Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...

(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cation­selective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; α­hemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic α­helix(502­522). In the second publication we investigated another predicted transmembrane α­helix(565­591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...

The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...

<p dir="ltr">This dissertation delves into two research projects that aim to characterize post-translational modifications in two distinct proteins, each originating from a different species – one from the eukaryotic sea slug Aplysia californica and the other from the bacterial pathogen Bordetella bronchiseptica.</p><p dir="ltr">Aplysia have an unusually large neuron and therefore serve as an excellent model for studying cell signaling regulating neuronal chemotaxis. Cortactin is an actin binding protein that is regulated by post-translational modifications, including acetylation and phosphorylation. Studies have shown that Src2 tyrosine kinase phosphorylates cortactin to regulate lamellipodia protrusion and filopodia formation in Aplysia bag cell neurons. However, these in vivo phenotypes have not been tested mechanistically in vitro. To this end, the goal of my thesis work was to validate in vivo observations. The following work describes the methodology we developed to purify homogenous non-phosphorylated proteins. Our collaborative results show that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro.</p><p dir="ltr"> Filamentation induced by cAMP (Fic) proteins constitute a recently characterized family of enzymes that are being recognized to regulate diverse cellular processes in bacteria and metazoans. While Fic proteins predominantly utilize adenosine triphosphate (ATP) to post-translationally modify target proteins via a covalent addition of AMP, two Fic proteins have been reported that utilize uridine triphosphate (UTP) and cytidine diphosphate-choline (CDP-choline) to alter the activity of their target. In this dissertation, we report the discovery of the first guanosine triphosphate (GTP) specific Fic protein – BB0907 (BbFic) from Bordetella bronchiseptica. BbFic displays weak to no binding to ATP; instead has a 10-fold increased preferential usage for GTP. We identify key residues involved in GTP recognition. Additionally, sequence similarity network (SSN) analyses reveal that BbFic represents a distinct clade of Fic proteins, highlighting BbFic as a representative new class of guanylyltransferase. Our discovery adds to the functional diversity of the growing Fic protein family and frames the groundwork for understanding Fic-mediated GMPylation as a novel signaling paradigm. </p><p dir="ltr">Taken together, my thesis work provides novel insights into biological consequences of Fic-mediated GMPylation in bacteria and Src-mediated phosphorylation in filopodia formation.</p><p><br></p>