is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular
capsular type K2 has been associated with high ...antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by
experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (
) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50 μg is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant
infections.
is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The
K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria
; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.
Abstract
The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the ...complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2′OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model.
To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2′OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection.
In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2′OMe oligomers.
Burkholderia cenocepacia is a multi-drug-resistant lung pathogen. This species synthesizes various virulence factors, among which cell-surface components (adhesins) are critical for establishing the ...contact with host cells. This work in the first part focuses on the current knowledge about the adhesion molecules described in this species. In the second part, through in silico approaches, we perform a comprehensive analysis of a group of unique bacterial proteins possessing collagen-like domains (CLDs) that are strikingly overrepresented in the Burkholderia species, representing a new putative class of adhesins. We identified 75 CLD-containing proteins in Burkholderia cepacia complex (Bcc) members (Bcc-CLPs). The phylogenetic analysis of Bcc-CLPs revealed the evolution of the core domain denominated “Bacterial collagen-like, middle region”. Our analysis remarkably shows that these proteins are formed by extensive sets of compositionally biased residues located within intrinsically disordered regions (IDR). Here, we discuss how IDR functions may increase their efficiency as adhesion factors. Finally, we provided an analysis of a set of five homologs identified in B. cenocepacia J2315. Thus, we propose the existence in Bcc of a new type of adhesion factors distinct from the described collagen-like proteins (CLPs) found in Gram-positive bacteria.
Microorganisms are exposed to harmful oxygen and nitrogen reactive species that are formed in the environment and produced by cells of the mammalian innate immunity. Iron-sulfur clusters, contained ...in proteins that largely sustain microbial life, are damaged by oxidative/nitrosative stresses causing the inactivation of the protein/enzyme. Depending on the extension of the damage, the functional recuperation of the Fe-S protein requires specialized machineries for repair or de novo assembly of the cluster. Repair of Iron Centres (RIC) are a family of di-iron proteins whose first member was identified in Escherichia coli (formely YtfE) as having an unprecedented role in the protection of Fe-S centres by delivering iron for the formation of Fe-S clusters1,2. We show that in human pathogens such as Staphylococcus aureus, Neisseria gonorrhoeae and Trichomonas vaginalis RIC confers protection against nitrogen and oxygen reactive species. Moreover, we demonstrate that RIC is required for full virulence of S. aureus upon infection of the wax moth larva Galleria mellonella3–5.
Abstract
The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the ...complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2′OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model.
To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2′OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection.
In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2′OMe oligomers.
Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare ...the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2′-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.
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•LNA-gapmers were able to significantly reduce C. albicans filamentation and the levels of EFG1 gene expression in vitro.•The inclusion of PS-linkage and palmitoyl-2′-amino-LNA chemical modification in the LNA-gapmers increasing the survival of G. mellonella.•LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.
This research investigates pH changes during the green synthesis of ZnO nanoparticles (NPs) and emphasises its importance in their physicochemical, antibacterial, and biological properties. Varying ...the synthesis pH from 8 to 12 using “Bravo de Esmolfe” apple extracts neither affected the morphology nor crystallinity of ZnO but impacted NP phytochemical loads. This difference is because alkaline hydrolysis of phytochemicals occurred with increasing pH, resulting in BE-ZnO with distinct phytocargos. To determine the toxicity of BE-ZnO NPs, Galleria mellonella was used as an alternative to non-rodent models. These assays showed no adverse effects on larvae up to a concentration of 200 mg/kg and that NPs excess was relieved by faeces and silk fibres. This was evaluated by utilising fluorescence-lifetime imaging microscopy (FLIM) to track NPs’ intrinsic fluorescence. The antibacterial efficacy against Staphylococcus aureus was higher for BE-ZnOsub.12 than for BE-ZnOsub.8; however, a different trend was attained in an in vivo infection model. This result may be related to NPs’ residence in larvae haemocytes, modulated by their phytocargos. This research demonstrates, for the first time, the potential of green synthesis to modulate the biosafety and antibacterial activity of NPs in an advanced G. mellonella infection model. These findings support future strategies to overcome antimicrobial resistance by utilizing distinct phytocargos to modulate NPs’ action over time.
Abstract
Objectives
To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus ...biofilms.
Methods
Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths.
Results
Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified.
Conclusions
Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.
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