An Investigation of the Function and Mechanism of Critical Protein WhiB1 in Mycobacterium tuberculosis

Huey-Xian Kelly Wong Author

04/05/2021 Added

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This video is a presentation of the findings of my investigation into the function and mechanism of critical protein WhiB1 in Mycobacterium tuberculosis over this past academic year.

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[00:00:01.832]Hello, my name is Huey-Xian Wong,
[00:00:03.942]though I typically go by Kelly,
[00:00:06.035]and my investigation this past academic
[00:00:08.421]year was into the function and mechanism
[00:00:10.381]of the critical protein and transcription
[00:00:12.441]factor WhiB1 in Mycobacterium
[00:00:14.284]tuberculosis. As a broad introduction
[00:00:17.250]to my investigation, we are interested
[00:00:19.570]in investigating the transcriptional
[00:00:21.453]regulation mechanisms at play in
[00:00:23.560]Mycobacterium tuberculosis because of the
[00:00:25.565]epidemic of tuberculosis as a significant
[00:00:28.052]issue in the status quo today. According
[00:00:30.405]to the World Health Organization in 2019,
[00:00:32.946]tuberculosis, caused by the pathogen
[00:00:35.101]Mycobacterium tuberculosis, was the
[00:00:37.307]leading cause of death from a single
[00:00:39.093]infectious agent. While
[00:00:40.337]treatment regimens
[00:00:41.337]have certainly been developed today,
[00:00:43.141]the problem is that
[00:00:44.177]the fight against
[00:00:45.043]the tuberculosis epidemic is complicated
[00:00:47.137]by the evolution of multi-drug
[00:00:48.934]resistant strains of Mycobacterium
[00:00:50.588]tuberculosis. Additionally, the
[00:00:52.485]pathogen has an adaptive ability to
[00:00:54.727]initiate essentially a state of dormancy,
[00:00:57.003]or enter into a state in which its
[00:00:58.982]metabolic processes are shut down under
[00:01:01.524]hypoxic or nutrient-starved stress often
[00:01:04.124]making many antibiotics or other treatment
[00:01:06.646]regimens ineffective since the pathogen
[00:01:08.941]is not in a state of active metabolism.
[00:01:11.254]We’d like to study how the pathogen is
[00:01:13.456]able to effectively enter into this state,
[00:01:15.598]evade typical treatment regimens, and
[00:01:17.838]adapt in such a versatile fashion to
[00:01:20.023]environmental stresses to persist. From
[00:01:23.520]here we narrow our focus to the WhiB-like
[00:01:25.620]family of proteins in Mycobacterium
[00:01:27.350]tuberculosis, which are widely conserved
[00:01:29.548]across Actinobacteria and have
[00:01:31.345]specifically been implicated in essential
[00:01:33.420]cellular processes in Mycobacterium
[00:01:35.579]tuberculosis. One member in particular,
[00:01:38.054]WhiB1, is a monomeric transcription factor
[00:01:40.505]that has been implicated in the
[00:01:42.028]pathogen’s response under nitric oxide or
[00:01:44.055]hypoxic environmental stress. Thus, WhiB1
[00:01:46.409]may have a primary role in the
[00:01:48.358]pathogen’s entry, maintenance, or
[00:01:50.190]exit from dormancy, which as we
[00:01:51.996]discussed, is a significant contributor to
[00:01:54.065]why the pathogen is so difficult to
[00:01:55.809]fight with traditional treatment regimens.
[00:01:58.214]However, the mechanism by which
[00:02:00.417]WhiB1 modulates transcriptional
[00:02:02.193]regulation in Mycobacterium tuberculosis
[00:02:04.198]is relatively unestablished as of yet.
[00:02:06.948]Recently, the Zhang lab did find however,
[00:02:09.429]that WhiB1 likely modulates
[00:02:11.338]transcriptional regulation in a
[00:02:13.085]noncanonical manner. While several
[00:02:15.210]possible models by which WhiB1 modulates
[00:02:17.190]transcriptional regulation were proposed
[00:02:19.382]by the Zhang group, we are most interested
[00:02:21.431]here in the possibility of an additional,
[00:02:23.433]unknown transcriptional regulator
[00:02:25.160]interacting with WhiB1 in complex with a
[00:02:27.483]Sigma Factor SigA to stabilize binding
[00:02:30.244]with RNA Polymerase holoenzyme and thus
[00:02:32.849]affect transcription. You can really see
[00:02:35.133]this in Figure 2, in which
[00:02:37.082]a possible schematic
[00:02:38.082]is illustrated in the lower figure, again
[00:02:40.187]with that unknown regulator interacting
[00:02:42.516]with the WhiB1-SigA complex to overall
[00:02:46.005]interact with RNAP and
[00:02:48.091]potentially activate
[00:02:49.091]specific promoters and
[00:02:50.627]transcriptional mechanisms. This really
[00:02:53.189]gives rise to our hypothesis
[00:02:54.561]though that there is an additional
[00:02:56.387]regulator at play that interacts with the
[00:02:58.612]WhiB1-SigA complex to influence
[00:03:00.685]transcriptional regulation.
[00:03:03.552]One more thing I’d like to give
[00:03:05.306]some background on before moving
[00:03:06.946]on is the DosR regulon. In addition to
[00:03:09.050]investigating WhiB1, and what additional
[00:03:11.493]regulator may interact in coordination
[00:03:13.438]with WhiB1 to control transcriptional
[00:03:15.541]regulation, we focused on this series
[00:03:17.558]of 49 genes implicated in the DosR
[00:03:19.586]regulon, which has been established
[00:03:21.887]to be a crucial component to modulating
[00:03:23.845]that dormancy response observed in
[00:03:25.510]tuberculosis and thus making it relevant
[00:03:27.478]to our investigation of WhiB1’s potential
[00:03:29.648]role in the dormancy or enduring
[00:03:31.709]hypoxic response in Mycobacterium
[00:03:33.458]tuberculosis.
[00:03:36.359]Now we move onto the experimental design
[00:03:38.650]and methodology we pursued in our
[00:03:40.797]investigation.
[00:03:41.630]We had to pursue an investigation that
[00:03:43.503]was largely "dry lab" due to the COVID-19
[00:03:45.691]pandemic restricting how much in-lab,
[00:03:47.699]in-person labwork was able to be conducted
[00:03:50.770]Prior to the pandemic, however,
[00:03:52.554]the Zhang lab had conducted an RNA
[00:03:54.467]sequence analysis of the WhiB1
[00:03:56.024]transcriptome in Mycobacterium
[00:03:58.003]smegmatis, a non-pathogenic model
[00:03:59.969]organism for studying Mycobacterium
[00:04:01.819]tuberculosis, analyzing which genes in the
[00:04:04.214]WhiB1 transcriptome demonstrated a change
[00:04:06.764]in expression levels upon significant
[00:04:08.819]mutation of WhiB1 itself. If a gene
[00:04:11.904]demonstrated a change in expression
[00:04:13.635]level in genes, it indicated a strong
[00:04:15.597]possibility of WhiB1 influencing or
[00:04:17.653]potentially interacting with these
[00:04:19.606]genes in a significant manner as we would
[00:04:21.834]expect an unknown regulator
[00:04:23.277]to do. Having the results of this RNA
[00:04:26.152]sequence analysis of WhiB1, we needed
[00:04:28.574]to further narrow down which of the
[00:04:30.456]genes demonstrating a change in expression
[00:04:32.537]levels may be a candidate for that
[00:04:34.425]unknown regulator co-controlling
[00:04:36.093]transcriptional regulation with WhiB1.
[00:04:38.429]This is really where the DosR
[00:04:40.154]regulon genes come in.
[00:04:41.634]As we stated previously, the DosR
[00:04:43.478]regulon is a critical component in the
[00:04:45.747]enduring hypoxic response or this
[00:04:47.610]phenotype of dormancy in tuberculosis.
[00:04:50.305]Thus, we reasoned that those genes
[00:04:52.121]that require the binding of DosR for
[00:04:53.922]induction of the hypoxic response may
[00:04:56.136]also interact or bind to WhiB1 in a
[00:04:58.020]similar manner to again induce that
[00:04:59.884]characteristic hypoxic dormancy response
[00:05:01.884]we’re interested in. We performed a
[00:05:03.897]literature review first and created a
[00:05:06.032]comprehensive list of 49
[00:05:07.901]Mycobacterium tuberculosis genes
[00:05:10.047]requiring the interaction with DosR for
[00:05:12.417]induction. However, the RNA sequence
[00:05:15.577]analysis we mentioned earlier, again,
[00:05:17.834]identified genes in Mycobacterium
[00:05:19.689]smegmatis rather than Mycobacterium
[00:05:21.610]tuberculosis. Thus, the next step of
[00:05:23.695]our research was in identifying all the
[00:05:25.646]Mycobacterium smegmatis
[00:05:27.156]orthologues, if they existed, of the 49
[00:05:29.690]identified Mycobacterium tuberculosis DosR
[00:05:32.583]genes we had already identified.
[00:05:36.165]As you can see in
[00:05:37.425]this included Figure 3, if there was not
[00:05:39.699]an established Mycobacterium smegmatis
[00:05:42.106]orthologue for the Mycobacterium
[00:05:43.819]tuberculosis gene already, we identified
[00:05:45.846]possible Mycobacterium smegmatis
[00:05:47.672]orthologues through
[00:05:48.772]SWISS-MODEL and BLAST sequence
[00:05:50.493]analysis, which identified Mycobacterium
[00:05:52.800]smegmatis homologues on the basis of
[00:05:54.915]sequence and structure homology, allowing
[00:05:57.110]us to identify likely Mycobacterium
[00:05:59.407]smegmatis orthologues.
[00:06:01.158]For example, in the case of Mycobacterium
[00:06:03.730]tuberculosis gene
[00:06:04.824]Rv0082, requiring the interaction of
[00:06:07.387]DosR for induction, the identified
[00:06:09.297]Mycobacterium smegmatis orthologue was
[00:06:11.848]MSMEG_2062, identified through a generated
[00:06:14.699]SWISS-MODEL as provided here
[00:06:17.428]as opposed to simple reference of the TB
[00:06:20.621]database or other established sources.
[00:06:24.198]Finally, after obtaining a master list
[00:06:26.098]of genes in Mycobacterium smegmatis that
[00:06:28.205]were associated with the DosR regulon,
[00:06:30.072]this list was cross-referenced with those
[00:06:32.083]genes listed as demonstrating a change
[00:06:34.025]in expression levels upon modification of
[00:06:36.025]WhiB1 as listed in the RNA-seq file.
[00:06:39.469]Ultimately, our results and data
[00:06:41.227]show and identify 13 genes that are strong
[00:06:43.573]candidates for playing a role as a
[00:06:45.299]regulator in coordination with WhiB1 to
[00:06:47.628]influence transcriptional regulation.
[00:06:49.827]These 13 smegmatis genes are tabulated
[00:06:52.221]in Table 1 and the gene product encoded
[00:06:54.273]was additionally included. What was
[00:06:56.338]interesting was of these final 13 gene
[00:06:58.980]candidates
[00:06:59.849]out of originally 49 genes in the DosR
[00:07:01.994]regulon, many code for a gene product that
[00:07:06.312]is a conserved hypothetical protein,
[00:07:08.672]whose function is not completely
[00:07:10.350]clear yet. In that sense, again, these
[00:07:12.268]genes could very well act in that
[00:07:14.149]yet-uncharacterized manner with
[00:07:15.737]WhiB1 as a transcriptional regulator.
[00:07:18.149]From here, we’d like to emphasize that
[00:07:20.201]our investigation, while fruitful in
[00:07:22.119]yielding these 13 gene candidates as
[00:07:24.508]possibly coding for a gene product that
[00:07:26.444]interacts with WhiB1 in a concerted
[00:07:28.512]manner, our data and conclusions
[00:07:31.285]still need to be tested
[00:07:32.855]under experimental conditions. The
[00:07:35.056]next steps will really be in performing
[00:07:36.962]a series of in vitro pull-down assays
[00:07:38.775]in laboratory to determine if any of
[00:07:40.552]these 13 gene candidates, when used as
[00:07:42.371]"bait" in pull-down assays, can
[00:07:44.018]demonstrate significant interaction with
[00:07:45.986]WhiB1-SigA complex
[00:07:47.690]upon pulling this protein complex down.
[00:07:50.730]So, that’s essentially the conclusion of
[00:07:52.763]my presentation, and I hoped you all
[00:07:54.921]enjoyed it. I’d like to acknowledge the
[00:07:56.951]amazing mentorship I received by
[00:07:58.730]my faculty mentor, Dr. Limei Zhang,
[00:08:00.882]as well as all the members of the
[00:08:02.515]Zhang group.
[00:08:03.445]I'd also obviously like to acknowledge the
[00:08:05.706]funding that UCARE has provided
[00:08:09.126]to me in allowing
[00:08:10.126]this investigation to proceed.
[00:08:13.053]I look forward to answering any
[00:08:14.944]questions in the future.
[00:08:17.049]Thank you, bye!

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An Investigation of the Function and Mechanism of Critical Protein WhiB1 in Mycobacterium tuberculosis

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