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

Numerous investigations exist about the alterations that oxygen free radicals can provoke on biomolecules; these modifications can be prevented and/or reversed by different antioxidants agents. On the other hand, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), a stable nitrogen synthetic radical, is used to evaluate the antioxidant capacity of medicinal herbal products; however, the structural changes that this radical provoke on the herbal active principles are not clear yet. In this work, we compared the redox reactivity of oxygen free radicals and DPPH radical on phospholipids and protein thiol groups present in rat liver microsomes. Cu2+/ascorbate was used as generator system of oxygen free radical and as antioxidant, an extract of Buddleja globosa's leaves. Cu2+/ascorbate provoked microsomal lipid peroxidation, microsomal thiols oxidation and oxygen consumption; all of these phenomena were inhibited by B. globosa extract. On the other hand, DPPH was bleached in different extension by the herbal extract and phosphatidyl choline; beside, DPPH decreased microsomal thiols content, but this phenomenon were not prevented by the herbal extract. Furthermore, DPPH did not induce oxygen consumption and neither modified the oxygen consumption induced by Cu2+/ascorbate. Distinct redox mechanisms may explain the differences between the reactivity of DPPH and oxygen free radicals on biomolecules, which is discussed.

DPPH and oxygen free radicals as pro-oxidant of biomolecules.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

ALS-linked protein disulfide isomerase variants cause motor dysfunction.

The parapineal organ is a midline-derived epithalamic structure that in zebrafish adopts a left-sided position at embryonic stages to promote the development of left-right asymmetries in the habenular nuclei. Despite extensive knowledge about its embryonic and larval development, it is still unknown whether the parapineal organ and its profuse larval connectivity with the left habenula are present in the adult brain or whether, as assumed from historical conceptions, this organ degenerates during ontogeny. This paper addresses this question by performing an ontogenetic analysis using an integrative morphological, ultrastructural and neurochemical approach. We find that the parapineal organ is lost as a morphological entity during ontogeny, while parapineal cells are incorporated into the posterior wall of the adult left dorsal habenular nucleus as small clusters or as single cells. Despite this integration, parapineal cells retain their structural, neurochemical and connective features, establishing a reciprocal synaptic connection with the more dorsal habenular neuropil. Furthermore, we describe the ultrastructure of parapineal cells using transmission electron microscopy and report immunoreactivity in parapineal cells with antibodies against substance P, tachykinin, serotonin and the photoreceptor markers arrestin3a and rod opsin. Our findings suggest that parapineal cells form an integral part of a neural circuit associated with the left habenula, possibly acting as local modulators of the circuit. We argue that the incorporation of parapineal cells into the habenula may be part of an evolutionarily relevant developmental mechanism underlying the presence/absence of the parapineal organ in teleosts, and perhaps in a broader sense in vertebrates.

Ontogenesis of the asymmetric parapineal organ in the zebrafish epithalamus.

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
DPPH and oxygen free radicals as pro-oxidant of biomolecules. Toxicology in vitro : an international journal published in association with BIBRA\| PubMed ID: 17888621	2008
ALS-linked protein disulfide isomerase variants cause motor dysfunction. The EMBO journal\| PubMed ID: 26869642	2016
Ontogenesis of the asymmetric parapineal organ in the zebrafish epithalamus. Frontiers in cell and developmental biology\| PubMed ID: 36568973	2022
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

Karina Palma

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