demonstrated its capability to induce in vivo cell death of HeLa cells. Both catalytic activity and death domain binding were found to be required for its complete biological function. As this redox protein toxin was shown to necessitate two Fe2+
ions per molecule, an iron-depleted environment during persistence may contribute to the inhibition of apoptosis, thus further supporting the hypothesis that apoptosis inhibition is an integral part of persistent infection or at least concurrent with transition to that state (11
We believe that consistent and severe downregulation of the ctcB-ctcC
gene pair also represents an interesting observation. It was predicted to encode highly conserved proteins with homology to bacterial two-component regulatory systems, and a possible role in late gene regulation during RB-to-EB transition was suggested (21
). As both ctcB
were downregulated in our persistence models from 24 h p.i. onward, and RB-to-EB differentiation was obviously not taking place, it is conceivable that the genes and/or proteins are involved in this late differentiation process during acute infection.
The considerable divergence among data from the different model systems suggests that, at least in vitro, there is no persistence model per se, but rather a number of different ways for chlamydiae to reach this state. Similarly, cytokine-mediated inhibition of intracellular chlamydial growth was found to involve multiple mechanisms, such as nitric oxide induction, tryptophan catabolism, and iron deprivation (19
). On the other hand, the effect of persistent chlamydial infection on the host cell response was also found to be dependent on the inducing reagent. While IFN-γ and penicillin treatment led to inhibition of C. pneumoniae
-induced gene expression in HeLa cells, continuous and even increasing responses were observed with the iron depletion model (29
Being the first molecular study on C. psittaci persistence, the present work demonstrates the capability of this pathogen to respond to simulated host challenges by altering mRNA expression of specific genes, which led mostly to strong inhibition or complete shutdown of mRNA expression in the persistent state. The reversibility of this phenomenon illustrates the efficiency of the chlamydial response and provides a principal clue for the explanation of a number of peculiarities, including chronicity of chlamydioses, failure of antibiotic therapy and immunoprophylaxis, and practical difficulties in isolating and culturing viable strains from infected tissue.