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Identification of type III secretion effectors in Aeromonas spp.
Jirsová, Anežka ; Kamanová, Jana (advisor) ; Lišková, Petra (referee)
Bacteria of the genus Aeromonas are Gram-negative, rod-shaped bacteria that are ubiquitous in the aquatic environment. The mesophilic species of this genus, represented by A. hydrophila, A. dhakensis, A. veronii and A. schubertii, are not only important pathogens in fish but also emerging pathogens in humans. They are associated with various gastrointestinal, systemic and wound infections. Their virulence has been described as multifactorial and associated with the presence of a type 3 secretion system. However, not much is known about the effector proteins that are introduced into the host cell by this system. The aim of this work was to characterize the two different type 3 secretion systems API-1 and API-2 of A. schubertii with respect to their cellular cytotoxicity and to identify the effector proteins that they transport into the host cell. To achieve these goals, a variety of methods were used, including microbiological techniques, molecular cloning, tissue culture techniques, SDS polyacrylamide gel electrophoresis, and mass spectrometry. Successful construction of mutant strains of A. schubertii with inactivated API-1 or API-2 and a strain with deletion of the gene encoding the negative regulator of API-1 enabled me to identify potential type 3 effector proteins of this species. Among the...
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BopN function in the Bordetella type III secretion system
Kincová, Veronika ; Kamanová, Jana (advisor) ; Večerek, Branislav (referee)
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...
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