Vp Pilus Indir
Elis Riebow
Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.
Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.
Conjugative pili allow for the transfer of DNA between bacteria, in the process of bacterial conjugation. They are sometimes called "sex pili", in analogy to sexual reproduction, because they allow for the exchange of genes via the formation of "mating pairs". Perhaps the most well-studied is the F-pilus of Escherichia coli, encoded by the F sex factor.
A sex pilus is typically 6 to 7 nm in diameter. During conjugation, a pilus emerging from the donor bacterium ensnares the recipient bacterium, draws it in close, and eventually triggers the formation of a mating bridge, which establishes direct contact and the formation of a controlled pore that allows transfer of DNA from the donor to the recipient. Typically, the DNA transferred consists of the genes required to make and transfer pili (often encoded on a plasmid), and so is a kind of selfish DNA; however, other pieces of DNA are often co-transferred and this can result in dissemination of genetic traits throughout a bacterial population, such as antibiotic resistance. The connection established by the F-pilus is extremely mechanically and thermochemically resistant thanks to the robust properties of the F-pilus, which ensures successful gene transfer in a variety of environments. [5] Not all bacteria can make conjugative pili, but conjugation can occur between bacteria of different species.[citation needed]
Fimbria (Latin for 'fringe', pl.: fimbriae) is a term used for a short pilus that is used to attach the bacterium to a surface, sometimes also called an "attachment pilus".[8] The term "fimbria" can refer to many different (structural) types of pilus, as many different types of pili have been used for adhesion, a case of convergent evolution.[3] The Gene Ontology system does not treat fimbriae as a distinct type of appendage, using the generic pilus (GO:0009289) type instead.
The Tra (transfer) family includes all known sex pili (as of 2010). They are related to the type IV secretion system (T4SS).[3] They can be classified into the F-like type (after the F-pilus) and the P-like type. Like their secretion counterparts, the pilus injects material, DNA in this case, into another cell.[10]
The development of attachment pili may then result in the development of further virulence traits. Fimbriae are one of the primary mechanisms of virulence for E. coli, Bordetella pertussis, Staphylococcus and Streptococcus bacteria. Their presence greatly enhances the bacteria's ability to attach to the host and cause disease.[28] Nonpathogenic strains of V. cholerae first evolved pili, allowing them to bind to human tissues and form microcolonies.[24][27] These pili then served as binding sites for the lysogenic bacteriophage that carries the disease-causing toxin.[24][27] The gene for this toxin, once incorporated into the bacterium's genome, is expressed when the gene coding for the pilus is expressed (hence the name "toxin mediated pilus").[24]
A number of pilin structures are available for both subclasses mainly for Gram-negative bacteria (13). They suggest an overall conserved architecture, with each pilin having an extended N-terminal domain (α1-N and α1-C) and a C-terminal globular head domain. α1-N is primarily hydrophobic and retains the pilin subunits in the inner membrane until assembly, whereas the α1-C is tightly packed against the head domain composed of several β-strands. The α/β loop connects the N-terminal helix to the β-sheet and is important for interactions between individual pilin subunits (10). Upon pilus assembly, α1-N forms the core of the assembled pilus, and α1-C is buried in the C-terminal head domain that forms the pilus surface. Characteristic for most pilins is also a disulfide-bonded loop (D-region) in the C-terminal domain, which is essential for pilus assembly (10). Most of the structural diversity among different pilins lies in the α/β loop, and the number and topology of β-strands are in the C-terminal domain. Notably, many of the available pilin structures are lacking the highly hydrophobic N-terminal domain (α1-N) making the truncated protein more soluble and easier to purify for later structural characterization.
Type IV pili are also produced by Gram-positive bacteria, including several Clostridium species (14), Ruminococcus albus (15), and Streptococcus species (9, 16, 17), but many molecular and structural aspects of pilus biogenesis in Gram-positive species remain unclear. Recently, DNA uptake in Streptococcus pneumoniae was shown to rely on the formation of a type IV pilus that is able to directly bind to DNA (9). This transformation pilus is assembled on the surface of competent bacteria and composed of the major pilin ComGC. Pneumococcal comGC is encoded in the comG operon that also encodes a putative ATPase (ComGA), which powers pilus assembly (9), a membrane-spanning protein (ComGB), and four minor pilins (ComGD, -E, -F, and -G) whose functions remain elusive.
S. pneumoniae assembles competence type IV pili composed of ComGC.A, electron micrograph of negatively stained competent S. pneumoniae T4RΔrrgA-srtD induced with CSP. Black arrows indicate the pilus. B, transformation frequency of S. pneumoniae T4R and R6 strain. The error bars represent standard deviation (S.D.) of a minimum of three independent experiments. C and D, immunogold electron microscopy to visualize pili on competent S. pneumoniae R6 using primary antibody specific to ComGC and secondary antibody conjugated to 6-nm gold particles. D, enlargement of the immunogold-labeled pilus. Black arrows indicate the pilus. E, electron micrograph of a competence pilus in strain R6 stained with anti-ComGC antibody and protein A coupled to 10-nm gold particles. F, two-dimensional PAGE to assess multimerization of mature ComGC. A pilus preparation of T4 WT or ΔC strain was run on a 12% native gel (1D, first dimension). A piece of gel corresponding to one lane of the gel was cut and placed horizontally on top of a second SDS-PAGE (2D, second dimension). After migration, gels were immunoblotted with anti-ComGC antibody. Arrows indicate ComGC and protein multimers.
For that reason, we decided to do immunogold labeling of ComGC in competent R6 bacteria and used primary polyclonal ComGC antibodies, raised against the purified protein or an anti-peptide antibody, and secondary antibody labeled with 6-nm gold particles. We frequently found gold particles labeling the entire type IV pilus suggesting that ComGC is the major pilin protein (Fig. 1, C and D). We also stained competent R6 bacteria with primary polyclonal ComGC antibody followed by incubation with protein A coupled to 10-nm gold particles. In this way the pilus is less frequently labeled with gold particles; however, the underlying pilus filament is clearly visible (Fig. 1E).
Pneumococcal ComGC shares many features of canonical type IV pilins. Full-length ComGC has a well-defined conserved prepilin cleavage motif, an invariant Glu residue at position 5 after the cleavage site, and it is processed by PilD. The structure of soluble ComGC, provided here, is the first example of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and reveals new structural features. Similar to previously described type IV pilins, ComGC has a predicted extended N-terminal α-helix but differs otherwise significantly as follows: 1) ComGC is exclusively α-helical; 2) the head group is much smaller; 3) the α1-C helix is separated from the transmembrane helix by a flexible linker that is largely unfolded in solution; and 4) ComGC contains no cysteines. Overall, ComGC is shorter than other type IV pilins and highly dynamic in solution, which may be an important feature for pilus assembly and function.
The assembly of pilin monomers into a model of the fully formed pilus has primarily been based on negative staining and electron cryo-micrographs, where individual monomers are fixed in a favorable multimeric organization and fitted into the obtained electron density (26, 31). These structural models serve as important frameworks for understanding pilus dimensions, appearance, and surface, but as a consequence of the low structural similarity of ComGC, primarily in the head group, we were not able to reliably predict ComGC assembly. Our data suggest that soluble monomeric ComGC will not adopt secondary or tertiary folds similar to other type IV pilins, but we cannot rule out that additional conformational states (i.e. helical rearrangements) will be favored during assembly or in the mature pilus structure.
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