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Characterization of a TatA/TatB binding site on the TatC component of the Escherichia coli twin arginine translocase

Emmanuele Severi, Mariana Bunoro Batista, Adelie Lannoy​, Phillip J Stansfeld, Tracy Palmer

In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A 'polar' cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

Microbiology. February 2023

Structural basis for membrane attack complex inhibition by CD59

Emma C. Couves, Scott Gardner, Tomas B. Voisin, Jasmine K. Bickel, Phillip J. Stansfeld, Edward W. Tate & Doryen Bubeck

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.

Nature Communications. February 2023

Wed 15 Mar 2023, 07:51 | Tags: Cells & Development HDC

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome directed bacterial cell shape

Carmina Micelli, Yunfei Dai, Nicole Raustad, Ralph R. Isberg, Christopher G. Dowson, Adrian J. Lloyd, Edward Geisinger , Allister Crow and David I. Roper 

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infectionsHere, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria.

PNAS. February 2023.

Press Release

Tue 07 Mar 2023, 08:02 | Tags: Microbiology & Infectious Disease HDC

Supramolecular Organisation and Dynamics of Mannosylated Phosphatidylinositol Lipids in the Mycobacterial Plasma Membrane

Chelsea M. Brown, Robin A. Corey, Axelle Grélard, Ya Gao, Yeol Kyo Choi, Emanuel Luna, Martine Gilleron, Nicolas Destainville, Jérôme Nigou, Antoine Loquet, Elizabeth Fullam, Wonpil Im, Phillip J. Stansfeld, Matthieu Chavent

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually.. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM (phosphatidyl-myoinositol mannosides) lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

PNAS. January 2023

Fri 10 Feb 2023, 08:21 | Tags: Microbiology & Infectious Disease HDC

Genomic Assembly of Clinical Candida glabrata (Nakaseomyces glabrata) Isolates Reveals within-Species Structural Plasticity and Association with In Vitro Antifungal Susceptibility

Irene Stefanini, Emily Stoakes, Houdini H T Wu, Li Xu-McCrae, Abid Hussain, John Moat, Christopher G Dowson, Miruna D David, Chrystala Constantinidou

The opportunistic human pathogen Candida glabrata has become an increasingly important threat to human health, with infections globally characterized by high mortality rates and multidrug resistance. Previous comparative genetics studies conducted on limited numbers of strains only revealed partial resolution of chromosomal settings. In this study, by combining short- and long-read genome sequencing, phenotypic characterization, and comparative genomics over a large set of strains, we detected strict relationships between large chromosomal rearrangements and phylogenetic clades, genes subjected to different selective pressures, and new sets of genes associated with resistance to antifungals. Overall, these results not only provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology but may also lay the foundations for the future development of tailored therapeutic approaches.

Microbiology Spectrum. December 2022

Wed 18 Jan 2023, 07:58 | Tags: Microbiology & Infectious Disease HDC

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

Ruochen Ouyang, Ana Rita Costa, C. Keith Cassidy, Aleksandra Otwinowska, Vera C. J. Williams, Agnieszka Latka, Phill J. Stansfeld, Zuzanna Drulis-Kawa, Yves Briers, Daniël M. Pelt, Stan J. J. Brouns & Ariane Briegel

The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Å and 3.0 Å resolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

Nature Communications. November 2022

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