Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Benn, Georgina; Pyne, Alice L. B.; Ryadnov, Maxim G.; Hoogenboom, Bart W.

doi:10.1039/c9an01185d

Cited by 24 publications

(17 citation statements)

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“…Bacteria were grown as previously described and resuspended in HEPES buffered saline (HBS, 20 mM HEPES/120 mM NaCl at pH 7.4) to OD600 = 1.0 (1x10 9 bacteria/ml). 100 μl of bacteria were immobilized on cleaned glass slides (Corning/Sigma Aldrich) covered with Vectabond ® (Vector Laboratories, USA) as described previously for 30 minutes at RT [ 54 ]. During all steps, care was taken in preventing the droplet from drying out.…”

Section: Methodsmentioning

confidence: 99%

Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

et al. 2020

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An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

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Section: Methodsmentioning

confidence: 99%

Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

et al. 2020

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“…Importantly, the immobilization step should not affect the integrity of the microorganism surface in a chemical or structural way. Most bacteria do not adhere well to solid surfaces or glass; thus, different techniques are required for proper bacterial cell immobilization, including (i) drying of the sample, (ii) passing suspension through the filters to stock the cells into the pores [ 36 ], (iii) soft gels [ 37 ], or (iv) surface coatings that assure charge drawing immobilization [ 36 , 38 ]. If there is a need for high-resolution imaging, it is advantageous to employ mica surface characterized by good adsorption and low background noise [ 39 ].…”

Section: Introductionmentioning

confidence: 99%

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

et al. 2020

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Despite the hope that was raised with the implementation of antibiotics to the treatment of infections in medical practice, the initial enthusiasm has substantially faded due to increasing drug resistance in pathogenic microorganisms. Therefore, there is a need for novel analytical and diagnostic methods in order to extend our knowledge regarding the mode of action of the conventional and novel antimicrobial agents from a perspective of single microbial cells as well as their communities growing in infected sites, i.e., biofilms. In recent years, atomic force microscopy (AFM) has been mostly used to study different aspects of the pathophysiology of noninfectious conditions with attempts to characterize morphological and rheological properties of tissues, individual mammalian cells as well as their organelles and extracellular matrix, and cells’ mechanical changes upon exposure to different stimuli. At the same time, an ever-growing number of studies have demonstrated AFM as a valuable approach in studying microorganisms in regard to changes in their morphology and nanomechanical properties, e.g., stiffness in response to antimicrobial treatment or interaction with a substrate as well as the mechanisms behind their virulence. This review summarizes recent developments and the authors’ point of view on AFM-based evaluation of microorganisms’ response to applied antimicrobial treatment within a group of selected bacteria, fungi, and viruses. The AFM potential in development of modern diagnostic and therapeutic methods for combating of infections caused by drug-resistant bacterial strains is also discussed.

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“…Bacteria were grown as previously described and resuspended in HEPES buffered saline (HBS, 20 mM HEPES/120 mM NaCl at pH 7.4) to OD600 = 1.0 (1×10 9 bacteria/ml). 100 µl of bacteria were immobilized on cleaned glass slides (Corning/Sigma Aldrich) covered with Vectabond ® (Vector Laboratories, USA) as described previously for 30 minutes at RT (45). During all steps, care was taken in preventing the droplet from drying out.…”

Section: Methodsmentioning

confidence: 99%

Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

Doorduijn

Bardoel

Heesterbeek

et al. 2019

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15An important effector function of the human complement system is to directly kill Gram-16 negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when 17 surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and 18 multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. 19 Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by 20 surface-bound convertases. In this study we aimed to understand why local assembly of MAC 21 pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is 22 required to form bactericidal MAC pores. Binding experiments with fluorescently labelled C6 23 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving 24 experiments and atomic force microscopy revealed that this rapid interaction between C7 and 25 C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete 26 MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial 27 pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. 28 While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring 29 of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. 30 Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are 31 assembled and how bacteria evade MAC-dependent killing. 33When bacteria invade the human body, they are attacked by the immune system that tries to clear 34 the infection. Gram-negative bacteria can be directly killed by the complement system, a family 35 of proteins in blood and bodily fluids, that forms toroid-shaped Membrane Attack Complex 36 (MAC) pores into bacterial membranes (1-4). MAC assembly is initiated when recognition 37 molecules, such as antibodies and lectins, bind to bacterial surface structures and recruit early 38 complement components to the target cell surface. This triggers activation of the complement 39 system, a proteolytic cascade that labels the surface with convertase enzymes that cleave the 40 central components C3 and C5 (5). Conversion of C5 into C5b, by C5 convertases, is a critical 41 first step for the unidirectional step-wise assembly of the MAC pore ( Fig. 1A). Upon formation 42 of C5b, C6 rapidly binds metastable C5b to form a stable C5b6 complex (6). Subsequent 43 association of C7 converts the hydrophilic C5b6 complex into lipophilic C5b-7 that uses an 44 exposed membrane-binding domain of C7 to anchor to the target membrane (7,8). Membrane-45 bound C5b-7 subsequently recruits one copy of C8 and up to 18 copies of C9, which oligomerize 46 and form of a 1.2 MDa transmembrane MAC pore (C5b-9) (9-11). 47Although the molecular steps of MAC assembly have been extensively studied on erythrocytes 48 and model membranes (8,9,(12)(13)(14)(15), the mechanisms by which complement f...

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Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Abstract: Different sample preparations are compared, to facilitate atomic force microscopy (AFM) of live Gram-negative bacteria. The obtained resolution is sufficient to resolve the proteinaceous network in the outer membrane.

Cited by 24 publications

References 41 publications

Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

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