One ring to rule them all : Identification and characterization of the type IV pili secretin associated protein TsaP and analysis of the type IV secretion system of Neisseria gonorrhoeae

Over the years, N. gonorrhoeae has evolved and acquired different mechanisms to protect itself against a variety of antibiotics and chemotherapeutic agents. One reason for the rapid spread of antibiotic resistance in gonococci is the highly effective horizontal gene transfer. The transferred DNA is...

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Main Author: Siewering, Katja
Contributors: Søgaard-Andersen, Lotte (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2014
Biologie
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Summary:Over the years, N. gonorrhoeae has evolved and acquired different mechanisms to protect itself against a variety of antibiotics and chemotherapeutic agents. One reason for the rapid spread of antibiotic resistance in gonococci is the highly effective horizontal gene transfer. The transferred DNA is either provided directly via conjugation, or via the environment via autolysis or the gonococcal type IV secretion system (T4SS), which secretes ssDNA into the extracellular milieu. DNA uptake from the environment in Neisseria involves the type IV pili (T4P) and the competence system, transporting the DNA across the outer and the inner membrane, respectively. Functional characterization of the type IV secretion system and DNA uptake system and thus the type IV pili machinery in N. gonorrhoeae could provide starting points in the exploration of new therapeutic strategies. To better understand the transcriptional regulatory network of the type IV secretion system of N. gonorrhoeae transcriptional mapping of genes essential for DNA secretion was performed. This revealed that genes essential for DNA secretion are encoded within four different operons. Additional analysis of a region, which is not essential for DNA secretion, encoding the single-stranded DNA binding protein SsbB and the topoisomerase TopB showed that these genes are significantly more highly transcribed then genes that are involved in DNA secretion, such as the coupling protein TraD and the relaxase TraI. To investigate whether the single-stranded DNA, which is secreted via the T4SS encoded within the GGI facilitates biofilm formation, biofilm formation of N. gonorrhoeae strains were analyzed in continuous flow-chamber systems by confocal laser scanning microscopy. This showed that the ssDNA secreted via the T4SS plays a role in the early stages of biofilm formation. In Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which was named TsaP, the T4P secretin-associated protein, was identified as a widely conserved component that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the outer membrane in a PilQ-dependent manner. In addition, we identified that TsaP contains apart from the LysM domain, two FlgT-like domains and a linker region, which is specific for Neisseria spp. We could show that the linker domain plays an important role in pilus biogenesis in the β-proteobacterium N. gonorrhoeae. In order to determine if TsaP directly interacts with PilQ via the B2 domain, PilQ and TsaP of N. gonorrhoeae and M. xanthus were heterologously expressed and purified. Characterization of the heterologously expressed and purified proteins showed that TsaP is able to form SDS-stable complexes, resembling a ring-like structure, and that it might interact with PilQ, forming a double ring structure. In general, we propose that TsaP anchors the secretin to the PG to enable the secretin to withstand the forces generated during pilus extension and retraction. Because T4P play an important role in the pathogenesis of many bacteria and TsaP is found in all bacteria that express T4aP and plays an important role in T4aP biogenesis, it might be an important future drug target.
Physical Description:119 Pages
DOI:https://doi.org/10.17192/z2017.0048