Functional analysis of actin-like protein MreB and alternative sigma factor RpoN in chlamydial developmental cycle

Chlamydia trachomatis is an obligate intracellular pathogen of immense public health impact and is responsible for diverse disease states leading to blindness, sterility, atherosclerosis etc. in humans. Its unique biphasic developmental cycle is essentially linked to its pathogenesis. MreB, an actin-like protein, is known to be present in most rod-shaped organisms as a part of the cell elongation machinery. The role of MreB in Chlamydia is unknown, even though this gene is conserved. Recent studies have attributed diverse roles to MreB in different organisms. Our study focuses on the possible role of MreB in Chlamydia trachomatis using different approaches and techniques. To understand the functional role of MreB, we sought two strategies, in vitro and in vivo analysis. Purified recombinant chlamydial MreB was subjected to a polymerization reactions using ATP in the presence and absence of a known inhibitor of MreB polymerization (A22) and measured using laser light scattering. We observed that MreB scatters light even in the absence of ATP almost at a similar rate as in its presence. The presence of A22 increased the rate of light scattering as opposed to inhibiting it. When the MreB sequence from Chlamydia trachomatis was compared to that of other organisms, some amino acid substitutions in the conserved regions of the nucleotide binding pocket which also coincides with the A22 binding sites were evident in chlamydial MreB which could contribute to this inexplicable phenomenon. Chlamydial A22 mutant had a mutation outside the nucleotide binding pocket, confirming the possibility of other targets of A22 in Chlamydia. Quantitative RT-PCR at different time points through the developmental cycle of Chlamydia trachomatis revealed that the expression of MreB increases at later time points proposing its role in RB to EB conversion. Confocal images from immunofluorescence assay showed that MreB appeared as distinct puncta concentrated at the center of the inclusion which increased in number with time. Summarizing these observations, it could be said that MreB is involved in the RB to EB conversion in Chlamydia. Chlamydia is known to code for an alternative sigma factor, RpoN, but the role is unknown. We performed in vitro studies to elucidate the role of sigma 54 (RpoN) in Chlamydia trachomatis L2/434/Bu. Using EMSA it was observed that in the presence of RNA core polymerase, chlamydial sigma 54 could bind to the predicted RpoN promoters. The quantitative RT-PCR analysis showed a pattern which confirms its role in transcription of the predicted targets CT652.1 and CT683. Elucidating its role in vivo using modified shuttle vector is the next step to study its role in the chlamydial developmental cycle