Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms. Here, we uncover the mechanism by which these pili build three-dimensional (3D) biofilm architectures. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners. This antiparallel arrangement ensures that contacts form primarily between pili from interacting cells rather than pili from the same cell. With a remarkably short helical repeat, archaic chaperone-usher pili spontaneously establish a high density of junctions that determines the biofilm's 3D architecture. Our findings may help develop new therapies against multidrug-resistant bacterial infections by targeting pilus-pilus interactions.

Adhesive pili assembled through the chaperone–usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria 1 – 3 . Archaic chaperone–usher pathway pili, the most diverse and widespread chaperone–usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens 1 , 4 , 5 . However, their architecture and assembly–secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii —a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.

Adhesive pili are hair-like appendages assembled via the chaperone-usher pathway (CUP) that mediate host tissue colonization and biofilm formation of Gram-negative bacteria 1-3. Archaic CUP pili, the most diverse and widespread CUP adhesins, are promising vaccine and drug targets due to their prevalence in the most troublesome multidrug-resistant (MDR) pathogens 1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the 3.4 Å resolution cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii, a notorious MDR nosocomial pathogen. In contrast to the thick helical tubes of the classical CUP pili, archaic pili assemble into a conceptually novel ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed now for the first time in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight MDR bacterial infections.

Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as "lactosylceramide-binding". Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.

Acinetobacter baumannii-a leading cause of nosocomial infections-has a remarkable capacity to persist in hospital environments and medical devices due to its ability to form biofilms. Biofilm formation is mediated by Csu pili, assembled via the "archaic" chaperone-usher pathway. The X-ray structure of the CsuC-CsuE chaperone-adhesin preassembly complex reveals the basis for bacterial attachment to abiotic surfaces. CsuE exposes three hydrophobic finger-like loops at the tip of the pilus. Decreasing the hydrophobicity of these abolishes bacterial attachment, suggesting that archaic pili use tip-fingers to detect and bind to hydrophobic cavities in substrates. Antitip antibody completely blocks biofilm formation, presenting a means to prevent the spread of the pathogen. The use of hydrophilic materials instead of hydrophobic plastics in medical devices may represent another simple and cheap solution to reduce pathogen spread. Phylogenetic analysis suggests that the tip-fingers binding mechanism is shared by all archaic pili carrying two-domain adhesins. The use of flexible fingers instead of classical receptor-binding cavities is presumably more advantageous for attachment to structurally variable substrates, such as abiotic surfaces.

Adhesive pili are external component of fibrous adhesive organelles and help bacteria attach to biotic or abiotic surfaces. The biogenesis of adhesive pili via the chaperone-usher pathway (CUP) is independent of external energy sources. In the classical CUP, chaperones transport assembly-competent pilins in a folded but expanded conformation. During donor-strand exchange, pilins subsequently collapse, producing a tightly packed hydrophobic core and releasing the necessary free energy to drive fiber formation. Here, we show that pilus biogenesis in non-classical, archaic, and alternative CUPs uses a different source of conformational energy. High-resolution structures of the archaic Csu-pili system from Acinetobacter baumannii revealed that non-classical chaperones employ a short donor strand motif that is insufficient to fully complement the pilin fold. This results in chaperone-bound pilins being trapped in a substantially unfolded intermediate. The exchange of this short motif with the longer donor strand from adjacent pilin provides the full steric information essential for folding, and thereby induces a large unfolded-to-folded conformational transition to drive assembly. Our findings may inform the development of anti-adhesion drugs (pilicides) to combat bacterial infections.

High-temperature corrosion resistance of ferritic stainless steels (Fe–Cr based alloys) is built upon the formation of protective Cr-rich oxide scale. However, Cr vaporization limits the use of Fe–Cr alloys under extreme service conditions; in particular, it has been identified as the most significant failure mechanism in solid-oxide fuel cells (SOFCs). Our study focusses on the initial stages of oxide scale formation on ferritic stainless steels and shows that the Cr vaporization can be controlled via the alloy composition and heat treatments. In this work, we investigate the influence of heat treatment on the initial stages of oxidation of two Ti–Nb stabilized ferritic stainless steels (EN 1.4509 and EN 1.4521) at 650 °C by synchrotron light mediated X-ray photoelectron spectroscopy (XPS) and photoemission electron microscopy (PEEM). The high degree of alloying makes these alloys suitable for high temperature applications, but also renders the alloys prone to microstructural changes that can affect the growth of protective oxide scale. As a demonstration of this, we show that the heat treatment induced precipitation of (FeCrSi)2(MoNb)-type Laves phase results in less pronounced surface segregation and oxidation of minor alloying elements (Mo, Mn, Nb, Ti, Si). Most significantly, the diffusion of Mn and the formation of low volatile (MnCr)3O4 spinel oxide at the surface above Cr2O3 are strongly suppressed. 1. Ali-Löytty, H. et al. Int. J. Hydrog. Energy 37, 14528–14535 (2012). 2. Ali-Löytty, H. et al. Int. J. Hydrog. Energy 38, 1039–1051 (2013). 3. Ali-Löytty, H. et al. Corros. Sci. 112, 204–213 (2016). 4. Ali-Löytty, H. et al. Submitted to Corros. Sci.

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit. Bacterial RNA polymerase (RNAP) is crucial for cellular gene expression and a validated target for antimicrobial drugs. Here, Malinen et al. explore the effects of the CBR class of RNAP inhibitors on the E. coliRNAP transcription cycle and provide detailed mechanistic insight into their antibacterial action.