Genus-Specific Antibiotics Targeted to Gram-Positive Primase

Clarissa Mason Author

07/31/2021 Added

25 Plays

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A comparison of the primase and helicase structures of gram-positive and negative bacteria and how that applies to a novel antibacterial therapy

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[00:00:01.030]Hello. My name is Clarissa Mason
[00:00:02.640]and I am studying a genus-specific antibiotics targeted
[00:00:05.040]to gram-positive primase
[00:00:06.140]with Jessica Periago and Dr. Mark Griep.
[00:00:09.110]Deaths from bacterial infections are on the rise
[00:00:11.040]as a result of the antibiotic resistance crisis.
[00:00:13.340]According to the CDC, an average of 35,000 people die
[00:00:16.200]every year from bacterial infections in the United States.
[00:00:19.170]Of those people, 20,000 die
[00:00:20.630]from methicillin-resistant staphylococcus aureus,
[00:00:22.694]meaning that MRSA deaths make up 56.5% of deaths
[00:00:25.732]from bacterial infections.
[00:00:27.695]These infections occur when harmful bacteria infiltrate
[00:00:30.690]the human body systems and grow exponentially.
[00:00:33.010]The concept of bacterial replication is relatively simple.
[00:00:36.640]Each cell goes through mitosis,
[00:00:38.090]the cellular reproduction cycle that begins
[00:00:39.940]with the replication of the genetic material.
[00:00:42.875]During the last phase of mitosis,
[00:00:44.990]the cell then splits into two identical daughter cells.
[00:00:47.465]In bacteria, this process occurs rapidly
[00:00:49.788]and it's a very effective mechanism
[00:00:51.580]for the spread of an infection throughout the body.
[00:00:53.601]Typically, bacterial infections will be treated
[00:00:56.340]with antibiotics like methicillin.
[00:00:58.640]These drugs are small molecules that target enzymes
[00:01:00.940]and fully grown bacterial cells to cause cell death
[00:01:03.090]and kill the infection.
[00:01:04.304]When methicillin is used against staph,
[00:01:06.720]infections that do not yet have resistance,
[00:01:08.589]the antibiotics specifically targets
[00:01:10.510]the production of peptidoglycan,
[00:01:12.150]a structural protein in the bacterial cell membrane.
[00:01:14.246]When the production of this protein is inhibited,
[00:01:16.760]the cell ruptures and the infection is stopped.
[00:01:19.000]The problem we are facing is that of antibiotic resistance.
[00:01:21.622]Bacteria are able to transfer genetic information
[00:01:24.590]to one another after death through plasmids.
[00:01:26.807]The information in these plasmids becomes a part
[00:01:29.200]of the genetic code of the living bacteria,
[00:01:30.880]and it allows them to fight the antibiotic
[00:01:32.320]that killed the other bacteria in the colony.
[00:01:34.252]To address this problem,
[00:01:35.830]we are developing a novel and a bacterial therapy
[00:01:37.660]that will target the replication process
[00:01:39.300]instead of the full grown cells.
[00:01:40.798]Though our therapy will not necessarily
[00:01:42.500]cause immediate cell death
[00:01:43.580]in the way that antibiotics would,
[00:01:45.010]it will stop the bacteria from replicating
[00:01:46.860]and effectively stop the infection from spreading,
[00:01:49.390]so the immune system can fight it off.
[00:01:52.340]We began by looking at the confirmations of primase
[00:01:54.455]and interfaces at which the residues interact
[00:01:56.680]with helicase and the inhibitor.
[00:01:59.140]The primase structure
[00:01:59.980]for geobacillus stearothermophilus has two confirmations:
[00:02:02.580]open and closed.
[00:02:03.725]In the figure, the open confirmation is illustrated
[00:02:05.459]in the top structures.
[00:02:07.500]Interfaces A and B represent
[00:02:08.618]the helicase' primase interaction,
[00:02:11.260]in which one primase spines to the NTD
[00:02:13.220]of two different helicase proteins.
[00:02:15.766]When this interface forms,
[00:02:17.380]bacterial replication can proceed.
[00:02:19.092]The closed confirmation does not bind to helicase,
[00:02:21.762]and has a groove for a small molecule
[00:02:23.850]that can move equilibrium
[00:02:24.980]to favor the close confirmation.
[00:02:27.350]Our goal is to find a small molecule to bind
[00:02:29.290]to interface C, represented by the pink triangle,
[00:02:31.680]which would block the second helicase protein
[00:02:33.420]from binding to interface B.
[00:02:36.350]This summer, we've been analyzing the primase
[00:02:37.947]and helicase sequences
[00:02:39.170]with multiple, medically relevant bacteria
[00:02:41.040]to determine whether they have a similar 3D structure.
[00:02:43.582]Previous studies in our lab have led
[00:02:45.674]to the determination of the primase structure
[00:02:47.770]of geobacillus stearothermophilus,
[00:02:49.420]which was also found to have a very similar structure
[00:02:51.300]to that of staphylococcus aureus.
[00:02:53.053]In addition, our lab also discovered a small molecule
[00:02:55.270]that bound preferentially to the closed confirmation
[00:02:57.580]of staph aureus' primase.
[00:02:59.212]Our hypothesis is that the primase structures
[00:03:01.530]of other bacteria will fold, similar to that
[00:03:03.210]of staph and geobacillus.
[00:03:05.500]Using this principle,
[00:03:06.484]we created multiple sequence alignment,
[00:03:08.930]using the cholesterol omega program.
[00:03:11.083]And then, created phylogenetic trees to analyze
[00:03:13.580]the relationships between the gram-positive
[00:03:15.140]and gram-negative bacterial replication enzyme sequences.
[00:03:18.120]The major deciding factor in the choice
[00:03:20.120]of the bacteria being considered in the study
[00:03:21.790]was the tree shown here for primase sequence.
[00:03:24.390]There was a very distinct split in the tree
[00:03:26.380]between our chosen gram-negative
[00:03:27.820]and gram-positive sequences.
[00:03:30.050]The same was observed with the helicase tree,
[00:03:32.060]with the same organisms on each side of the divide,
[00:03:34.510]with the gram-positive and gram-negative bacteria.
[00:03:37.610]This observation allowed us to conclude
[00:03:39.220]that there's a distinctive difference
[00:03:40.430]in the primase sequences
[00:03:41.440]between the different types of bacteria.
[00:03:43.570]Once we had the trees, we also began looking
[00:03:45.820]at some preliminary homology models of the primase
[00:03:48.023]and helicase 3D structures.
[00:03:50.250]We found that the bacteria of opposite categories
[00:03:52.120]were not good templates for each other
[00:03:53.410]in these homology models.
[00:03:54.810]In our attempt to fold gram-negative primase
[00:03:56.700]on the gram-positive template,
[00:03:58.380]we also concluded that the gram-negative primase
[00:04:00.298]likely does not adopt the close confirmation
[00:04:02.950]in the same way that the gram-positive primase enzymes do,
[00:04:05.740]suggesting that interface B does not form,
[00:04:07.990]and cannot be a target for this therapy.
[00:04:10.270]From there on out,
[00:04:11.103]we began focusing in more on the gram-positive bacteria.
[00:04:15.780]The following sequence alignment shows
[00:04:17.410]the chosen gram-positive bacteria,
[00:04:19.150]primase C terminal dominant sequences.
[00:04:21.397]This part of the primase protein
[00:04:23.080]is typically very species-specific,
[00:04:25.080]as opposed to the high conservation observed
[00:04:26.860]in other domains of the protein.
[00:04:28.343]We identified the amino acids
[00:04:30.380]that were targeted in staph aureus in the previous study,
[00:04:32.720]and examined both the alignment and those 3D structures
[00:04:35.340]from the homology models based on the staph
[00:04:36.664]and geobacillus' templates at the three interfaces.
[00:04:40.360]Interface A is shown here,
[00:04:41.930]both in the alignment
[00:04:42.770]and on the models of staph in purple.
[00:04:44.900]Interface B in orange, and interface C in green.
[00:04:48.520]Throughout all three of these interfaces,
[00:04:50.120]there is very low conservation.
[00:04:51.860]However, when looking at the alignment,
[00:04:53.390]we found much higher sequence conservation
[00:04:55.730]among the individual genera.
[00:04:57.212]The small molecule found previously matched
[00:04:59.730]to specific tyrosine in interface B.
[00:05:01.830]This residue was found only in staph bacteria
[00:05:04.570]and geobacillus stearothermophilus.
[00:05:06.824]Any bacteria that does not have this tyrosine
[00:05:08.947]would probably not bind to the inhibitor as well,
[00:05:11.710]as the tyrosine provides a crucial hydrophobic interaction.
[00:05:17.020]In the open confirmation, interface B
[00:05:18.670]is positioned on the longer helix,
[00:05:20.540]closer to the five helix bundle in the primase structure.
[00:05:23.009]As the close confirmation forms,
[00:05:24.870]interface C incorporates residues near interface B,
[00:05:27.780]as shown in the second 3D structure.
[00:05:30.250]This prevents the helicase from binding to interface B
[00:05:32.571]in the close confirmation,
[00:05:34.160]as the inhibitor will block this interface.
[00:05:37.030]These discoveries led us to the conclusion
[00:05:38.920]that interface C may be a viable drug target
[00:05:40.970]for a genus-specific antibiotic
[00:05:42.480]that targets DnaG primase's and staphylococcus bacteria.
[00:05:46.530]As we continue with this study,
[00:05:47.930]we plan to find and document small molecules
[00:05:50.580]that will fit in the groove found
[00:05:52.330]in the staphylococcus bacteria.
[00:05:53.980]There will be effective inhibitors,
[00:05:55.210]as well as analyze the phyletic differences
[00:05:57.150]in the zinc binding domain of the DnaG primase
[00:05:59.850]in both the gram-negative and gram-positive bacteria
[00:06:01.920]to learn more about the target molecule
[00:06:03.410]for this novel antibacterial therapy.
[00:06:05.775]Thank you to the UNL summer UCARE program
[00:06:08.150]and the Nebraska EPSCOR grant program
[00:06:09.880]for their support in this project.
[00:06:11.680]Thank you for your time and have a great day.

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Genus-Specific Antibiotics Targeted to Gram-Positive Primase

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