Background
Keratinocytes are the first port of entry for Chlamydia trachomatis of the lymphogranuloma venereum (LGV) biovar which causes LGV. However LGV pathogenesis and gene expression studies are usually performed in cells which are not the native host cells.
Methods
To investigate the effect of temperature on the timing of the expression of genes that control chlamydial differentiation and replication in keratinocytes. HaCaT cells were grown in 12-well plates, inoculated with chlamydia LGV strains L2 434 and US151 and with G strain E. The cells were incubated at 33oC or 37oC in a humidified environment with 5 % CO2. At 2, 12, 24, 36 and 48 hours post-infection (hpi) for cells incubated at 37°C and in addition at 60 and 72 hpi for those incubated at 33°C, total RNA was harvested and converted to cDNA which was then analyzed by real time PCR for the expression of groEL-1, incB, pyk, hctA and omcB. Transmission electron microscopy (TEM) was performed to confirm the stage of the developmental cycle that the organism was at inside the keratinocytes
Results
RT-PCR analysis showed that the early cycle genes were expressed throughout the cycle at 37oC and 33oC as expected. At 37oC, the mid and late cycle genes were also expressed throughout the cycle for all the strains tested. At 33oC, the mid cycle genes showed gene expression at different time points compared to 37oC. However, the expression of late cycle genes in L2 434 and E at 33oC was in keeping with the published literature, with the exception of L2 US151 which showed gene expression from the early time points.
Conclusion
Mid and late-cycle chlamydial gene expression in HaCaT cells differs from published research conducted in HeLa cells at 37oC. Ttemperature has an effect on the level of chlamydial gene expression when grown in keratinocytes. At 2 hours post infection chlamydia still retains its EB structural conformation at 37oC. The L2 reference strain 434 differs from the clinical L2 US151as indicated by the difference in the gene expression pattern. This study also showed chlamydial gene expression differs between clinical LGV and G isolates at 33oC.
Keywords: Chlamydia trachomatis; Gene expression; Life cycle; Temperature
