super-resolution-imaging-proteus-mirabilis-biofilm-expansion

Super-resolution Imaging of <em>Proteus mirabilis</em> Biofilm by Expansion Microscopy

Accessing detailed visual information and quantitative data from microbiological samples using conventional optical microscopy is limited by the ...

University of Chile

Biomedical Neuroscience Institute BNI

 comprises two species and more than 2500 serovars with marked differences in host specificity, and is responsible for a wide spectrum of diseases, ranging from localized gastroenteritis to severe life-threatening invasive disease. The initiation of the host inflammatory response, triggered by many Pathogen-Associated Molecular Patterns (PAMPs) that  possesses, recruits innate immune cells in order to restrain the infection at the local site. Neutrophils are known for killing bacteria through oxidative burst, amid other mechanisms. Amongst those mechanisms for controlling bacteria, the release of Extracellular Traps (ETs) represents a newly described pathway of programmed cell death known as ETosis. Particularly, Neutrophil Extracellular Traps (NETs) were first described in 2004 and since then, a number of reports have demonstrated their role as a novel defense mechanism against different pathogens. This released net-like material is composed of cellular DNA decorated with histones and cellular proteins. These structures have shown ability to trap, neutralize and kill different kinds of microorganisms, ranging from viruses and bacteria to fungi and parasites.  was one of the first microorganisms that were reported to be killed by NETs and several studies have confirmed the observation and deepened into its variants. Nevertheless, much less is known about their counterparts in other immune cells, e.g. Macrophage Extracellular Traps (METs) and -induced MET release has never been reported so far. In this work, we observed the production of METs induced by  serovar Typhimurium and recorded their effect on bacteria, showing for the first time that macrophages can also release extracellular DNA traps upon encounter with  Typhimurium. Additionally we show that METs effectively immobilize and reduce  survival in a few minutes, suggesting METs as a novel immune-mediated defense mechanism against  infection. Of note, this phenomenon was confirmed in primary macrophages, since MET release was also observed in bone marrow-derived macrophages infected with . The evidence of this peculiar mechanism provides new incipient insights into macrophages´ role against  infection and can help to design new strategies for the clinical control of this transcendental pathogen.

 Typhimurium Triggers Extracellular Traps Release in Murine Macrophages.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Role of Proteus mirabilis flagella in biofilm formation.

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization.

Urinary tract infections associated with the placement of indwelling urinary catheters are a significant concern in hospital settings, as they are linked to an increased risk of severe infections and complications due to biofilm formation. These infections are primarily caused by uropathogens such as Escherichia coli (UPEC). UPEC possesses peritrichous flagella, which facilitates its motility, adhesion to surfaces, and biofilm formation. Understanding the development of UPEC communities is essential for developing effective treatment and eradication strategies. In this study, we characterized the biofilm formation of a clinical non-motile UPEC strain under both static and dynamic culture conditions that simulate the urinary catheter environment. We developed a dynamic culture system coupled with light sheet fluorescence microscopy (LSFM) to quantify the stages of biofilm formation over time. Our results demonstrate that flagella play a crucial role in the initial phase of biofilm formation. The non-motile strain exhibited a delay in the adhesion phase compared to motile strains but ultimately formed biofilms of similar volume during subsequent stages. These findings highlight the significance of flagella in dynamic biofilm formation models and provide valuable insights for modeling the evolution of bacterial communities in nosocomial environments using LSFM.

Article	Year
Typhimurium Triggers Extracellular Traps Release in Murine Macrophages. Frontiers in cellular and infection microbiology\| PubMed ID: 33981627	2021
Role of Proteus mirabilis flagella in biofilm formation. Revista Argentina de microbiologia\| PubMed ID: 37076397	2023
Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization. Journal of microbiological methods\| PubMed ID: 38561125	2024
Delayed biofilm formation in non-motile uropathogenic Escherichia coli strain in static and dynamic growth conditions. Microbial pathogenesis\| PubMed ID: 40412736	2025

Jorge Jara-Wilde

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