Widening the landscape of Shigella flexneri virulence factors: the key roles of YbjX, VirK and the AcrAB efflux pump

Shigella flexneri is a Gram-negative bacterium belonging to the Enterobacteriaceae family and, being one of the leading causes of shigellosis (bacillary dysentery) worldwide, it is a pathogen of concern considering the burden it poses on communities and healthcare systems, especially in developing countries. Similarly to other animal and human pathogens, the frequency of the emergence of multidrug-resistant strains is rising and antibiotic-based therapeutic approaches are rapidly becoming ineffective. The lack of treatments and the unavailability of a vaccine, whose development suffers from the high number of Shigella serotypes and the economic investment that would be required, calls for the development of alternative strategies that could prevent the emergence of resistant strains and restore our capability of taming Shigella infections. A possible approach would be hampering the virulence of Shigella, targeting key factors required for the complex and multifaceted process deployed by Shigella for the invasion and the colonisation of the colonic epithelium. Most of the virulence factors, like the master regulators of virulence or the type 3 secretion system and its effectors, are encoded by genes on a large virulence plasmid (pINV) that defines the Shigella/EIEC (Enteroinvasive Escherichia coli) pathogroup. The pINV has been obtained multiple and independent times during the evolution that led commensal bacteria towards the acquisition of a pathogenic lifestyle. The infectious strategy of Shigella requires stages of intracellular life to reach the final goal of colonising the colonic epithelium. After surviving the harsh environment of the gastrointestinal tract, the interspecific competition with the gut microbiota and the thick mucus layer, Shigella can enter M (microfold) cells by transcytosis and reach the resident macrophages. The bactericidal effect of the macrophagic phagocytic function is rapidly avoided by the escape from the phagosome and the induction of cell death. Thereby, the pathogen reaches the subepithelial region and is finally able to invade epithelial cells by their basolateral side. This process, mediated by the reorganisation of the host cell cytoskeleton, results in the formation of bacteria-containing vacuoles (BCVs) that, again, are rapidly ruptured. Bacterial movement in the cytoplasm of the host epithelial cells is achieved by the polymerisation of actin “comet tails” at one pole of Shigella and allows for the spreading to secondary cells, where double membrane BCVs are formed and destroyed by Shigella. The result of this complex chain of events is the induction of a strong inflammatory response and the destruction of the epithelium. In this thesis, I present two studies whose aim is the same: widening the landscape of S. flexneri virulence factors. This work and similar investigations may provide targets for the development of alternative therapeutic approaches directed against the virulence of Shigella and related pathogens. The approaches chosen for my work are complementary: looking at poorly characterised proteins, whose functions have yet to be discovered, and also at well-known players of bacterial physiology, that may be involved in more processes than previously believed. Hence, the first chapter of this thesis focuses on the characterisation of YbjX and VirK, two structurally similar proteins encoded by a gene on the chromosome of Shigella and on the virulence plasmid, respectively. The results I obtained highlight the involvement of these two cytoplasmic proteins in the integrity of the envelope and, possibly, in the synthesis/modification of LPS. Additionally, they are involved in the evasion of the host response both during the infection of macrophages and epithelial cells. However, YbjX and VirK are not redundant and a rescue function for YbjX was hypothesized. Additionally, by looking at other pathogens belonging to the Enterobacteriaceae family, it is tempting to suggest a convergent evolution of different pathogens towards the acquisition of VirK and the conservation of YbjX. The second chapter deals with the study of the involvement of the AcrAB-TolC multidrug resistance (MDR) efflux pump in the infection of epithelial cells by S. flexneri is presented. The AcrAB-TolC is, along with the AcrB structural homologue AcrD, the only MDR efflux pump belonging to the RND superfamily conserved between E. coli and Shigella. Gene decay is a typical feature of pathogens’ genomes, whose shedding of genes that were unnecessary or deleterious for the pathogenic lifestyle is common and well-studied. Hence, the conservation of acrAB genes suggests the relevance of their functions. However, this efflux pump is mainly studied for its importance in antibiotic resistance, given by the wide substrate profile and its abundance in the bacterial envelope. Data obtained in other pathogens, such as Salmonella, suggest the involvement of AcrAB in virulence, but before this study (partially published as Coluccia et al., 2023) there was no information regarding the virulence of Shigella flexneri. The data presented show that the lack of AcrAB significantly affects not only the survival of intracellular bacteria within epithelial cells, but also the ability of Shigella to spread across the epithelium. Moreover, specific roles of AcrA and AcrB, independently of the pump as a whole, are suggested and will be the objects of additional studies in the future. Finally, in the last section of the thesis, the publications are presented. In addition to the previously mentioned paper regarding AcrAB-TolC, two articles delve into the role of bacterial two-component systems in the virulence of Shigella (Pasqua et al., 2022) and into the development of histidine kinase inhibitors whose applications may include antivirulence treatments (Ishikawa et al., 2024). Overall, this thesis aims to highlight the importance of understanding the roles of all factors implied in the complex virulence mechanisms of bacterial pathogens, because both old and well-studied proteins or new and uncharacterised proteins provide a source of potential therapeutical targets.