Analysis of modulated gene expression in a model of Interferon-γ-induced persistence of Chlamydia trachomatis in HEp-2 cells
Introduction
Infection of the human urogenital mucosa by Chlamydia trachomatis, a Gram-negative, obligate intracellular bacterium, gives rise to acute inflammatory responses, and is the commonest of the sexually transmitted bacterial infections [1]. Symptomatic or asymptomatic infections of the genital tract may occur as a result of C. trachomatis serovars D to K. Infections may follow a chronic pathway, even though provoking an immune response [1], and persisting infections of the female reproductive tract lead to severe complications of pelvic inflammatory disease, ectopic pregnancy and infertility [1], [2].
Over recent years, it has been increasingly recognised that C. trachomatis, as well as other chlamydial species, can establish a state of in vitro [1], and in vivo [3], [4] persistence. This is implicated in the chronic disease progression caused by chlamydial species, including those of C. trachomatis in the upper genital tract of females [5], [6].
There is controversy with regard to the cellular and molecular changes that occur in the developmental cycle of chlamydia as a consequence of both the establishment and the maintenance of the persistent state in vitro and in vivo, but Interferon-γ (IFN-γ) is one of a number of factors known to trigger persistence in vitro [7], [8]. IFN-γ knockout mouse studies confirm that IFN-γ is crucial in the resolution and clearance of C. trachomatis infections [9]. Furthermore, following infection of monocytic cells with chlamydia, spontaneous persistence occurs [10], [11]. Other factors able to induce chlamydial persistence in vitro include exposure to antibiotics [12], [13], iron limitation [14] and lack of various nutrients [15], [16]. The establishment of C. trachomatis persistence in epithelial cell lines in vitro using IFN-γ has been found to be indirectly due to a reduction in availability of tryptophan, an essential metabolite for the organism [2], [17], and general depletion of the host cell nutrients needed by these obligate intracellular bacteria elicits major changes in their developmental cycle [8], [16].
Following infection, C. trachomatis elementary bodies (EBs) are contained within an inclusion and differentiate into reticulate bodies within about 24 h. By 48–72 h, and after several cycles of multiplication of the reticulate bodies by binary fission, progeny EB are formed [18]. The persistent state of C. trachomatis in cell lines in vitro is characterised morphologically by the development of abnormal or aberrant inclusion body formation, by a disruption or cessation in the chlamydia developmental cycle and by a loss or reduction in infectivity [14], [17]. At the transcriptional level, attempts have been made to characterise gene expression that is consistently altered as a state of persistence develops in cells infected with chlamydial species in vitro [14], [19], [20], [21], [22]. However at present, no set pattern of proteomic and/or transcripteomic change has consistently been recognised as associated with the persistent chlamydial state [14], [19], [20], [21]. In part this is due to the great variety of in vitro cell systems available for the culture of chlamydial species, the methods employed to induce the persistent state, the varying sensitivity of chlamydial strains to IFN-γ and the varying stages of the chlamydial life cycle [14], [19], [23], [24]. The latter point may be possible to control for in vitro, however for in vivo studies, there will be inclusion bodies of varying stages of the chlamydial life cycle, whether in persistent states or not. It is clear, however, that infections require therapies directed not just against active disease, but against persistent states also [25].
In the present study, an investigation was carried out into the state of persistence of C. trachomatis, serovar E, established in HEp-2 cells in the presence of IFN-γ. The use of IFN-γ in these studies arises from work indicating that this natural cytokine is intimately involved in C. trachomatis infection, is active in human cells in vitro against these organisms [26], and is important in modulating C. trachomatis infection in a mouse model [27], [28]. IFN-γ is also reported to be increased in the seminal plasma of infertile males [29]. We chose to establish a model of C. trachomatis infection in the HEp-2 epithelial cell line, as these cells have been widely used by other workers as a control for normal, active C. trachomatis growth [30], [31], [32], and for studies of C. trachomatis persistence [13], [33]. In addition, HEp-2 cells have been used in the investigation of Chlamydophila psittaci [23] and Chlamydia pneumoniae [34], [35], [36] persistence.
The effect of IFN-γ on C. trachomatis gene expression was assessed at various times post-infection. The aims of the present study were to investigate C. trachomatis specific gene activities associated with either the establishment and/or the maintenance of C. trachomatis persistence in a cell culture system, and to attempt to correlate any observed morphological changes with changes to relative gene expression. We hypothesised that altered gene expression would be consistent with a delayed chlamydial life cycle and that sequential changes in gene expression throughout the normal chlamydial life cycle would be delayed in persistence. To highlight these changes, chlamydial genes thought to be expressed at all stages, and also at specific stages of the chlamydial life cycle were chosen, including some genes previously shown to be altered in persistence. DnaK, coding for HSP70, is expressed at steady-state levels throughout the chlamydial life cycle [37], [38] AmiA, is an N-acetylmuramoyl-l-alanine amidase involved in maintaining the integrity of the C. trachomatis cell envelope [8]. The genes ct604, and ct755, code for the HSP60 paralogs groEL2 and groEL3 respectively [39] while ftsW, and ftsK, code for proteins involved in cell division [21], [22]. Omp1, codes for the major outer membrane protein [40]. All transcripts were assessed with regard to the levels of their expression at various times post-infection by Real-Time RT-PCR, relative to 16S rRNA, considered as a housekeeping gene for the organism in these experiments.
Section snippets
C. trachomatis and cell cultures
C. trachomatis serovar E reference strain E/UW-5/CX, originally a kind gift from Professor Deborah Dean (Children’s Hospital, Oakland Research Institute, Oakland, USA) was used in all experiments, at a multiplicity of infection (moi) of 3.0 unless otherwise stated. The bacterium was propagated in flasks containing confluent monolayers of the human epithelial cell line, HEp-2 (ATCC-CCL23), known to be suitable for the growth of chlamydiae [41]. The cells were grown in EMEM + 10% (v/v) foetal
IFN-γ induces aberrant morphology of C. trachomatis inclusion bodies
The morphology of C. trachomatis grown in HEp-2 cells is shown in Fig. 1. Briefly, in the absence of IFN-γ, inclusion bodies were mainly small and round, with a relatively homogeneous appearance (Fig. 1a), whereas in the presence of IFN-γ many, but not all of the inclusion bodies were more irregular in outline, pleomorphic in shape, and appeared relatively larger and much less homogenous (Fig. 1b). The morphology of the inclusion bodies in IFN-γ-treated cells was the same irrespective of the
Discussion
There is considerable evidence to suggest that a state of persistence can arise during chlamydial infections both in humans, and in vitro using various cell culture systems [1]. Such chlamydial infections, including those associated with C. trachomatis, are by no means uncommon and appear to be the cause of several chronic debilitating diseases. However, the mechanisms that evoke and maintain the persistence of chlamydia in vivo remain elusive. In vitro, amongst a number of factors that may
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