• Characterization of Novel Factors Involved in Swimming and Swarming Motility in Salmonella enterica Serovar Typhimurium.

      Deditius, Julia Andrea; Felgner, Sebastian; Spöring, Imke; Kühne, Caroline; Frahm, Michael; Rohde, Manfred; Weiß, Siegfried; Erhardt, Marc; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
      Salmonella enterica utilizes flagellar motility to swim through liquid environments and on surfaces. The biosynthesis of the flagellum is regulated on various levels, including transcriptional and posttranscriptional mechanisms. Here, we investigated the motility phenotype of 24 selected single gene deletions that were previously described to display swimming and swarming motility effects. Mutations in flgE, fliH, ydiV, rfaG, yjcC, STM1267 and STM3363 showed an altered motility phenotype. Deletions of flgE and fliH displayed a non-motile phenotype in both swimming and swarming motility assays as expected. The deletions of STM1267, STM3363, ydiV, rfaG and yjcC were further analyzed in detail for flagellar and fimbrial gene expression and filament formation. A ΔydiV mutant showed increased swimming motility, but a decrease in swarming motility, which coincided with derepression of curli fimbriae. A deletion of yjcC, encoding for an EAL domain-containing protein, increased swimming motility independent on flagellar gene expression. A ΔSTM1267 mutant displayed a hypermotile phenotype on swarm agar plates and was found to have increased numbers of flagella. In contrast, a knockout of STM3363 did also display an increase in swarming motility, but did not alter flagella numbers. Finally, a deletion of the LPS biosynthesis-related protein RfaG reduced swimming and swarming motility, associated with a decrease in transcription from flagellar class II and class III promoters and a lack of flagellar filaments.
    • A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.

      Fabiani, Florian D; Renault, Thibaud T; Peters, Britta; Dietsche, Tobias; Gálvez, Eric J C; Guse, Alina; Freier, Karen; Charpentier, Emmanuelle; Strowig, Till; Franz-Wachtel, Mirita; et al. (2017-08)
      Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.
    • Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.

      Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; et al. (2016)
      The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells.
    • Strategies to Block Bacterial Pathogenesis by Interference with Motility and Chemotaxis.

      Erhardt, Marc; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Infections by motile, pathogenic bacteria, such as Campylobacter species, Clostridium species, Escherichia coli, Helicobacter pylori, Listeria monocytogenes, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella species, Vibrio cholerae, and Yersinia species, represent a severe economic and health problem worldwide. Of special importance in this context is the increasing emergence and spread of multidrug-resistant bacteria. Due to the shortage of effective antibiotics for the treatment of infections caused by multidrug-resistant, pathogenic bacteria, the targeting of novel, virulence-relevant factors constitutes a promising, alternative approach. Bacteria have evolved distinct motility structures for movement across surfaces and in aqueous environments. In this review, I will focus on the bacterial flagellum, the associated chemosensory system, and the type-IV pilus as motility devices, which are crucial for bacterial pathogens to reach a preferred site of infection, facilitate biofilm formation, and adhere to surfaces or host cells. Thus, those nanomachines constitute potential targets for the development of novel anti-infectives that are urgently needed at a time of spreading antibiotic resistance. Both bacterial flagella and type-IV pili (T4P) are intricate macromolecular complexes made of dozens of different proteins and their motility function relies on the correct spatial and temporal assembly of various substructures. Specific type-III and type-IV secretion systems power the export of substrate proteins of the bacterial flagellum and type-IV pilus, respectively, and are homologous to virulence-associated type-III and type-II secretion systems. Accordingly, bacterial flagella and T4P represent attractive targets for novel antivirulence drugs interfering with synthesis, assembly, and function of these motility structures.
    • Tumour-targeting bacteria-based cancer therapies for increased specificity and improved outcome.

      Felgner, Sebastian; Pawar, Vinay; Kocijancic, Dino; Erhardt, Marc; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-03)