Structure of Clostridium perfringens Type IV Pili

Alexander Meyer, Josephine Liess Author

04/01/2021 Added

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Clostridium perfringens is a Gram-positive anaerobic bacterium. Preliminary data suggest type IV pili are key for cell adhesion during C. perfringens infection. This study aims to identify the structure of C. perfringens Type IV pili, providing information about a poorly understood class of bacterial appendages.

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[00:00:01.050]Hi,
[00:00:01.500]my name is Alex Meyer and I'm a senior biochemistry major at the University of
[00:00:05.340]Nebraska Lincoln.
[00:00:06.740]And my name's Josie Liess.
[00:00:08.090]I'm a junior biology major at the University of Nebraska.
[00:00:11.570]Alex and I are working under Dr.
[00:00:13.040]Kurt Piepenbrink to explore the structure of Clostridium perfringens type IV
[00:00:16.550]pili.
[00:00:17.600]Our project focuses on a class of bacterial appendages called type IV pili.
[00:00:22.490]Type IV pili are thin hair-like fibers that can extend and retract from the
[00:00:26.450]surface of bacteria. There are built from small protein subunits called pilins,
[00:00:30.830]which wrap around one another in a helical fashion to produce the helical pilus
[00:00:35.270]fiber. Type IV pili are used for different functions in different bacteria,
[00:00:39.530]including motility, biofilm formation,
[00:00:42.080]natural competence and adherence to host cells.
[00:00:45.500]Type IV pili were originally characterized in Gram negative bacteria,
[00:00:48.710]like Escherichia coli and
[00:00:50.530]Salmonella species.
[00:00:51.530]Type IV pili have more recently been discovered in Gram-positive species
[00:00:55.010]as well.
[00:00:56.030]The first Gram-positive species in which type IV pili were characterized was
[00:00:59.540]Clostridioides difficile,
[00:01:01.310]where type IV pili are used for biofilm formation that can contribute to
[00:01:04.730]antibiotic resistance. C. difficile was previously placed in the Clostridia
[00:01:09.110]genius where all members of the genus have genes to produce type IV pili. However,
[00:01:14.090]C difficile has been recently reclassified due to its genetic divergence
[00:01:18.110]from other Clostridia species. Likewise,
[00:01:21.590]while other Clostridia possess genes for type IV pili
[00:01:25.250]they differ significantly from the genes found in the better understood C.
[00:01:29.000]difficile.
[00:01:30.260]Our project looks at one member of the
[00:01:32.680]Clostridia genus, Clostridium
[00:01:33.680]perfringens. C. perfringens is a Gram-positive bacteria well known as a
[00:01:38.060]food born pathogen that has developed antibiotic resistance to drugs like
[00:01:41.570]tetracycline and clindamycin. C. perfringens produces type IV pili as
[00:01:46.310]seen in this image,
[00:01:47.570]but preliminary data by Stephen Melville's group from Virginia Tech suggest that
[00:01:51.920]type IV pili in C. perfringens are less like the type IV pili in C.
[00:01:55.970]difficile and more similar to type IVa
[00:01:58.280]pili found in Neisseria which are responsible for adherence to host cells.
[00:02:03.020]Based on this,
[00:02:03.770]we hypothesized that C. perfringens type IV pili are more like those
[00:02:07.670]in Neisseria, which use their type IV pili for host cell adhesion.
[00:02:11.420]We're interested in exploring why C. perfringens has type IV pili that seem
[00:02:15.620]so different from its close relative C. difficile.
[00:02:18.500]We aim to solve the structure of type IV pili in C. perfringens to identify
[00:02:22.550]what differences exist between type IV pili in C. difficile in C.
[00:02:26.570]perfringens.
[00:02:27.620]This will help create a model for type IV pili in similar Clostridia
[00:02:30.740]species
[00:02:31.550]and will give more knowledge about a poorly understood class of bacterial
[00:02:34.910]appendages.
[00:02:36.380]We're focusing our study on the pilin protein PilA2
[00:02:39.170]from C. perfringens type IV pili,
[00:02:42.080]which is the protein that makes up the vast majority of the pilus fiber. We're
[00:02:45.740]currently exploring PilA2 from two different strains of C. perfringens,
[00:02:49.340]which are strain 13 and strain ATCC 13124.
[00:02:52.310]Strain 13 was chosen because it was the strain used by Dr.
[00:02:56.570]Stephen Melville in the preliminary preliminary data discussed earlier. Strain
[00:03:01.450]ATCC 13124
[00:03:02.920]was chosen because this is the type strain for C. perfringens.
[00:03:07.410]PilA2 is an insoluble protein,
[00:03:09.030]which makes crystallization efforts challenging. To overcome this,
[00:03:12.630]we have expressed both strains of PilA2
[00:03:14.760]as a fusion protein with surface entropy reduced maltose binding protein,
[00:03:18.840]where the maltose binding protein functions as a crystallization chaperone.
[00:03:22.920]While PilA2 does not crystallize well on its own,
[00:03:25.620]maltose binding protein can crystallize much more readily. By attaching PilA2
[00:03:29.810]to maltose binding protein.
[00:03:31.950]we can crystallize the maltose binding protein and the PilA2 will co-
[00:03:35.530]crystallize alongside it. This fusion protein, which we call MBPilA2
[00:03:39.900]was expressed in an E. coli host and isolated using nickel affinity
[00:03:44.370]chromatography and size exclusion chromatography.
[00:03:47.670]The strain 13 PilA2 was screened in several hundred conditions with crystals
[00:03:52.110]forming in a Hampton Research Index screening plate containing 0.1 M citric
[00:03:56.790]acid pH 3.5 and 2.0 M ammonium sulfate
[00:04:00.540]in the presence of maltotriose. Crystal diffraction was achieved with one of
[00:04:04.350]these initial crystals,
[00:04:05.700]though resolution was not sufficient to obtain structural data.
[00:04:10.020]You can see some of our crystals in the images here. Panels A, B and C
[00:04:14.070]show some of the crystals we produced,
[00:04:16.560]while panel D shows the diffraction data from one of our initial crystals.
[00:04:20.670]You'll notice that the diffraction pattern is somewhat ovular in shape.
[00:04:24.150]This is indicative of a phenomenon called anisotropy,
[00:04:28.320]which means the crystal is detracting differently in different dimensions.
[00:04:32.250]While the crystals we've been producing have not diffracted well enough to solve
[00:04:35.970]the structure of PilA2, the ability to produce crystals
[00:04:39.000]that diffract is worthwhile progress.
[00:04:41.880]An issue we're having with the strain 13
[00:04:43.650]MBPilA2 crystals is that they take a long time to form--often several weeks--
[00:04:48.360]and are difficult to replicate.
[00:04:50.160]We're currently in the process of optimizing crystal conditions for our MBPilA2
[00:04:53.850]construct by varying conditions, such as drop ratios, protein,
[00:04:58.380]concentration, pH, seeding, and temperature.
[00:05:02.370]While we work on producing more strain 13 crystals,
[00:05:04.830]we've also been working to crystallize PilA2 from the second strain of C.
[00:05:08.250]perfringens, strain ATCC 13124.
[00:05:11.220]We've successfully created crystals in a 2.4 M sodium malonate
[00:05:15.630]condition but crystals so far have been very small and needle-like and often
[00:05:19.470]form in haystack shaped clusters.
[00:05:22.020]We're in the process of optimizing these crystals using similar techniques,
[00:05:25.500]such as varying the protein concentration, adding additional compounds to
[00:05:28.840]the crystal crystallization condition and adding crystal seeds.
[00:05:33.630]Since we haven't been able to solve the crystal structure of PilA2
[00:05:36.810]yet we've been working on a theoretical model of the protein.
[00:05:40.410]We first used Robetta ab initio modeling to create five hypothetical structures
[00:05:44.790]of the PilA2 protein. To determine which structure was most reasonable,
[00:05:48.630]we collected
[00:05:49.230]PilA2 sequences from about 50 strains of C. perfringens in the NCBI database
[00:05:54.480]and aligned these sequences using Clustal Omega. We created a dendro-
[00:05:58.520]gram of these alleged PilA2 genes alongside our strain 13 PilA2
[00:06:02.450]sequence and some other known pilin genes like PilA1
[00:06:06.080]and PilA3.
[00:06:07.630]We used this dendrogram to determine which of our collected sequences were
[00:06:10.840]most similar to PilA2 and which were more similar to other pilin genes
[00:06:14.500]like PilA1.
[00:06:15.760]We excluded those sequences that were unlike PilA2 and used the remaining
[00:06:19.030]sequences to create a variation map of residues in PilA2.
[00:06:23.560]We used the variation map to determine which ab initio structures were most
[00:06:27.100]reasonable based on what structures possessed
[00:06:29.110]conserved residues in the interior portions of the protein.
[00:06:32.800]We selected two of the models as being most reasonable with one of those models
[00:06:37.150]shown here in panel A. In this model, conserved
[00:06:40.330]residues are shown in cyan and variable residues are shown in magenta.
[00:06:44.680]We see conservation in some areas that we would expect such as the internal
[00:06:48.760]alpha helix,
[00:06:49.480]which is buried in the interior of the pilus fiber in a complete pilus.
[00:06:53.770]We also see an alternating pattern of conservation in the beta strands. Panel
[00:06:58.030]B is a closeup of these strands where we see the residues that point outwards
[00:07:01.960]towards solvent are not conserved,
[00:07:03.970]while residues that point inwards towards the center of the protein are
[00:07:07.390]conserved.
[00:07:08.470]Panel C shows our ab initio model constructed into a complete pilus with the
[00:07:12.790]surface variation map shown for three pilins.
[00:07:15.790]We see here that portions of the pilin that set exposed on the surface of the
[00:07:19.570]pilus fiber are variable
[00:07:21.490]while portions buried within the pilus are conserved. Moving forward,
[00:07:25.900]We aim to continue optimizing crystal conditions for both the strain 13 and
[00:07:29.770]strain ATCC 13124
[00:07:31.900]proteins so that we can produce diffraction quality crystals.
[00:07:35.620]Once we've obtained diffraction data,
[00:07:37.210]we'll use this to solve the structure of PilA2 and adapt our model to
[00:07:40.510]accurately display the structure of the protein.

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Structure of Clostridium perfringens Type IV Pili

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