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 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

Electron Cryotomography of Bacterial Secretion Systems

In biology, function arises from form. For bacterial secretion systems, which often span two membranes, avidly bind to the cell wall, and contain hundreds of individual proteins, studying form is a daunting task, made possible by electron cryotomography (ECT). ECT is the highest-resolution imaging technique currently available to visualize unique objects inside cells, providing a three-dimensional view of the shapes and locations of large macromolecular complexes in their native environment. Over the past 15 years, ECT has contributed to the study of bacterial secretion systems in two main ways: by revealing intact forms for the first time and by mapping components into these forms. Here we highlight some of these contributions, revealing structural convergence in type II secretion systems, structural divergence in type III secretion systems, unexpected structures in type IV secretion systems, and unexpected mechanisms in types V and VI secretion systems. Together, they offer a glimpse into a world of fantastic forms—nanoscale rotors, needles, pumps, and dart guns—much of which remains to be explored

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

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 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

Which stabilization technique corrects anatomy best in patients with AC-separation? An experimental study

In ten human cadaveric shoulder specimens four different parameters were documented prior to, and after, dissecting all passive stabilizers. These included the vertical, horizontal and mediolateral acromioclavicular distance, as well as the clavicular rotation. In addition, the same parameters were documented after acromioclavicular (AC) reconstruction using eight different techniques. The results showed a good reconstruction of the vertical ac-distance. Most of the techniques, especially the coracoid-sling procedure, led to a significant anterior displacement of the clavicle in relation to the scapula. To a lesser degree, most of the conventional procedures also resulted in a lateralization of the acromion and/or clavicular rotation. A bone anchor system for distal fixation in the base of the coracoid process and a medialized hole in the clavicle restored anatomy best. This new technique therefore is recommended for anatomical AC-reconstruction