Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. Like in other life-threatening human pathogens, also in Shigella the regulation of virulence genes occurs at diverse levels and is modulated by environmental stimuli from the host. A central factor in this process is VirF, an activator belonging to the AraC family of transcriptional regulators whose expression is activated as soon as Shigella senses to have entered the host environment. Despite its crucial role as major player in the cascade of events leading to the activation of the virulent programme, its molecular characterization is insufficiently detailed and information on its mechanisms of action is still scarce.
The aim of this project is to investigate on new regulatory interactions involved in the optimization of the invasive programme of Shigella by deeper analyzing the structure and function of VirF. The project will be articulated into two research lines, which will coordinately exploit the specific competencies and know-how of the participating scientists and will be based on a multidisciplinary approach including bacterial genetics, molecular biology methodologies, and in vivo infection assays. In particular, we will analyze the capacity of VirF to form dimers or more complex aggregates and, following the recent identification by our group of a minor VirF form (VirF21), we will study its the role during the invasive process of Shigella.
AraC proteins are considered very interesting candidate targets in anti virulence strategies, due to their critical role in controlling virulence in pathogenic bacteria. Hence, a better understanding of the structure of the VirF proteins can be anticipated to positively impact on the development of new therapeutic approaches exploiting specific inhibitory compounds.
Each year Shigella is responsible for 125 million cases of illness, mainly in low income countries. Despite the enormous clinical relevance of Shigella infections and the emergence of multiresistance strains, no vaccine has been as yet released for public use (Anderson et al., 2016). Several recent studies have focused on the development of novel treatment strategies targeting virulence instead of bacterial viability, since this is regarded a highly effective approach to combat bacterial infections while minimizing the emergence of antibiotic resistances (Rasko and Sperandio, 2010). The expression of virulence factors is not required for cell viability and therefore bacterial pathogens should be subject to less selective pressure to develop resistance to inhibitors of virulence determinants. AraC proteins are considered very interesting candidate targets in anti virulence strategies due to their critical role in controlling virulence in pathogenic bacteria (Rasko and Sperandio, 2010). Specific inhibitors can affect AraC-mediated processes at different stages, such as self association, DNA binding and recruitment of RNA polymerase.
The complexity of the circuitry regulating virulence in Shigella highlights the relevance of the VirF protein to the successful development of the invasive programme. It is therefore not surprising that VirF is regarded as a very good antivirulence target, since its silencing prevents host cell invasion and intercellular spreading without affecting bacterial viability (Falconi et al., 1998). Recently, a high-throughput screening for small molecules, based on a VirF-driven beta-galactosidase reporter assay has been used to identify, among large collections of small molecules, those able to inhibit the activity of VirF (Emanuele and Garcia, 2015). While further studies will be required to structurally optimize the selected compounds and clarify their effectiveness in hampering VirF activity, these studies highlight the promising potential of a VirF-based antivirulence therapy.
VirF has been identified almost three decades ago, however several open questions still exist, such as its capacity to form dimers or more complex aggregates and the molecular mechanisms underlying its DNA binding specificity and its interactions with RNA polymerase. Through our recent studies on the transcription and translation of the virF gene , the complex nature of the regulation of VirF and of the genes under its control is emerging with intriguing detail. It will be therefore interesting to better define the structure of this protein, identify the key residues involved in protein protein interactions or in binding to DNA and eventually to RNA polymerase. Our recent observations on the co-existence of two forms of VirF, VirF30 and Vir21 (Di Martino et al., 2016), represent a turning point for the development of further investigations aimed at understanding how the minor form, besides controlling the transcription of the virF gene, also interferes with the dimerization and DNA binding of the functional VirF molecule. In the long run a better understanding of the structure of the VirF protein can be anticipated to positively impact on the development of new therapeutic approaches based on the use of specific inhibitory compounds.
Additional references :
Anderson et al., 2016, Front Cell Infect Microbiol 6:45
Emanuele and Garcia ,2015, PLoS ONE 10:e0137410
Rasko and Sperandio, 2010, Nat Rev Drug Discov 9: 117-28