Availability of iron ions impacts physicochemical properties and proteome of outer membrane vesicles released by Neisseria gonorrhoeae.

Outer membrane vesicles (OMVs) are bilayer structures released by bacteria for various purposes, e.g., response to environmental factors, bacterial communication, and interactions with host cells. One of the environmental variables bacteria need to react is the amount and availability of iron, a crucial element for bacteria biology. We have investigated the impact of the iron amount and availability on OMV secretion by pathogenic Neisseria gonorrhoeae, which, depending on the infection site, challenges different iron availability. N. gonorrhoeae releases OMVs in iron starvation and repletion growth environments. However, OMVs differed in physicochemical features and proteome according to iron amount and availability during the bacteria growth, as was analyzed by Liquid Chromatography-Tandem Mass Spectrometry, Infrared spectroscopy with a Fourier transform infrared spectrometer, and Atomic Force Microscopy. OMVs from iron starvation and repletion conditions had a higher variation in size, different flexibility, and different membrane protein and lipid components than OMVs isolated from control growth conditions. These OMVs also varied qualitatively and quantitatively in their total proteome composition and contained proteins unique for iron starvation and repletion conditions. Thus, the modulation of OMVs' properties seems to be a part of N. gonorrhoeae adaptation to surroundings and indicates a new direction of antigonococcal proceeding.

A Devonian crinoid with a diamond microlattice.

Owing to their remarkable physical properties, cellular structures, such as triply periodic minimal surfaces (TPMS), have multidisciplinary and multifunctional applications. Although these structures are observed in nature, examples of TPMS with large length scales in living organisms are exceedingly rare. Recently, microstructure reminiscent of the diamond-type TPMS was documented in the skeleton of the modern knobby starfish Protoreaster nodosus. Here we report a similar microlattice in a 385 Myr old crinoid Haplocrinites, which pushes back the origins of this highly ordered microstructure in echinoderms into the Devonian. Despite the low Mg2+/Ca2+ ratio of the 'calcite' Devonian sea, the skeleton of these crinoids has high-Mg content, which indicates strong biological control over biomineralogy. We suggest that such an optimization of trabecular arrangement additionally enriched in magnesium, which enhances the mechanical properties, might have evolved in these crinoids in response to increased predation pressure during the Middle Palaeozoic Marine Revolution. This discovery illustrates the remarkable ability of echinoderms, through the process of evolutionary optimization, to form a lightweight, stiff and damage-tolerant skeleton, which serves as an inspiration for biomimetic materials.

Phase-variable Type I methyltransferase M.NgoAV from Neisseria gonorrhoeae FA1090 regulates phasevarion expression and gonococcal phenotype.

The restriction-modification (RM) systems are compared to a primitive, innate, prokaryotic immune system, controlling the invasion by foreign DNA, composed of methyltransferase (MTase) and restriction endonuclease. The biological significance of RM systems extends beyond their defensive function, but the data on the regulatory role of Type I MTases are limited. We have previously characterized molecularly a non-canonical Type I RM system, NgoAV, with phase-variable specificity, encoded by Neisseria gonorrhoeae FA1090. In the current work, we have investigated the impact of methyltransferase NgoAV (M.NgoAV) activity on gonococcal phenotype and on epigenetic control of gene expression. For this purpose, we have constructed and studied genetic variants (concerning activity and specificity) within M.NgoAV locus. Deletion of M.NgoAV or switch of its specificity had an impact on phenotype of N. gonorrhoeae. Biofilm formation and planktonic growth, the resistance to antibiotics, which target bacterial peptidoglycan or other antimicrobials, and invasion of human epithelial host cells were affected. The expression of genes was deregulated in gonococcal cells with knockout M.NgoAV gene and the variant with new specificity. For the first time, the existence of a phasevarion (phase-variable regulon), directed by phase-variable Type I MTase, is demonstrated.

Polyelectrolyte Membrane Nanocoatings Aimed at Personal Protective and Medical Equipment Surfaces to Reduce Coronavirus Spreading.

The study of the surface of membrane coatings constructed with adsorbed coronavirus (COV) was described to test their suitability for the antiviral activity for application in personal protective and medical equipment. The nanocoating based on polyethyleneimine (PEI) or polystyrene sulfonate (PSS) with metallic nanoparticles incorporated was investigated using the AFM technique. Moreover, the functioning of human lung cells in a configuration with the prepared material with the adsorbed coronavirus was studied using microscopic techniques and flow cytometry. The mean values of the percentage share of viable cells compared with the control differed by a maximum of 22%. The results showed that PEI and PSS membrane layer coatings, modified with chosen metallic nanoparticles (AuNPs, AgNPs, CuNPs, FeNPs) that absorb COV, could support lung cells' function, despite the different distribution patterns of COV on designed surfaces as well as immobilized lung cells. Therefore, the developed membrane nanocoatings can be recommended as material for biomedical applications, e.g., medical equipment surfaces to reduce coronavirus spreading, as they adsorb COV and simultaneously maintain the functioning of the eukaryotic cells.

A Composite Membrane System with Gold Nanoparticles, Hydroxyapatite, and Fullerenol for Dual Interaction for Biomedical Purposes.

Background: Wound dressing plays a vital role in post-operative aftercare. There is the necessity to develop dressings for application on the border of soft and hard tissue. This study aimed to develop multifunctional polyelectrolyte layers enhanced by hydroxyapatite nanoparticles, gold nanoparticles (AuNPs), and/or fullerenol nanocomposites to achieve a wound dressing that could be applied on the bone-skin interface. Methods: Constructed shells were examined using TEM, STEM, and EDX techniques. The human osteoblasts or fibroblasts were immobilized within the shells. The systems morphology was assessed using SEM. The functioning of cells was determined by flow cytomery. Moreover, the internalization of AuNPs was assessed. Results: Involvement of fullerenol and/or hydroxyapatite nanoparticles influenced the immobilized cell systems morphology. Membranes with fullerenol and hydroxyapatite nanoparticles were observed to block the internalization of AuNPs by immobilized hFOB cells. Conclusions: The designed bilayer membranes incorporating fullerenol, and bacteriostatic elements, prevented the internalization of AuNPs by hFOB cells and ensured the proper counts and morphology of eukaryotic cells. The developed material can be recommended for dressings at the bone-skin interface.

Nanocomposite Membrane Scaffolds for Cell Function Maintaining for Biomedical Purposes.

Nanocomposite multilayered membrane coatings have been widely used experimentally to enhance biomedical materials surfaces. By the selection of reliable components, such systems are functionalized to be adjusted to specific purposes. As metal nanoparticles can reduce bacterial cell adhesion, the idea of using gold and silver nanoparticles of unique antimicrobial properties within membrane structure is outstandingly interesting considering dressings facilitating wound healing. The study was aimed to explore the interface between eukaryotic cells and wound dressing materials containing various nanoelements. The proposed systems are based on polyethyleneimine and hydroxyapatite thin layers incorporating metallic nanoparticles (silver or gold). To examine the structure of designed materials scanning electron and transmission electron microscopies were applied. Moreover, Fourier-transform infrared and energy-dispersive X-ray spectroscopies were used. Additionally, water contact angles of the designed membranes and their transport properties were estimated. The functioning of human fibroblasts was examined via flow cytometry to assess the biocompatibility of developed shells in the aspect of their cytotoxicity. The results indicated that designed nanocomposite membrane scaffolds support eukaryotic cells' functioning, confirming that the elaborated systems might be recommended as wound healing materials.

Polyelectrolyte Membrane with Hydroxyapatite and Silver Nanoparticles as a Material for Modern Wound Dressings.

Modern wound dressings not only play a covering role but also facilitate the function of the wound, contributing to a faster healing process. In this paper, we present a polyelectrolyte system with nanosized elements that could stimulate the growth of eukaryotic cells while providing antimicrobial properties, which may be recommended as a potential dressing material. The proposed platform consisted of polyethyleneimine, hydroxyapatite, and silver nanoparticles and was characterized using various macroscopic techniques. The constructed membrane scaffold was evaluated with immobilized WEHI 164 cells as a model system for cells sustained at the interface of bone and skin. Moreover, the bacteriostatic function of the designed membrane material was evaluated using different bacterial strains.

Lactobacillus crispatus and its enolase and glutamine synthetase influence interactions between Neisseria gonorrhoeae and human epithelial cells.

Neisseria gonorrhoeae, an obligatory human pathogen causes the sexually transmitted disease gonorrhea, which remains a global health problem. N. gonorrhoeae primarily infects the mucosa of the genitourinary tract, which in women, is colonized by natural microbiota, dominated by Lactobacillus spp., that protect human cells against pathogens. In this study, we demonstrated that precolonization of human epithelial cells with Lactobacillus crispatus, one of the most prevalent bacteria in the female urogenital tract, or preincubation with the L. crispatus enolase or glutamine synthetase impairs the adhesion and invasiveness of N. gonorrhoeae toward epithelial cells, two crucial steps in gonococcal pathogenesis. Furthermore, decreased expression of genes encoding the proinflam-matory cytokines, TNFα and CCL20, which are secreted as a consequence of N. gonorrhoeae infection, was observed in N. gonorrhoeae-infected epithelial cells that had been preco-lonized with L. crispatus or preincubated with enolase and glutamine synthetase. Thus, our results indicate that the protection of human cells against N. gonorrhoeae infection is a complex process and that L. crispatus and its proteins enolase and glutamine synthetase can have a potential role in protecting epithelial cells against gonococcal infection. Therefore, these results are important since disturbances of the micro-biota or of its proteins can result in dysbiosis, which is associated with increased susceptibility of epithelium to pathogens.

Virulence properties of Campylobacter jejuni are enhanced by displaying a mycobacterial TlyA methylation pattern in its rRNA.

Campylobacter jejuni is a bacterial pathogen that is generally acquired as a zoonotic infection from poultry and animals. Adhesion of C. jejuni to human colorectal epithelial cells is weakened after loss of its cj0588 gene. The Cj0588 protein belongs to the type I group of TlyA (TlyAI ) enzymes, which 2'-O-methylate nucleotide C1920 in 23S rRNA. Slightly longer TlyAII versions of the methyltransferase are found in actinobacterial species including Mycobacterium tuberculosis, and methylate not only C1920 but also nucleotide C1409 in 16S rRNA. Loss of TlyA function attenuates virulence of both M. tuberculosis and C. jejuni. We show here that the traits impaired in C. jejuni null strains can be rescued by complementation not only with the original cj0588 (tlyA I ) but also with a mycobacterial tlyA II gene. There are, however, significant differences in the recombinant phenotypes. While cj0588 restores motility, biofilm formation, adhesion to and invasion of human epithelial cells and stimulation of IL-8 production in a C. jejuni null strain, several of these properties are further enhanced by the mycobacterial tlyA II gene, in some cases to twice the original wild-type level. These findings strongly suggest that subtle changes in rRNA modification patterns can affect protein synthesis in a manner that has serious consequences for bacterial pathogenicity.

Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent.

Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage-vB_BbrP_BB8-of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae.

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes' encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

Gold Nanoparticle-Modified Poly(vinyl chloride) Surface with Improved Antimicrobial Properties for Medical Devices.

Despite the significant technological progress achieved in the past decades in the medical field, device-related infections carry a heavy social and economic burden. Surface modification of medical equipment is one of the most interesting approaches employed to improve the antibacterial activity of a material. Herein, we developed a process for the gold nanoparticle modification of a poly(vinyl chloride) laryngeal tube, which typically serves as an airway management device. In our study, we focused specifically on increasing the antimicrobial properties of the material while maintaining its biocompatibility. We applied two different modification methods to the poly(vinyl chloride) laryngeal tube. An increase in the antimicrobial activity of the surface was observed for both methods. In addition, the adsorption of bacterial cells on the material surface was assessed. We determined that the number of colonies cultured in the presence of the gold nanoparticle-modified samples or absorbed to the material surface decreased significantly compared with the control group. The trend was observed for both Gram-positive and Gram-negative strains. Moreover, it was established that the designed material did not exhibit a lethal impact on a control cell line. Finally, we noted discrepancies in the growth of bacteria cultured in the presence of modified or unmodified PVC material as well as differences in cell adherence to its surface. The proposed poly(vinyl chloride) modifications are most effective against Gram-positive bacteria, especially L. monocytogenes. Nevertheless, it ought to be emphasized that due to their different properties, each strain requires an individual approach.

Evaluation of selected phenotypic features among Campylobacter sp. strains of animal origin.

A total of 43 Campylobacter isolates from poultry, cattle and pigs were investigated for their ability to form biofilm. The studied strains were also screened for motility, adhesion and invasion of Caco-2 cells as well as extracellular DNase activity. The relation between biofilm formation and selected phenotypes was examined. Biofilm formation by the tested strains was found as irrespective from their motility and not associated with colonization abilities of human Caco-2 cells. Results of our study show that Campylobacter isolates from various animal sources are able to form biofilm and invade human Caco-2 cells in vitro.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm-3 (41.3±3.8µWcm-2) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices.

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni.

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2'-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2'-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2'-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2'-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.

Type III Methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 Regulates Biofilm Formation and Interactions with Human Cells.

Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many restriction modification (RM) systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngoAXmod gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a twofold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wild-type (wt) strain under standard grow conditions. Genes with changed expression levels encoded mostly proteins involved in cell metabolism, DNA replication and repair or regulating cellular processes and signaling (such as cell wall/envelop biogenesis). As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain. Live biofilm observations showed that the biofilm formed by the gonococcal ngoAXmod gene mutant is more relaxed, dispersed and thicker than the one formed by the wt strain. This more relaxed feature of the biofilm, in respect to adhesion and bacterial interactions, can be involved in pathogenesis. Moreover, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci [adhesion index = 0.672 (±0.2) and 2.15 (±1.53), respectively]; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells [invasion index = 3.38 (±0.93) × 10(5) for mutant and 4.67 (±3.09) × 10(4) for the wt strain]. These results indicate that NgoAX knock-out cells have lower ability to attach to human cells, but more easily penetrate inside the host cells. All these data suggest that the NgoAX methyltransferase, may be implicated in N. gonorrhoeae pathogenicity, involving regulation of biofilm formation, adhesion to host cells and epithelial cell invasion.

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence.