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Bacteria produce a plethora of specialized metabolites (SM), with the ecological function of most of them not known. A major group of SM are peptides derived from nonribosomal peptide synthetases (NRPS). In entomopathogenic bacteria of the genus Xenorhabdus, PAX (peptide-antimicrobial-Xenorhabdus) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in Xenorhabdus doucetiaeand found the majority bound to the cells. We derivatized PAX with fluorophores and show binding to cells when added externally using super-resolution microscopy. Externally added PAX in X. doucetiaeand E. colias well as inducible PAX production in X. doucetiaeshowed a protective effect against various antimicrobial peptides (AMPs) from insects, where they are used as a defense mechanism against pathogens. Because AMPs are often positively charged, our results suggest a PAX-induced repulsive force due to positive charge at the bacterial cell wall.

We demonstrate stimulated emission depletion (STED) microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multicolor, and live-cell STED microscopy.

ABSTRACTBacterial pathogens exploit eukaryotic pathways for their own end. Upon ingestion, Salmonella entericaserovar Typhimurium passes through the stomach and then catalyzes its uptake across the intestinal epithelium. It survives and replicates in an acidic vacuole through the action of virulence factors secreted by a type three secretion system located on Salmonellapathogenicity island 2 (SPI-2). Two secreted effectors, SifA and SseJ, are sufficient for endosomal tubule formation, which modifies the vacuole and enables Salmonellato replicate within it. Two-color, superresolution imaging of the secreted virulence factor SseJ and tubulin revealed that SseJ formed clusters of conserved size at regular, periodic intervals in the host cytoplasm. Analysis of SseJ clustering indicated the presence of a pearling effect, which is a force-driven, osmotically sensitive process. The pearling transition is an instability driven by membranes under tension; it is induced by hypotonic or hypertonic buffer exchange and leads to the formation of beadlike structures of similar size and regular spacing. Reducing the osmolality of the fixation conditions using glutaraldehyde enabled visualization of continuous and intact tubules. Correlation analysis revealed that SseJ was colocalized with the motor protein kinesin. Tubulation of the endoplasmic reticulum is driven by microtubule motors, and in the present work, we describe how Salmonellahas coopted the microtubule motor kinesin to drive the force-dependent process of endosomal tubulation. Thus, endosomal tubule formation is a force-driven process catalyzed by Salmonellavirulence factors secreted into the host cytoplasm during infection.IMPORTANCEThis study represents the first example of using two-color, superresolution imaging to analyze the secretion of Salmonellavirulence factors as they are secreted from the SPI-2 type three secretion system. Previous studies imaged effectors that were overexpressed in the host cytoplasm. The present work reveals an unusual force-driven process, the pearling transition, which indicates that Salmonella-induced filaments are under force through the interactions of effector molecules with the motor protein kinesin. This work provides a caution by highlighting how fixation conditions can influence the images observed.