Bioinformatic identification and characterization of cytokinetic regulators in Mtb
Date
2013
Authors
Crew, Rebecca M., author
Slayden, Richard, advisor
Jackson, Mary, committee member
Hanneman, William, committee member
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Abstract
A fundamental lack of understanding of Mtb regulation during latent tuberculosis infections (LTBI), which comprises the vast majority of tuberculosis cases, has hindered global eradication efforts. To elucidate mechanisms associated with transition to the non-replicating persistent (NRP) state associated with LTBI, we set out to identify regulators involved in cell division control in Mtb. Bioinformatic analysis identified rv1708 as encoding a MinD-like protein putatively involved in septum placement, and rv2216 as encoding a potential SOS-associated cell division inhibitor, SulA. Bioinformatic-based assessments of orthology revealed a differential lineage than anticipated for the proteins encoded by both open reading frames (ORFs). We describe these two novel regulators in Mtb here for the first time. It was found that Rv1708 lacks regions vital for MinD function and shows greater similarity with the Soj protein from Bacillus sp. involved in the regulation of sporulation and timing of division. Due to these similarities we have re-named Rv1708 as SojMtb. Significantly, SojMtb shows potential as a cytokinetic and dormancy regulator both by homology, morphology, and growth kinetic analysis. Overexpression of sojMtb attenuates growth and elicits filamentation characteristic of a disruption in early division, similar to Soj activity in other organisms. Given the role of Soj in the control of dormancy phenotypes in Bacillus sp. we believe SojMtb serves as an important regulator during dormancy transitions in mycobacteria, as associated with the development of LTBI. Although Rv2216 was initially identified by homology to SulA proteins, analysis of orthology indicates greater similarity with a separate group of widely conserved yet poorly defined cell division regulatory proteins. Thusly, we have re-name rv2216 as cdr for cell division regulator. Cdr proteins share limited similarity to SulA: enough to be mis-identified in organisms lacking a true SulA but insufficient to infer similar functionalities. Cdr proteins are present in hundreds of organisms through different walks of life, yet this work presents the first characterization of their effects on cellular activity. Induction of the SOS response by Mitomycin C treatment did not induce cdr expression, supporting our classification Cdr proteins separate from SulA. Overexpression of cdr resulted in a bimodal increase in cell length without an apparent effect on growth kinetics, suggesting Cdr stimulation of cellular elongation relative to division. Profiling of cell cycle discriminant genes in response to cdr overexpression corroborates this hypothesis, showing an induction of late division events associated with the production of new plasma membrane and cell wall components. Sub-cellular localization studies using an inducible Cdr-GFP fusion protein revealed cell cycle-dependent localization to the inner membrane at sites involved in cell wall and plasma membrane growth and remodeling. Furthermore, global transcriptional analysis revealed a unique profile of adaptive programs associated with hypoxia-associated NRP, de novo lipid synthesis and phospholipid/triacylglycerol turnover. These processes are required for normal growth and promote homeostasis during times of stress by preventing and repairing oxidative damage to membrane constituents in diverse organisms. Importantly, Cdr represents a novel regulatory class of proteins with broad representation in all classifications of life, potentially involved in division and stress responses associated with dormancy, and is described here for the first time in Mtb. The foundation provided here, both for SojMtb and Cdr, provides insight into the regulatory mechanisms employed during NRP transitions associated with LTBI, and will aid in the development and implementation of more targeted studied in the future.
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Subject
functional genomics
tuberculosis
rv2216
rv1708