Adhesion of Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly(vinyl chloride) (PVC) from endotracheal intubation devices

Pseudomonas aeruginosa pneumonia is a life threatening complication in mechanically ventilated patients that requires the ability of the bacteria to adhere to, and colonize the endotracheal intubation device. New strategies to prevent or reduce these nosocomial infections are greatly needed. We report here the study of a set of P. aeruginosa clinical isolates, together with specific mutants, regarding their adhesion on native and chemically modified poly(vinyl chloride) (PVC) surfaces from endotracheal intubation devices. The adhesion of the different strains to untreated PVC varied widely, correlating with several physico-chemical characteristics known to influence the attachment of bacteria to inert surfaces. The adhesion patterns were compared to the calculations obtained with the DLVO theory of colloidal stability. These results illustrate the importance of testing different clinical isolates when investigating bacterial adhesion. Oxygen plasma treatment of the PVC pieces yielded a hydrophilic surface and reduced the number of adhering bacteria by as much as 70%. This reduction is however unlikely to be sufficient to prevent P. aeruginosa colonization of endotracheal intubation devices. (C) 2002 Elsevier Science Ltd. All rights reserved.

Characterization of Pseudomonas aeruginosa mechanosensing through label-free imaging of type IV pili

Lorenzo Anton-Louis Talà

Bacteria are ubiquitously found in all sorts of environment. They're found in the ocean, soil, or even in our guts or on our skin. Independently of their niche, they can transition from a planktonic state were they freely swim in an aqueous environment to a surface-associated state where they form multicellular communities known as biofilms. It is known that bacteria change their physiology when associated with surfaces. This is many times accompanied with a different transcriptional state compared to planktonic populations. Pseudomonas aeruginosa is an opportunistic pathogen often involved in nosocomial infections that can lead to chronic infection in immunocompromised patients. P. aeruginosa infections are often associated with a surface-associated biofilm lifestyle. In addition, it uses a host contact-dependent toxin secretion system during pathogenicity. It also possesses a surface-specific motility system known as twitching. These three examples are all physiological adaptation to surface contact. All these are a result of a change in gene expression upon surface contact.To power twitching motility, P. aeruginosa uses long and thin extracellular filaments called type IV pili (TFP). P. aeruginosa extends and retracts its TFP to pull itself forward on surfaces. Beyond their function in motility, TFP extension, attachment on a solid substrate and retraction are required for a surface-specific transcriptional response in a process called mechanosensing. A chemotaxis-like two component system called the Chp system also mediates this surface-dependent response. The activated Chp system upregulates transcription of virulence related genes including genes involved in TFP biogenesis. However, little is known about how the Chp system is activated by TFP, and how TFP respond to mechanosensing. The aim of this thesis is to characterize the mechanisms of mechanosensation in P. aeruginosa. In order to achieve this, one must first better characterize the mechanical input signal generated by TFP attachment events. This is an experimental challenge as TFP fibers cannot be imaged with conventional microscopes. In this thesis I present an approach to the investigation of P. aeruginosa mechanosensation by leveraging interferometric scattering microscopy (iSCAT) that I optimized for label-free detection of TFP during live cell imaging. I also show the power of iSCAT microscopy to detect and analyze TFP and their dynamic behavior on surfaces.By combining iSCAT to fluorescence microscopy, I could then track intracellular regulatory proteins and simultaneous TFP activity. Combining these measurements with single cell motility assays and protein localization, I was able to highlight signaling feedback loops between TFP and the Chp chemotaxis system that are critical to motility and mechanosensing. This feedback system allows for cell-polarization that directs twitching motility, which we term mechanotaxis.Finally, based on new biophysical and structural evidence, I propose a model for the mechanisms of mechanosensing by TFP and the Chp system. Here, my preliminary data show that the PilA monomers that are disassembled from TFP during retraction interact with the Chp chemosensor PilJ. Due to TFP attachment, the flux of PilA towards PilJ is different in liquid vs surfaces. I propose a mechanism wherein PilJ senses this flux imbalance.

EPFL2022

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Sandor Kasas, Giovanni Longo, Petar Stupar

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

Royal Society of Chemistry2015

Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster

Andrew Jonathan Bretscher, Jan Paul Dudzic, Bruno Lemaitre, Claudia Melcarne, Elodie Julie Virginie Ramond

Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivoRNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1(1);eater(1) haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake.

WILEY2019

Characterization of Pseudomonas aeruginosa mechanosensing through label-free imaging of type IV pili

Lorenzo Anton-Louis Talà

EPFL2022