A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Role of KCNK3 Dysfunction in Dasatinib-associated Pulmonary Arterial Hypertension and Endothelial Cell Dysfunction.

Pulmonary arterial (PA) hypertension (PAH) is a severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (Potassium channel subfamily K member 3; coding for outward K+ channel) variant in a patient with dasatinib-associated PAH and investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control human PA smooth muscle cells (hPASMCs) and human pulmonary endothelial cells (hPECs), we evaluated the consequences of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp experiments revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to PA constriction by decreasing KCNK3 function and expression. In control hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that a loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Mutation of SOCS2 induces structural and functional changes in mammary development.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.

Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility†.

The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

Comparison of goat and cow milk-derived extracellular vesicle miRNomes.

miRNAs present in milk are mainly found in extracellular vesicles (EVs), which are nanosized membrane vesicles released by most of the cell types to ensure intercellular communication. The majority of the studies performed so far on these vesicles have been conducted on human and cow's milk and focused on their miRNA content. The objectives of this study were to profile the miRNA content of purified EVs from five healthy goats and to compare their miRNome to those obtained from five healthy cows, at an early stage of lactation. EV populations were morphologically characterized using Transmission Electron Microscopy and Nanoparticle Tracking Analysis. The presence of EV protein markers checked by Western blotting and the absence of contamination of preparations by milk proteins. The size distribution and concentration of bovine and goat milk-derived EVs were similar. RNA-sequencing were performed, and all sequences were mapped to the cow genome identifying a total of 295 miRNAs. This study reports for the first-time a goat miRNome from milk EVs and its validation using cow miRNomes.

Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food.

The high incidence of foodborne diseases caused by pathogenic bacteria raises concerns worldwide and imposes considerable public healthcare challenges. This is especially observed with dormant spores of Bacilli, which can often survive treatments used by the food industry to kill growing bacteria. The early and rapid detection of bacterial spores is essential to ensure food safety. Commercial availability of such a test will present a high potential for food sector. We present a point-of-need colorimetric assay for detection of Bacillus cytotoxicus spores in food. The detection principle is based on spore-enhanced peroxidase-like catalytic activity of gold nanoparticles. The sensing platform consists of a microtube containing gold nanoparticles (AuNPs), and magnetic particles (MPs), both conjugated with specific aptamer BAS6R that recognize B. cytotoxicus spores. Upon the addition of the sample, spores were determined as present by the enhanced color change of the solution, due to the oxidation of tetramethylbenidine (TMB) with H2O2. The assay was evaluated by the naked eye (on/off) and quantitatively with use of a spectrophotometer. BAS6R@AuNPs aptasensor coupled to BAS6R@MPs proved to be highly sensitive, achieving the naked-eye limit of detection as low as 102 cfu/mL in water and milk, and 104 cfu/mL in mashed potatoes. Moreover, discrimination between spores of B. cytotoxicus and B. subtilis as well as bacterial vegetative cells was achieved in contaminated food samples, providing a good selectivity. This work provides a promising proof of concept for the development of instrument-free, low-cost and rapid assay for Bacillus cytotoxicus spore detection, which is able to compete in sensitivity with conventional costly and time-consuming laboratory analyses.

PBP2b Mutations Improve the Growth of Phage-Resistant Lactococcus cremoris Lacking Polysaccharide Pellicle.

Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.

Biomineralization of magnetic nanoparticles in stem cells.

Iron is one of the most common metals in the human body, with an intrinsic metabolism including proteins involved in its transport, storage, and redox mechanisms. A less explored singularity is the presence of magnetic iron in the organism, especially in the brain. The capacity of human stem cells to biosynthesize magnetic nanoparticles was recently demonstrated, using iron released by the degradation of synthetic magnetic nanoparticles. To evidence a magnetic biomineralization in mammalian cells, it is required to address the biosynthesis of magnetic nanoparticles in cells supplied exclusively with non-magnetic iron salt precursors. Herein, mouse and human mesenchymal stem cells were incubated with ferric quinate for up to 36 days. By optimizing the concentration and culture time, and by measuring both total intracellular iron content and cellular magnetic signals, the biosynthesis of magnetic nanoparticles was found to occur from 14 days of continuous iron incubation and was correlated with important doses of intracellular iron. The local electronic structure and chemical environment of intracellular iron were further characterized by XAS spectroscopy at the Fe K-edge, showing a total conversion of Fe2+ to Fe3+ when using ferrous salts (ascorbate and sulfate), and a transformation towards ferrihydrite as well as a small proportion of a magnetic phase.

Hierarchical superparamagnetic metal-organic framework nanovectors as anti-inflammatory nanomedicines.

Among a plethora of drug nanocarriers, biocompatible nanoscale metal-organic frameworks (nanoMOFs) with a large surface area and an amphiphilic internal microenvironment have emerged as promising drug delivery platforms, mainly for cancer therapy. However, their application in biomedicine still suffers from shortcomings such as a limited chemical and/or colloidal stability and/or toxicity. Here, we report the design of a hierarchically porous nano-object (denoted as USPIO@MIL) combining a benchmark nanoMOF (that is, MIL-100(Fe)) and ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (that is, maghemite) that is synthesized through a one-pot, cost-effective and environmentally friendly protocol. The synergistic coupling of the physico-chemical and functional properties of both nanoparticles confers to these nano-objects valuable features such as high colloidal stability, high biodegradability, low toxicity, high drug loading capacity as well as stimuli-responsive drug release and superparamagnetic properties. This bimodal MIL-100(Fe)/maghemite nanocarrier once loaded with anti-tumoral and anti-inflammatory drugs (doxorubicin and methotrexate) shows high anti-inflammatory and anti-tumoral activities. In addition, the USPIO@MIL nano-object exhibits excellent relaxometric properties and its applicability as an efficient contrast agent for magnetic resonance imaging is herein demonstrated. This highlights the high potential of the maghemite@MOF composite integrating the functions of imaging and therapy as a theranostic anti-inflammatory formulation.

Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease.

OBJECTIVE: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity. METHODS: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale. RESULTS: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin. CONCLUSIONS: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

Naked-eye detection of Staphylococcus aureus in powdered milk and infant formula using gold nanoparticles.

Nonspecific binding of proteins from complex food matrices is a significant challenge associated with a biosensor using gold nanoparticles (AuNPs). To overcome this, we developed an efficient EDTA chelating treatment to denature milk proteins and prevent their adsorption on AuNPs. The use of EDTA to solubilize proteins enabled a sensitive label-free apta-sensor platform for colorimetric detection of Staphylococcus aureus in milk and infant formula. In the assay, S. aureus depleted aptamers from the test solution, and the reduction of aptamers enabled aggregation of AuNPs upon salt addition, a process characterized by a color change from red to purple. Under optimized conditions, S. aureus could be visually detected within 30 min with the detection limit of 7.5 × 104 CFU/mL and 8.4 × 104 CFU/mL in milk and infant formula, respectively. The EDTA treatment provides new opportunities for monitoring milk contamination and may prove valuable for biosensor point-of-need applications.

In Vivo Assimilation of CuS, Iron Oxide and Iron Oxide@CuS Nanoparticles in Mice: A 6-Month Follow-Up Study.

Nanoparticles (NPs) are at the leading edge of nanomedicine, and determining their biosafety remains a mandatory precondition for biomedical applications. Herein, we explore the bioassimilation of copper sulfide NPs reported as powerful photo-responsive anticancer therapeutic agents. The nanoparticles investigated present a hollow shell morphology, that can be left empty (CuS NPs) or be filled with an iron oxide flower-like core (iron oxide@CuS NPs), and are compared with the iron oxide nanoparticles only (iron oxide NPs). CuS, iron oxide@CuS and iron oxide NPs were injected in 6-week-old mice, at doses coherent with an antitumoral treatment. Cu and Fe were quantified in the liver, spleen, kidneys, and lungs over 6 months, including the control animals, thus providing endogenous Cu and Fe levels in the first months after animal birth. After intravenous NPs administration, 77.0 ± 3.9% of the mass of Cu injected, and 78.6 ± 3.8% of the mass of Fe, were detected in the liver. In the spleen, we found 3.3 ± 0.6% of the injected Cu and 3.8 ± 0.6% for the Fe. No negative impact was observed on organ weight, nor on Cu or Fe homeostasis in the long term. The mass of the two metals returned to the control values within three months, a result that was confirmed by transmission electron microscopy and histology images. This bioassimilation with no negative impact comforts the possible translation of these nanomaterials into clinical practice.

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension.

INTRODUCTION: A reduction in pulmonary artery relaxation is a key event in the pathogenesis of pulmonary arterial hypertension (PAH). Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in airway epithelial cells plays a central role in cystic fibrosis; CFTR is also expressed in pulmonary arteries and has been shown to control endothelium-independent relaxation. AIM AND OBJECTIVES: We aimed to delineate the role of CFTR in PAH pathogenesis through observational and interventional experiments in human tissues and animal models. METHODS AND RESULTS: Reverse-transcriptase quantitative PCR, confocal imaging and electron microscopy showed that CFTR expression was reduced in pulmonary arteries from patients with idiopathic PAH (iPAH) and in rats with monocrotaline-induced pulmonary hypertension (PH). Moreover, using myography on human, pig and rat pulmonary arteries, we demonstrated that CFTR activation induces pulmonary artery relaxation. CFTR-mediated pulmonary artery relaxation was reduced in pulmonary arteries from iPAH patients and rats with monocrotaline- or chronic hypoxia-induced PH. Long-term in vivo CFTR inhibition in rats significantly increased right ventricular systolic pressure, which was related to exaggerated pulmonary vascular cell proliferation in situ and vessel neomuscularisation. Pathologic assessment of lungs from patients with severe cystic fibrosis (F508del-CFTR) revealed severe pulmonary artery remodelling with intimal fibrosis and medial hypertrophy. Lungs from homozygous F508delCftr rats exhibited pulmonary vessel neomuscularisation. The elevations in right ventricular systolic pressure and end diastolic pressure in monocrotaline-exposed rats with chronic CFTR inhibition were more prominent than those in vehicle-exposed rats. CONCLUSIONS: CFTR expression is strongly decreased in pulmonary artery smooth muscle and endothelial cells in human and animal models of PH. CFTR inhibition increases vascular cell proliferation and strongly reduces pulmonary artery relaxation.

The nucellus: between cell elimination and sugar transport.

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.

CEP250 is Required for Maintaining Centrosome Cohesion in the Germline and Fertility in Male Mice.

Male gametogenesis involves both mitotic divisions to amplify germ cell progenitors that gradually differentiate and meiotic divisions. Centrosomal regulation is essential for both types of divisions, with centrioles remaining tightly paired during the interphase. Here, we generated and characterized the phenotype of mutant mice devoid of Cep250/C-Nap1, a gene encoding for a docking protein for fibers linking centrioles, and characterized their phenotype. The Cep250 -/- mice presented with no major defects, apart from male infertility due to a reduction in the spermatogonial pool and the meiotic blockade. Spermatogonial stem cells expressing Zbtb16 were not affected, whereas the differentiating spermatogonia were vastly lost. These cells displayed abnormal γH2AX-staining, accompanied by an increase in the apoptotic rate. The few germ cells that survived at this stage, entered the meiotic prophase I and were arrested at a pachytene-like stage, likely due to synapsis defects and the unrepaired DNA double-strand breaks. In these cells, centrosomes split up precociously, with γ-tubulin foci being separated whereas these were closely associated in wild-type cells. Interestingly, this lack of cohesion was also observed in wild-type female meiocytes, likely explaining the normal fertility of Cep250 -/- female mice. Taken together, this study proposes a specific requirement of centrosome cohesion in the male germline, with a crucial role of CEP250 in both differentiating spermatogonia and meiotic spermatocytes.

Efficient incorporation and protection of lansoprazole in cyclodextrin metal-organic frameworks.

Lansoprazole (LPZ) is an acid pump inhibitor, which readily degrades upon acidic or basic conditions and under heating. We investigated here LPZ stability upon incorporation in particles made of cyclodextrin metal-organic frameworks (CD-MOFs). LPZ loaded CD-MOFs were successfully synthesized, reaching high LPZ payloads of 23.2 ± 2.1 wt%, which correspond to a molar ratio of 1:1 between LPZ and γ-CD. The homogeneity of LPZ loaded CD-MOFs in terms of component distribution was confirmed by elemental mapping by STEM-EDX. Both CTAB, the surfactant used in the CD-MOFs synthesis, and LPZ compete for their inclusion in the CD cavities. CTAB allowed obtaining regular cubic particles of around 5 µm with 15 wt% residual CTAB amounts. When LPZ was incorporated, the residual CTAB amount was less than 0.1 wt%, suggesting a higher affinity of LPZ for the CDs than CTAB. These findings were confirmed by molecular simulations. Vibrational circular dichroism studies confirmed the LPZ incorporation inside the CDs. Solid-state NMR showed that LPZ was located in the CDs and that it remained intact even after three years storage. Remarkably, the CD-MOFs matrix protected the drug upon thermal decomposition. This study highlights the interest of CD-MOFs for the incorporation and protection of LPZ.

Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew.

The nanoparticles produced by magnetotactic bacteria, called magnetosomes, are made of a magnetite core with high levels of crystallinity surrounded by a lipid bilayer. This organized structure has been developed during the course of evolution of these organisms to adapt to their specific habitat and is assumed to resist degradation and to be able to withstand the demanding biological environment. Herein, we investigated magnetosomes' structural fate upon internalization in human stem cells using magnetic and photothermal measurements, electron microscopy, and X-ray absorption spectroscopy. All measurements first converge to the demonstration that intracellular magnetosomes can experience an important biodegradation, with up to 70% of their initial content degraded, which is associated with the progressive storage of the released iron in the ferritin protein. It correlates with an extensive magnetite to ferrihydrite phase transition. The ionic species delivered by this degradation could then be used by the cells to biosynthesize magnetic nanoparticles anew. In this case, cell magnetism first decreased with magnetosomes being dissolved, but then cells remagnetized entirely, evidencing the neo-synthesis of biogenic magnetic nanoparticles. Bacteria-made biogenic magnetosomes can thus be totally remodeled by human stem cells, into human cells-made magnetic nanoparticles.

Bacillus subtilis Regulators MntR and Zur Participate in Redox Cycling, Antibiotic Sensitivity, and Cell Wall Plasticity.

The Bacillus subtilis MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of mntR and zur deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in ΔmntR and Δzur cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg2+ accumulation in the ΔmntR mutant. Phenotypic and morphological analyses of ΔmntR and Δzur mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure.IMPORTANCE Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.

In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.