Molecular Mechanism Of S. Flexneri VirB Protein In Transcription Activation Of The Virulence Genes

Bacillary dysentery remains the leading public health threat, especially in developing country. Shigella, a Gram-negative facultative intracellular pathogen of humans, is the primary causative agent of bacillary dysentery. Shigella can be subdivided into four species, S. flexineri, S dysenteriae, S. sonnei and S. boydii, in which most of the pathogenicity studies were carried out on the prevalent S. flexineri. We previously completed the genome sequencing of the Shigella flexneri2a301strain in2001, the most prevalent strain in China, providing the complete genetic background for further studies. This project presents the first microbial genome decoded by chinese scientists.The genomic sequence of S. flexneri is highly homologous to the non-pathogenic E. coli. The virulence of S. flexneri depends on the virulence genes clustered within the so called "entry region", a31-Kb segment on the virulence plasmid (～230-Kb). These genes encode a variety of proteins, including invasins, structural proteins forming the sophisticated type Ⅲ secretion system and the effector proteins. The vast quantity of the virulence genes products obviously presents a great metabolic burden for the bacteria; therefore, the bacteria has developed a sophisticated regulatory system to control the expression of the virulence genes, comprising of the plasmid encoded regulatory system and the chromosome encoded global gene regulation.When the growth conditions are not ideal for invasion, the transcription of the virulence genes is repressed by a chromosomally encoded heat-stable nucleoid structural protein (H-NS). It is believed that H-NS can bind the AT-rich DNA segment near the promoters, forming the repressive nucleoprotein structures that function as the obstacles to impede the movements of RNA polymerase, thereby silent the transcription. In response to proper environmental signals, i.e. pH value, temperature and osmolarity similar to the environment in the lower intestine of the host, the H-NS mediated transcriptional repression can be alleviated by the plasmid encoded regulatory system. A transcriptional cascade is then initiated with the activation of virF gene expressing an AraC-like protein VirF that in turn activates the transcription of the virB regulatory gene. The gene product VirB protein consequently relieves the H-NS mediated transcriptional repression, leading to the activation of the virulence genes on the plasmid. VirB also activates the transcription of its own gene and feeds back positively onto the transcription of the virF upstream regulatory gene.VirB play an central role in transcriptional regulation of the virulence genes; however, the molecular basis for the alleviation of H-NS mediated promoter repression remained unclear. The amino acid sequence homology analysis reveals that VirB shares little homology with the conventional transcriptional regulators, but the protein is homologues to the ParB like proteins involving the plasmid partitioning. VirB can specifically bind the cis-acting site upstream the promoter region, which share the key elements with the ParS site recognized by the ParB-like proteins. Therefore, the mode of VirB-DNA interaction must be related to ParB-ParS interactions. We speculate that VirB was reassigned exclusively to the regulatory roles during the evolution, and many key structural features of its ancestor were well preserved.To understand molecular basis underlying recognition of the cis-acting site by VirB and its role in transcriptional activation of the virulence genes, we employed the methods of structural biology, biophysics&biochemistry and bacteriology to investigate the mode of VirB-DNA interaction.We first solved the crystal structure of VirB core complexed by the cis-acting site upstream of icsB promoter icsB-WT (26bp), VirB core complexed by the cis-acting site upstream of icsP promoter icsP-WT (31bp). The crystal structures reveal that the overall structure of VirB core is related to ParB-like proteins, containg a typical HTH domain. The crystal structures show that VirB Core domain binds directly to DNA major groove of the cis-acting site, presenting the structural determinant for DNA sequence specificity. VirB core provides11stable intermolecular hydrogen bonds or salt bridges to the DNA backbone phosphates and base, facilitating readout of DNA sequence. We found that the hydrogen bonds donated by the side chain of R167to the guanosine base at the3rd position of the Box2in the cis-acting sites present the only DNA base reading by VirB. The structural comparison of VirB-DNA complex to the ParB-ParS like complexes, We found VirB binds only one of the half sites in the inverted repeats,ParB binds two of the half sites simultaneously.which inform the most difference between VirB and ParB for DNA recognizion We next carried out the mutagenesis studies, confirming that10residues out of the11residues providing contacts to DNA are essential for the activity of VirB in transcriptional activation, thereby established the structure basis for DNA sequence specificity and the correlation of the sequence recognition and the function of VirB. We further analyzed the structure of DNA double helices of icsB-WT and icsP-WT, and discovered the conformation of the DNA bound by VirB deviates from the standard DNA. The distortion of DNA involves the bending of helical axis and the narrowing of the DNA minor groove. The conformational changes observed in DNA demonstrated the capability of VirB of introducing distortion of DNA double helix, a capability not shared by ParB-like proteins.We performed fluorescence anisotropy experiments to study the binding capability of VirB variants to a fluorescently labeled DNA cis-acting site.the results showed that both N-and C-terminal portion of VirB contribute significantly to the DNA binding affinity, which is independent of DNA sequence. The cooperative DNA binding capability of VirB owns to its C-terminal domain that mediates the oligomerization of the protein, suggesting that the high-order VriB oligomer is the functional assembly providing multiple sites for DNA binding.Our findings suggest a model for VirB-mediated promoter activation.The C-terminal domain of VirB facilitates the oligomerization and assembles the functional high-order VirB oligomer. The HTH domain of VirB recognizes a specific cis-acting site upstream the promoter, thus anchors the VirB oligomer to DNA. The binding of VirB HTH domain induces the distortion at the AT-rich segment in VirB binding site, which may subsequently promote the adjacent DNA strand bending toward the VirB oligomer, therefore assists the nearby DNA strand to be loaded on the other DNA binding site on the oligomer. Thereby, the DNA wrapping around VirB oligomer initiates. Due to that the specific VirB binding site is limited, the subsequent DNA strand loading requires nonspecific DNA binding capability of VirB. The high-order VirB oligomer combines multiple DNA binding sites from the VirB monomers, therefore a considerable long DNA strand could be accommodated. The DNA wrapping around VirB oligomer subsequently remodels the DNA upstream the promoter and destabilizes the repressive H-NS-DNA complexes, such as the H-NS-DNA-H-NS bridges. Eventually, the trapped RNA polymerase is then released and the transcription resumes.Shigella is not only an important pathogen for humans but also a model organism for the research of the bacteria pathogenicity. The molecular mechanism underlying the virulence gene regulation is one of the key question to be addressed in the field of pathogenic bacteria, severing a important target for drug design. Our studies have provided the structural basis for the recognition of the cis-acting site by VirB, thus, proposed a model for the relieving of H-NS mediated transcriptional repression. Our works provided the basis for the understanding of virulence gene regulation and a framework for the structure-based inhibitor design.