7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

PD Dr.

Chair

The exchange of central parts of the bacterial type III secretion system is required for assembly and function of the syste

Membranes and Transport

Important bacterial pathogens manipulate eukaryotic target cells by injecting effector proteins through type III secretion systems (T3SS). Recent in situ observations revealed that these large molecular machines, also called injectisomes, are amazingly dynamic and adaptive entities. We recently found that the cytosolic components of the T3SS form a mobile network that shuttles effector proteins from the cytosol to the export machinery (Wimmi et al, Nature Microbiol 2024). In contrast to these soluble components, the transmembrane rings anchoring the injectisome are stably associated – with one exception. Using live cell microscopy, single particle tracking and functional assays, we found that SctD, which forms the inner membrane ring of the T3SS, exchanges subunits in secreting injectisomes. To decipher the biological function of the unexpected mobility of this central structural component of the T3SS, we analyzed its role in assembly and function of the injectisome. Based on molecular dynamics modeling, we engineered SctD to allow to tune its exchange rate. The experiments showed that lower exchange leads to decreased secretion of effectors, revealing a direct correlation between protein exchange and the function of the T3SS. Our findings uncover a new aspect of the molecular function and regulation of the T3SS, which may be applicable to other secretion systems and molecular machines.

Oral Presentation

Prof. Dr.

Functional dissection of transport processes during glycopeptide antibiotic production

Glycopeptide antibiotics (GPAs) - e.g., vancomycin - are natural products that have been used for many years as a last resort and are an important group of agents to fight bacterial infections by inhibiting cell wall biosynthesis. GPAs are modified heptapeptides that are synthesized by non-ribosomal peptide synthetases (NRPS) from proteinogenic and non-proteinogenic amino acids. However, their modification results in a variety of different structures. In addition to all the biosynthetic proteins, we noticed that a transport-related protein was always co-encoded in the associated biosynthetic gene cluster (BGC). Interestingly, we found that in all 89 BGCs we analyzed, this was an ABC transporter. In A. balhimycina, the transporter Tba is essential for the export of the GPA balhimycin. However, a detailed functional analysis of its specificity and interactions in the cellular context is not yet available, which is the primary goal of our work.We performed a phylogenetic analysis of GPA ABC transporters in the context of other transporters encoded by producer strains and found that they form a distinct clade. We have established a balhimycin export assay that allows the investigation of the specificity of different GPA transporters in vivo by HPLC-MS and bioassays. This indicates substrate specificity of different GPA transporters, despite high similarity in sequence and structure. Furthermore, MD simulation and mutagenesis studies led us to suggest, which parts of the GPA are important for the interaction with the transporter. To confirm this in vitro, we are trying to purify Tba. Additionally, our data indicate a regulatory mechanism of the balhimycin production in absence of the native transporter. We have therefore used different omics approaches to investigate it further. So far, we have ruled out another regulation at the transcriptional level. Finally, we are trying to understand the role of GPA production and export in a cellular context by using MS-based methods to identify protein-protein interactions after proximity biotinylation and immunoprecipitation.

Characterization of the membrane phosphodiesterase NbdA of Pseudomonas aeruginosa.

The molecule c-di-GMP is a second messenger that controls various processes such as virulence and biofilm formation in bacteria (1). Synthesis and degradation of c-di-GMP is mediated by two types of enzymes, diguanylate cyclases (DGC) and phosphodiesterases (PDE) (1, 2). Pseudomonas aeruginosa PAO1, an opportunistic pathogen and biofilm model organism, encodes more than 40 c-di-GMP modulating proteins (3). In total, 22 of these proteins are predicted to be membrane bound. Until now, the biochemical characterization of these proteins has been limited mostly to their soluble cytosolic domains (4).The phosphodiesterase NbdA comprises a membrane bound MHYT domain, predicted to be a sensor for NO, CO or O2 (5), and a cytosolic part consisting of a degenerated diguanylate cyclase and a functional phosphodiesterase domain. In vivo assays showed the phosphodiesterase activity of NbdA, that could be confirmed in vitro with the purified cytosolic part of the protein (4). We established the production and purification of the full- length protein from E. coli using the nanodisc forming polymer DIBMA. We obtained pure and enzymatically active NbdA and characterized the binding of heme to the full-length protein. Furthermore, to study putative interaction partners of the full-length protein NbdA, we analyzed Pseudomonas membranes by mass spectrometry and complexome profiling (6). Several identified interaction partners were subsequently confirmed by bacterial two hybrid analysis. With this work we shed light on another part of the complex c-di-GMP signaling network of Pseudomonas aeruginosa.(1) Hengge, R. (2009) Nature Rev. Microbiol. 7: 263–273.(2) Römling, U. et al. (2005). Mol. Microbiol. 57: 629–639.(3) Valentini, M.&amp; Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.(4) Li, Y. et al. (2013) J. Bacteriol., 195 (16), 3531-3542.(5) Galperin, M. Y. et al. (2001) FEMS Microbiol. Lett 205 (1): 17–23.(6) Scherhag, A. et al. (2023) microLife. 4:uqad028.

Raum 13

Oral Session

Oral Sessions

Oral Session
Membranes and Transport

Appointment

Chair

Topic

Programme

Oral SessionMembranes and Transport

Appointment

Chair

Topic

Programme

Oral Session
Membranes and Transport