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The interferons are a family of related antiviral proteins, produced by mammalian cells in response to infection (including chlamydial infection) and other stimuli. The interferons also act as intercellular messengers, altering the function of many different kinds of cell. Thus, they are useful in the treatment of hairy cell leukaemia and other kinds of cancer.

Interferon is usually abbreviated by the letters ifn supplemented by a Greek letter to indicate the particular family. Thus, interferon alpha is produced by epithelial cells, the cells lining the moist surfaces of the body, whereas interferon gamma (ifng) is produced by cells of the cellular immune system (T cells and NK cells ), and is the key effector of the protective Th1 cellular immune response to chlamydial infection. Our concern here is with the effect of ifng on chlamydial development and persistence.

It has been known for a long time that ifn was capable of inhibiting infection with bacteria, such as chlamydiae, that of necessity multiply inside host cells. Essentially, ifng binds to target molecules (receptors) on the surface of uninfected epithelial cells, triggering changes which include the depletion of the amino acid tryptophan in the targeted cell. Tryptophan, one of the essential "building blocks" of protein, is also an essential constituent of chlamydial proteins. Thus a shortage of cellular tryptophan leads to (reversible) inhibition of the chlamydial developmental cycle at the RB stage, prior to their conversion into infectious EB. In the laboratory ifng can induce incomplete chlamydial development, which, as we have seen, is associated with persistent infection 

It is easy to imagine a situation, in which a vigorous chlamydial infection challenges the cellular immune system which responds, among other ways, with the production of ifng from T lymphocytes. The ifn gamma brings about a reversible inhibition of chlamydial development, which means that there is now less stimulus to sustain the cellular immune response. So, ifng production, wanes. As ifn gamma levels fall below a critical threshold, the tryptophan constraint is released enabling productive chlamydial development with the production of infectious EB to start again. Thus the stage is set for renewal of the balancing act between chlamydial replication and ifng production. Clinically, the theory is attractive because it explains, superficially at least, why chlamydial infections are characterised by intermittent bouts of activity and chlamydial shedding. This concept is generally well known and has been extensively reviewed. [For a review of earlier work see Beatty et al., 1994 or Ward, 1999; also: evolution and persistence].

Tryptophan synthase and organotropism of C. trachomatis

Since ifng leads to a depletion of intracellular tryptophan pools, it follows that those chlamydiae capable of synthesizing tryptophan from other sources will be at an advantage. C. trachomatis organisms having a functional tryptophan synthase trpBA gene are able to synthesize tryptophan from indole. Caldwell et al., 2003 found that all ocular isolates of C. trachomatis tested had inactivating mutations in trpAB making them sensitive to ifng, whereas all genital serovars encoded the functional enzyme. Interestingly ocular serovars (serovar B) isolated from the genital tract had a functioning tryptophan synthase. It was suggested that tryptophan synthase is an important determinant of organotropism among C. trachomatis serovars, with chlamydiae in the genital tract in possession of tryptophan synthase able to escape ifng-mediated eradication by synthesizing tryptophan from indole provided by other members of the vaginal microbial flora.

Effect of ifng on chlamydial gene transcription.

Using ifng of chlamydial infected epithelial cells as a model of chlamydial persistence, a number of studies have tried to determine whether persistent versus productive infection is associated with differences in chlamydial gene transcription. Byrne et al., 2001 used RT-PCR to investigate C. pneumoniae gene transcription in HEp 2 cells in parallel with electron microscopic observation. Real-time PCR was used to assess chlamydial chromosome replication. In the presence of moderate levels of ifng, C. pneumoniae chromosome numbers were able to increase several fold, though there was less accumulation at high ifng doses, and this was associated with the presence of morphologically aberrant forms.  Chlamydial primary rRNA transcripts were present in all ifng -treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in ifng -treated and untreated cells. However, the expression of ftsK and ftsW, associated with bacterial cell division, was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high doses. It was concluded that persistent infection associated with ifng permitted production of transcripts for DNA replication-related, but not cell division-related, activities [Byrne et al., 2001]. 

Gerard et al., 2001 compared C. trachomatis serovar K gene expression by real time PCR in productive infection in Hep-2 cells (48 hour period) versus more chronic infection (7 days) in human monocytes. They considered the latter to be a model of persistent infection. The also looked at chlamydial gene expression in samples from patients with chronic chlamydial infection. In Hep 2 cells,  significant accumulation of chlamydial chromosome began about 12 hours post infection. In artificially infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days one to seven post infection, but these were only weakly expressed in patient samples. Chlamydial chromosome number continued to increase during the 7 day monocyte infection, although the rate of accumulation was lower than during active growth in Hep 2. However, transcripts of ftsK and ftsW associated with bacterial cell wall division were detected only at the first day post infection in infected monocytes but not thereafter; they were absent in all patient samples. Consistent with the work of Byrne et al., 2001, gene products required for chlamydial DNA replication were expressed during the longer term infection monocytes, but transcription of genes required for chlamydial cell division were severely downregulated, possibly explaining the observed attenuation of new infectious elementary body production during chlamydial persistence [Gerard et al., 2001]. 

In a similar study of infected monocytes, of cells from the synovia of patients with reactive arthritis and acutely infected Hep 2 cells, the same group examined the expression of gene transcripts associated with energy metabolism.  The genes targeted were:  (a) chlamydial primary rRNA transcripts and adt1 mRNA and; (b) chlamydial mRNA encoding enzymes of the glycolysis (pyk, gap, pgk) and pentose phosphate (gnd, tal) pathways, the TCA cycle (mdhC, fumC), electron transport system (cydA, cydB), and sigma factors (rpoD, rpsD, rpoN). Primary rRNA transcripts and adt1 mRNA were present in each infected preparation and patient sample. In actively infected Hep-2 cells, all energy transduction-related genes were expressed by approximately 11 h post-infection. In monocytes, pyk, gap, pgk, gnd, tal, cydA mRNA were present in one to two day infected cells but were absent at 3 days and therefter after. Transcripts of  cydB, mdhC, fumC were expressed throughout the five days post-infection. Lack of mRNA from sigma factor genes did not explain transcriptional down-regulation in longer term monocyte infection. Analyses of RNA from synovial tissues were similar to the monocytes. It was suggested that, in the first phase of active chlamydial infection, ADP/ATP exchange provides the necessary energy for metabolism. During active growth, glycolysis could supplement host-provided ATP. In the monocytes host provided ATP was the primary energy source. As would be expected, the metabolic rate of C. trachomatis in monocytes was lower than in actively growing cells [Gerard et al., 2002].      

Finally,  Molestina et al., 2002 used a proteomics approach to analyse the translation of various C. pneumoniae proteins in ifng treated cells.  Treatment with 50 international units of ifng per ml caused a marked upregulation of major outer membrane protein, heat shock protein 60, and of proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), or  type III secretion (SctN). However [and contrary to earlier reports by others for C. trachomatis], no significant decreases in chlamydial protein expression were observed due to ifng treatment. It was considered that the upregulation of certain C. pneumoniae proteins might allow the organism to resist the inhibitory effects of ifng while retaining basic chromosome replication function [Molestina et al., 2002].

[MEW comments: In these studies, ifng treatment, or chlamydial infection in monocytes versus Hep 2 cells, are being used as models of persistence. This tells us something interesting as to how chlamydiae respond in vitro when stressed by infg from the Th1 response. However it may be premature to regard this as being a model of persistence per se.  With the non ifng model used by Gerard and colleagues I have further difficulties, since to compare a short term infection in Hep 2 with a medium term infection in something as different as a monocyte seems to me like comparing chalk and cheese. The use of patient samples is commendable, relevant but fraught with problems, including what the relevant controls should be. These are good and interesting studies in a difficult area. However, I believe caution is necessary at this early stage before we equate too many modelsl to persistence, whatever that may be?!  It is conceivable one might end up chasing self fulfilling laboratory artifacts. A consensus of some radically different approaches is needed. I like the proteomics approach generally because ultimately it's translated protein products we are interested in. Thoughts to the discussion forum please].

Effects of ifng on apoptosis

Two recent papers suggest that inhibition of chlamydial induced apoptosis might be a mechansim of persistent infection [Dean & Powers, 2001; Perfettini et al., 2002]. The first of these studies compared apoptotic responses among HeLa 229 epithelial cells acutely or persistently [ifng again], infected with C. trachomatis A/Har-13/OT. Chlamydial infection caused a significant reduction in apoptosis and this was caspase independent. However, there was otherwise little difference between acute and ifng-persistent infection in cellular resistance to apoptotic stimuli. Notwithstanding, the authors considered that chlamydial disregulation of apoptosis and ensuing persistence might be a major mechanism for the scarring sequelae of trachoma and of genital tract infection [Dean & Powers, 2001]. Ifng concentrations that lead to the development of a heterogenous population of normal and aberrant chlamydial vacuoles, cause a decrease in apoptosis in cells containing aberrant inclusions. This effect was at lest  in part du to expression of host cell indoleamine 2,3-dioxygenase activity, since inhibition of apoptosis could be partially reversed by exogenous tryptophan. Apoptotic cells can be observed in the genital tract of wild type mice infected with C. muridarum but occur in greater numbers in ifng deficient mice. It was suggested that diminished apoptosis of epithelial host cells in the knock out mice might lead to a greater likelihood of persisting infection [Perfettini et al., 2002]. 

[MEW comment: Other studies indicate that the effect of chlamydiae on apoptosis is much more complex than this, with multiple pathways involving both caspase dependent and independent mechanisms, both stimulation and depression [see: apoptosis paradox]. In vivo increased apoptosis might be the result of increased chlamydial multiplication in mice unable to mount the necessary protective ifng response. More work on in vivo models is required before a clear picture of the intertwined roles of ifng, persistence, apoptosis, scarring and tissue damage emerges].

[MEW] August 2003

NEXT: Persistent infection in animals.

References

Beatty, W. L., Morrison, R. P.& Byrne, G. I. (1994). Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microbiology Reviews 58, 686 - 699. [Good review to the earlier literature].

Byrne, G. I., Ouellette, S. P., Wang, Z., Rao, J. P., Lu, L., Beatty, W. L. & Hudson, A. P. (2001). Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells. Infection and Immunity 69, 5423 - 5429. Full article  

Caldwell, H. D., Wood, H., Crane, D., Bailey, R., Jones, R. B. et al., (2003). Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates. Journal of Clinical Investigation 111, 1757 - 1769. Full article

Dean, D. & Powers, V. C. (2001). Persistent Chlamydia trachomatis infections resist apoptotic stimuli. Infection and Immunity 69, 2442 - 2247. Full article   

Gerard, H. C., Freise, J., Wang, Z., Roberts, G., Rudy, D., Krauss-Opatz, B. et al., (2002). Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection. Microbes and Infection 4, 13 - 22.

Gerard, H. C., Krausse-Opatz, B., Wang, Z., Rudy, D., Rao, J. P., Zeidler, H. et al., (2001). Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection. Molecular Microbiology 41, 731 - 741.

Molestina, R. E., Klein, J. B., Miller, R. D., Pierce, W. H., Ramirez, J. A. & Summersgill, J. T. (2002). Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells. Infection and Immunity 70, 2976 - 2981.

Perfettini, J. L., Darville, T., Dautry-Varsat, A., Rank, R. G. & Ojcius, D. M. (2002). Inhibition of apoptosis by gamma interferon in cells and mice infected with Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis). Infection and Immunity 70, 2559 - 2565.

Ward, M. E. (1999). Mechanisms of chlamydial induced disease. In: Chlamydia. Intracellular biology, pathogenesis and immunity. (Stephens R. S. ed) pp 171 - 210. American Society of Microbiology, Washington DC. ISBN 1-55581-155-8

NEXT: Persistent infection in animals.