Investigation into Potential Interactions between the Unique Transcription Factor WhiB1 and Rv2699c in Mycobacterium tuberculosis
Huey-Xian Kelly Wong
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08/03/2021
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Description
While WhiB1 is known to be an essential monomeric transcription factor highly implicated in the Mycobacterium tuberculosis (Mtb) stress response, the mechanism of transcriptional regulation by WhiB1 remains elusive. The research investigated in this study hopes to advance knowledge underlying the novel regulatory mechanism of WhiB1 in Mtb for broad applications in the epidemic of tuberculosis. In particular, we investigate the potential structure and function of Rv2699c in this role. An in-depth understanding of the pathogen’s ability to persist in hosts and Mtb’s ability to persist through its complication regulatory system will prelude the development of exciting new treatments in fight the Mtb epidemic.
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- [00:00:00.390]My name is Huey-Xian Wong though I typically
- [00:00:03.930]go by Kelly and my investigation this past academic year was into
- [00:00:08.100]the function and mechanism of the critical protein and transcription factor.
- [00:00:12.090]WhiB1 in Mycobacterium tuberculosis,
- [00:00:15.030]and how it may potentially interact with the protein RV 2 6, 9, 9 C to
- [00:00:19.770]co-regulate gene expression.
- [00:00:22.650]As a broad introduction to my investigation,
- [00:00:25.170]we are particularly interested in determining transcriptional regulation
- [00:00:28.830]mechanisms in Mycobacterium
- [00:00:30.260]tuberculosis because
- [00:00:31.260]of the significant issue that the epidemic of tuberculosis continues to
- [00:00:34.890]pose today. According to the World Health Organization,
- [00:00:38.520]tuberculosis caused by the pathogen Mycobacterium tuberculosis is a leading
- [00:00:42.600]cause of death from a single infectious
- [00:00:45.570]agent. The pathogen has a remarkably adaptive ability to initiate a wide
- [00:00:49.590]variety of survival mechanisms, such as dormancy,
- [00:00:52.110]often making many antibiotics or other modern treatments and effective since the
- [00:00:56.130]pathogen is not in a normal state of gene expression and active metabolism.
- [00:01:01.050]We'd like to study how the pathogen is able to effectively enter into this state
- [00:01:05.490]through changing its gene expression and thereby adapt in such a versatile
- [00:01:09.300]fashion to stressors. From here,
- [00:01:12.750]we narrow our focus to the WhiB-like family of proteins in mycobacterium
- [00:01:15.900]tuberculosis,
- [00:01:16.890]which has specifically been implicated in essential cellular processes in
- [00:01:20.130]Mycobacterium tuberculosis. One member in particular,
- [00:01:23.400]WhiB1, is a monomeric transcription factor that has been implicated in the
- [00:01:27.150]pathogen's response to nitric oxide stress.
- [00:01:29.880]Particularly WhiB1 is predicted to have a primary role in the pathogens,
- [00:01:33.750]entry, maintenance, and exit from dormancy. However,
- [00:01:38.220]the mechanism by which WhiB1 modulates transcriptional regulation is
- [00:01:41.610]relatively unclear still today, which becomes a broad research question:
- [00:01:45.570]How does WhiB1 mechanistically regulate transcription in Mycobacterium
- [00:01:49.440]tuberculosis?
- [00:01:51.360]What is known is that WhiB1 tightly interacts with the region four of the Sigma
- [00:01:55.980]factor A to promote interactions with RNA polymerase holoenzyme,
- [00:01:59.640]which really is unusual because transcription factors do not typically recruit
- [00:02:03.390]Sigma factor
- [00:02:04.050]A particularly for activation of transcription. From here, while several
- [00:02:08.970]possible models by which WhiB1-SigA
- [00:02:10.920]modulates transcriptional regulation have been proposed,
- [00:02:13.620]we are most interested in the possibility of an additional unknown
- [00:02:16.980]transcriptional regulator interacting with WhiB1, potentially in complex with
- [00:02:20.640]Sigma factor [inaudible] SigA, to again
- [00:02:24.000]stabilize or promote interactions with RNA
- [00:02:25.950]polymerase holoenzyme and thus affect transcription. In particular,
- [00:02:30.990]one protein [inaudible] becomes the focus of our investigation at hand
- [00:02:36.030]per online protein modeling software Raptor X.
- [00:02:38.730]We were able to determine the coevolution profile of Rv2699c with WhiB1.
- [00:02:43.740]On the right here,
- [00:02:44.610]you see the predicted model of Rv2699c and to the left,
- [00:02:48.930]you can see the probability contract contact matrix that shows the significant
- [00:02:52.950]co-evolution of Rv2699c along the horizontal edge here with WhiB1
- [00:02:57.840], which is along the vertical edge here.
- [00:03:01.180]The darker regions in the probability contact matrix particular identified a
- [00:03:04.810]conserved domain in Rv2699c, consisting of four cysteines and one histidine
- [00:03:09.850]residue that are not only highly conserved, denoting
- [00:03:13.000]its significance to Rv2699c,
- [00:03:15.040]but also likely co-evolved with Rv2699c. This co-evolution profile thus
- [00:03:19.540]suggested to us that Rv 2 6, 9, 9 C,
- [00:03:22.060]which has been so far uncharacterized, may in fact act in a coordinated manner
- [00:03:26.110]with WhiB1 and SigA to co-regulate essential transcriptional
- [00:03:29.380]processes in Mycobacterium tuberculosis.
- [00:03:33.910]Now we dive into the experimental design and methodology we pursued in our
- [00:03:38.590]investigation.
- [00:03:39.850]The primary methodology included a series of molecular cloning experiments to
- [00:03:43.540]first materialize the proteins of interest,
- [00:03:45.640]namely a SigA-WhiB1 construct and Rv 2 6, 9, 9 C construct.
- [00:03:49.570]We chose to integrate a peptide linker to ensure the complex of SigA to
- [00:03:52.870]WhiB1, as it is the protein complex that is relevant to this investigation,
- [00:03:57.160]and additionally integrated tags such as the six histidine tag for later
- [00:04:00.550]purification methods,
- [00:04:02.050]The subsequent genes encoding SigAc82Btip-Linker-WhiB1
- [00:04:05.770]were cloned into the expression plasmid pET28a and the gene
- [00:04:10.600]encoding the putative mycobacterium tuberculosis transcription factor of
- [00:04:14.320]interest Rv 2 6, 9, 9 C was cloned into the expression plasmid pCDF-1b
- [00:04:19.240]by Genscript.
- [00:04:22.570]Utilizing those aforementioned tags,
- [00:04:24.760]namely the six histidine tag, affinity purification methodology was first used
- [00:04:28.780]to purify the expressed proteins. For 6His-Rv2699c, significant impurities
- [00:04:33.310]appeared to be present in initial SDS-PAGE analyses and size exclusion
- [00:04:36.940]gel filtration
- [00:04:37.840]additionally followed to purify protein samples of further impurities. Here
- [00:04:41.830]the Superdex200 gel filtration elution curve is pictured and the major peak
- [00:04:45.790]here indicative of the desired
- [00:04:48.040]6His-Rv2699c protein was collected and analyzed via UV vis
- [00:04:52.120]spectrometry, again, as seen here in figure three.
- [00:04:57.370]In addition, throughout purification procedures,
- [00:04:59.530]SDS page analyses were conducted for measures of purity of collected protein
- [00:05:03.370]samples. As can be seen here,
- [00:05:07.930]the SDS-PAGE analysis of purified 6His-Rv2699c
- [00:05:12.680]particularly posed a few problems, namely,
- [00:05:15.090]the shift upwards of the expected band for the final protein sample.
- [00:05:18.840]But, largely we were able to confirm the presence of 6His-Rv2699c
- [00:05:22.950]from purification procedures.
- [00:05:24.930]We suspect that there may be some potential oligomerization of the 6His-Rv2699c
- [00:05:29.400]that's causing the band to shift upward of where it is expected,
- [00:05:31.890]but further tests are being performed now to confirm this possibility.
- [00:05:37.080]Now having purified samples of each protein,
- [00:05:39.240]we move on to an analysis of Rv2699c
- [00:05:41.430]itself by x-ray fluorescence spectroscopy.
- [00:05:44.280]We were primarily interested in conducting an elemental analysis
- [00:05:47.100]first of Rv2699c, particularly
- [00:05:49.350]to investigate if there was any zinc binding ability of the cysteines in the
- [00:05:52.260]conserved domain of Rc2699c
- [00:05:53.340]Here in the x-ray emission
- [00:05:58.070]analysis of the purified 6His-Rv2699c,
- [00:06:00.140]a notable peak is seen here, and was confirmed as characteristic of zinc based on
- [00:06:04.160]zinc's atomic structure and its associated reference values for x-ray emission
- [00:06:07.520]spectroscopy. Additionally,
- [00:06:09.680]in the X-ray absorption analysis of the purified 6His-Rv2699c
- [00:06:13.790]as seen in figure seven,
- [00:06:17.120]the near edge, or where the absorption curve changed
- [00:06:20.390]the fastest, of the absorption curve were determined to be around
- [00:06:24.380]9,662 electrovolts. This again, based on reference curves,
- [00:06:28.880]not only confirmed the presence of zinc,
- [00:06:30.620]but support the presence of zinc in relation to sulfur
- [00:06:33.350]as opposed to solvent atoms, which again
- [00:06:35.480]strongly suggests the interaction of zinc with the sulfur atoms characteristic
- [00:06:39.090]in cysteine, such as those in the conserved domain of Rv2699c.
- [00:06:45.260]This largely concludes this summer's investigation.
- [00:06:49.400]And from gathered data,
- [00:06:50.630]we were able to determine strong evidence of the zinc binding ability of
- [00:06:54.290]Rv2699c,
- [00:06:55.820]which gives insight into the structure and potentially function of this un
- [00:06:59.690]characterized potential transcription factor.
- [00:07:02.180]This project is only really just the beginning, as from here further studies
- [00:07:05.660]into characterization of Rv2699c itself via protein crystallography are
- [00:07:10.400]planned. In addition,
- [00:07:11.690]we'd like to further analyze how Rv2699c may interact with WhiB1 and/or
- [00:07:16.340]SigA via methods such as analytical column and pull down essays.
- [00:07:20.570]That's essentially the conclusion of my presentation,
- [00:07:22.700]so I hope you all enjoyed it.
- [00:07:24.680]I'd like to acknowledge the amazing mentorship I received by my faculty mentor,
- [00:07:28.520]Dr. LiMei Zhang and the assistance of all the members of the Zhang group, [inaudible]
- [00:07:33.020]who all assisted me with this project.
- [00:07:36.410]Feel free to reach out with any further questions as I would love to discuss,
- [00:07:40.310]but otherwise, bye.
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