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Structural-functional aspects of the Bordetella pertussis adenylate cyclase toxin
Březinová, Karolína ; Bumba, Ladislav (advisor) ; Brzobohatá, Hana (referee)
Whooping cough (pertussis) is a highly infectious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. Even though the vaccination rate of the population is high, pertussis is one of the most widespread vaccine-preventable diseases. The bacterium produces a variety of virulence factors that facilitate the process of colonization of the ciliated epithelium and infection of the upper respiratory tract. Among the most important virulence factors is the adenylate cyclase toxin (CyaA). This toxin belongs to the so- called RTX (Repeat-In-ToXins) proteins, which are released from the bacterium using the Type 1 secretion apparatus (T1SS). CyaA is a multifunctional toxin, showing both hemolytic and cytotoxic activity. The cytotoxic activity is caused by the N-terminal adenylyl cyclase (AC) domain, which is translocated across the cytoplasmic membrane into the cell cytosol, where upon interaction with calmodulin catalyzes the uncontrolled conversion of adenosine triphosphate (ATP) to cyclic adenosine-3',5'-monophosphate (cAMP). CyaA is recognized by the integrin receptor CD11b/CD18 (also known as complement receptor type 3), which is primarily found on phagocytic cells of the host organism. This work focuses on the structural- functional aspects of the CyaA toxin and summarizes...
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Mechanisms underlying subversion of host immunity by Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Černý, Jan (referee) ; Filipp, Dominik (referee)
Bordetella pertussis is a Gram-negative human-adapted pathogen of the respiratory tract and the causative agent of the whooping cough (pertussis) illness. The bacterium produces a number of virulence factors, of which adenylate cyclase toxin (ACT) and pertussis toxin (PT) play important roles in manipulation of host immune response and establishment of the early catarrhal stage of infection. Although the toxins exert their cytotoxic activity by elevation of intracellular cAMP levels, both are distinct from each other in terms of their structures, mechanisms of secretion and cell intoxication, as well as in their ability to modulate the adaptive immune response of the host. The aim of this thesis was to determine the structure- function relationship underlying the mechanism of the Type I secretion system (T1SS)- mediated secretion of ACT and to decipher the immunomodulatory properties of ACT and PT in the course of B. pertussis infection. Integrative structural biology approaches revealed that the RTX domain of ACT consists of a contiguous assembly of five Ca2+ -loaded β-roll blocks, whose co-secretional folding constitute an intramolecular Brownian ratchet that prevents backsliding of the translocating polypeptide in the T1SS conduit, thus accelerating the secretion of ACT from bacterial cells by a...
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The role of RTX domain in the activity of adenylate cyclase toxin from Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.
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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David ; Bumba, Ladislav (advisor) ; Novák, Petr (referee) ; Řehulka, Pavel (referee)
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Substrate specificity of histone deacetylases
Ustinova, Kseniya ; Bařinka, Cyril (advisor) ; Bumba, Ladislav (referee) ; Obšil, Tomáš (referee)
In the cell, tubulin undergoes post-translational modifications that create functionally distinct microtubules and mark them for specialized functions. Acetylation of Lys40 of α-tubulin is one of such post-translational modifications controlled by the activity of histone deacetylase 6 (HDAC6). The Lys40 acetylation is a hallmark of stable microtubules, it protects them from mechanical aging, influences cell motility as well as axonal branching and maintenance of neuronal processes. Tubulin stands out as the most prominent physiological substrate for HDAC6. Being a multidomain cytosolic protein, HDAC6 is involved in the myriad of cellular processes and is a promising target for the treatment of cancer and neurodegenerative diseases. The understanding of the mechanisms of HDAC6 interactions with its substrates, especially with tubulin, can open avenues for the development of new treatment strategies exploiting highly selective HDAC6 inhibitors. In this thesis, we have investigated the molecular basis of tubulin recognition by HDAC6. We provided a detailed kinetic analysis showing the HDAC6 deacetylation rate of free tubulin is 1500-fold faster than microtubules. Additionally, we have shown that amino acids of the flexible Lys40 loop (except P1 and P-1) make a minor contribution to the substrate...
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Development of an opsonophagocytic assay for the measurement of functional antibody activity against Bordetella pertussis
Brázdilová, Ludmila ; Bumba, Ladislav (advisor) ; Dráber, Peter (referee)
The Gram-negative pathogen bacterium Bordetella pertussis is the infectious agent causing pertussis or whooping cough. The infection is dangerous to infants, often being deadly if untreated. Since whole-cell pertussis vaccines have been replaced by acellular pertussis vaccines, pertussis has become the most prevalent vaccine-preventable disease in developed countries. Therefore, the development of a new generation of pertussis vaccines has become a high priority. Opsonophagocytic assays are one method used to assess the efficacy of new vaccines. The main objective of the thesis is to develop opsonophagocytic killing and uptake assays for the measurement of functional antibody activity against Bordetella pertussis. Neutrophils from mice and humans were isolated by three different methods and used for the assessment of different human and mouse sera in opsonophagocytic killing and uptake assays. Different experimental conditions were tested, including multiplicity of infection and serum dilutions. The opsonophagocytic uptake assay proved to discriminate between naïve and immune sera. Serum from mice vaccinated with the whole-cell pertussis vaccine enhanced opsonophagocytic uptake of B. pertussis cells into neutrophils, while serum from mice immunized with the acellular pertussis vaccine did not....
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The role of RTX domain in the activity of adenylate cyclase toxin from Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.
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Mechanism of secretion of adenylate cyclase toxin from Bordetella pertussis via Type I secretion system (TISS)
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
Type I secretion system in Gram-negative bacteria translocates proteins from the cytoplasm to the extracellular medium in a single step across both membranes. The membrane-spanning channel is made up of just three proteins - an ATPase in the inner membrane, a membrane fusion protein and a specific outer membrane protein. This work provides a summary of current knowledge concerning the structure of the secretion system, as well as the assembly of the trans-envelope complex and the mechanism of protein secretion. The role of substrate folding on secretion is highlighted. It deals to some extent with the properties of the substrates translocated by the type I secretion system, with emphasis on the adenylate cyclase toxin of Bordetella pertusis, the agent causing whooping cough.
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Adenylate-cyclase toxin of Bordetella pertussis as a marker for the study of the complement receptor CD11b/CD18 endocytosis.
Chvojková, Věra ; Bumba, Ladislav (advisor) ; Černý, Jan (referee)
Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.
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