[PhD Seminar] Structure and Dynamics of Pathogenic Scaffold Proteins; Metalloenzymes in microbial defense mechanisms against oxygen and nitric oxide & A targeted approach to control Streptococcus pneumoniae infection

Guillem, Maria and Catarina will present their work in the Auditorium in anticipation of the upcoming 13^th ITQB NOVA PhD Students' Meeting. 

Guillem Hernandez: Pathogens have evolved sophisticated mechanisms to subvert the host during infection, often by delivering effectors into host cells. Such effectors can act as scaffold/hub proteins that display protein disorder and host-like interactions. Two examples of this sophistication are the Nucleocapsid Protein (NProtein) from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) cause of the recent pandemic; and the Translocated intimin receptor (Tir) from Enterohemorrhagic (EHEC) or Enteropathogenic (EPEC) Escherichia coli, which cause gut infections such as hemorrhagic colitis or the hemolytic-uremic syndrome.
NProtein is one of the four main structural proteins from β-coronaviruses, with a well-conserved architecture consisting of two globular-like domains flanked and connected by intrinsically disordered regions. NProtein is known to facilitate RNA packaging into virions and, inside the host cell, it recruits host-factors to help in the viral RNA synthesis by forming biocondensates. The mechanism by which NProtein self-assembles to pack viral RNA and form biocondensates inside the host remains unknown, thus, it is a potential therapeutic target.
Regarding Tir, it is the first bacterial effector to be delivered into the host’s cytosol being essential for EPEC/EHEC infection. From there, it migrates and integrates into the host’s membrane adopting a hairpin-like topology with two transmembrane domains flanking an exocellular domain responsible for bacterial attachment while leaving two (predicted) disordered domains (N- and C-terminal) into the host cytoplasm where they interact with multiple host proteins to orchestrate pathogenic-driven signaling. Since Tir is essential to start the infection, knowing its molecular and atomic description becomes instrumental for therapeutic developments.
My PhD research aims to employ a combination of hybrid structural biology and biochemical approaches to raise some knowledge over these two scaffold proteins and reveal clues to their behavior in the infection.

Maria Martins: Oxygen and nitric oxide, and derived species, play key roles in life. While some organisms need them for survival, others use them as defence mechanisms against pathogens. Flavodiiron proteins (FDPs) are key players in microbial defence against oxidative/nitrosative stress and in survival within the host. My PhD project targeted the FDPs from Clostridiales, the phylogenetic group with larger diversity of FDP classes and one of the major components of the human gut microbiome.
The main aim of my work was to understand the determinants of their reactivity towards O2/NO, establish their catalytic mechanism, and the function of multiple extra domains in several FDPs. A plethora of biochemical, spectroscopic, and kinetics methods, complemented by in vivo studies, was used. Altogether, this project unraveled key properties of four FDP classes contributing to the understanding of the physiological role of these complex multidomain enzymes in strictly anaerobic organisms like Syntrophomonas wolfei and in the human pathogen Clostridioides difficile.

Catarina Candeias: Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. Nonetheless, it colonizes asymptomatically the nasopharynx of healthy people. Colonization precedes infection and is necessary for transmission. Current strategies for prevention and treatment of pneumococcal disease are effective but have limitations. This project aims to identify commensal bacteria that could act as biotherapeuticals to contain pneumococcal colonization, as a means to prevent infection. Previously, an in vitro screen identified seven strains (A-G) with inhibitory activity against S. pneumoniae. To understand if the inhibition would be maintained in more complex settings, in vitro and in vivo experiments were performed and the results obtained will be presented.