Antibacterial Activity of ZnO Nanoparticles in a Staphylococcus-aureus-Infected Galleria mellonella Model Is Tuned by Different Apple-Derived Phytocargos.

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-ZnO12 than for BE-ZnO8; 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.

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

In-Silico Analysis Highlights the Existence in Members of Burkholderia cepacia Complex of a New Class of Adhesins Possessing Collagen-like Domains.

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.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

Core-shell polycationic polyurea pharmadendrimers: new-generation of sustainable broad-spectrum antibiotics and antifungals.

The efficacy of conventional antimicrobials is falling to critical levels and raising alarming concerns around the globe. In this scenery, engineered nanoparticles emerged as a solid strategy to fight growing deadly infections. Here, we show the in vitro and in vivo performance of pharmadendrimers, a novel class of engineered polyurea dendrimers that are synthetic mimics of antibacterial peptides, against a collection of both Gram-positive and Gram-negative bacteria and fungi. These nanobiomaterials are stable solids prepared by low-cost and green processes, display a dense positively charged core-shell, and are biocompatible and hemocompatible drugs. Mechanistic data, corroborated by coarse-grained molecular dynamics simulations, points towards a fast-killing mechanism via membrane disruption, triggered by electrostatic interactions. Altogether this study provides strong evidence and support for the future use of polyurea pharmadendrimers in antibacterial and antifungal nanotherapeutics.

Insights into the Antimicrobial Activities and Metabolomes of Aquimarina (Flavobacteriaceae, Bacteroidetes) Species from the Rare Marine Biosphere.

Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the 'Sponge Microbiome Project' dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.

Impact of Ca2+-Induced PI(4,5)P2 Clusters on PH-YFP Organization and Protein-Protein Interactions.

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P2 concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P2 clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P2 is likely not defined solely by a simple (monomeric PI(4,5)P2)/(protein bound PI(4,5)P2) equilibrium, but instead depends on complex protein interactions with PI(4,5)P2 clusters. The insertion of PI(4,5)P2-binding proteins within these clusters can putatively modulate protein-protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca2+ on the organization and protein-protein interactions of PI(4,5)P2-binding proteins. We show that, in giant unilamellar vesicles presenting PI(4,5)P2, the membrane diffusion properties of pleckstrin homology (PH) domains tagged with a yellow fluorescent protein (YFP) are affected by the presence of Ca2+, suggesting direct interactions between the protein and PI(4,5)P2 clusters. Importantly, PH-YFP is found to dimerize in the membrane in the absence of Ca2+. This oligomerization is inhibited in the presence of physiological concentrations of the divalent cation. These results confirm that cation-dependent PI(4,5)P2 clustering promotes interactions between PI(4,5)P2-binding proteins and has the potential to dramatically influence the organization and downstream interactions of PI(4,5)P2-binding proteins in the plasma membrane.

Enhanced antibacterial activity of Rosehip extract-functionalized Mg(OH)2 nanoparticles: An in vitro and in vivo study.

The development of nanoparticles as antimicrobial agents against pathogenic bacteria has emerged as one of the leading global healthcare challenges. In this study, Mg(OH)2 NPs with controlled morphology and nanometric size, using two distinct counterions, chloride or nitrate, have been synthesized using Rosehip (RH) extract that has privileges beyond conventional chemical and physical methods. Various physicochemical techniques were used to characterize the RH-functionalized Mg-based NPs. They exhibited a spherical shape with a diameter of ~10 nm, low crystallinity compared to non-functionalized NPs, high polyphenol content, and negative zeta potential in three different media (H2O, TSB, and cell medium). The resulting RH-functionalized Mg-based NPs also exhibited an increased antibacterial activity against Gram-positive (S. Epidermis and S. aureus) and Gram-negative (E. Coli) bacteria compared to those prepared in pure water (0 % RH), an effect that was well evident with low NPs contents (250 µg/mL). A preliminary attempt to elucidate their mechanism of action revealed that RH-functionalized Mg-based NPs could disrupt cellular structures (bacterial cell wall and cytoplasmic membrane) and damage the bacterial cell, as confirmed by TEM imaging. Noteworthy is that Mg-based NPs exhibited higher toxicity to bacteria than to eukaryotic cells. More significantly, was their enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model, when infected with S. aureus bacteria. Overall, our findings indicate that well-engineered Rosehip magnesium-based nanoparticles can be used as a green non-cytotoxic polyphenolic source in different antibacterial applications for the biomedical industry.

Cationic lipid-based formulations for encapsulation and delivery of anti-EFG1 2' OMethylRNA oligomer.

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.

RNase R, a New Virulence Determinant of Streptococcus pneumoniae.

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. Therefore, the identification of new virulence determinants and the understanding of the molecular mechanisms behind pathogenesis have become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we used Galleria mellonella as an infection model to demonstrate that the presence of RNase R increases the pneumococcus virulence. Larvae infected with the RNase R mutant show an increased expression level of antimicrobial peptides. Furthermore, they have a lower bacterial load in the hemolymph in the later stages of infection, leading to a higher survival rate of the larvae. Interestingly, pneumococci expressing RNase R show a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Concomitantly, we observed a lower number of mutant bacteria inside larval hemocytes and a higher susceptibility to oxidative stress when compared to the wild type. Together, our results indicate that RNase R is involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalization and/or replication inside the larval hemocytes.

Third generation cephalosporin-resistant Klebsiella pneumoniae thriving in patients and in wastewater: what do they have in common?

BACKGROUND: Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3rd generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses. RESULTS: Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin. CONCLUSIONS: It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.

Anti-EFG1 2'-OMethylRNA oligomer inhibits Candida albicans filamentation and attenuates the candidiasis in Galleria mellonella.

EFG1 is a central transcriptional regulator of filamentation that is an important virulence factor of Candida albicans. This study serves to assess in vivo the applicability of the anti-EFG1 2'-OMethylRNA oligomer for inhibiting C . albicans filamentation and to attenuate candidiasis, using the Galleria mellonella model. For that, larvae infected with a lethal concentration of C. albicans cells were treated with a single dose and with a double dose of the anti-EFG1 2'OMe oligomer (at 40 and 100 nM). The anti-EFG1 2'OMe oligomer toxicity and effect on larvae survival was evaluated. No evidence of anti-EFG1 2'OMe oligomer toxicity was observed and the treatment with double dose of 2'OMe oligomer empowered larvae survival over 24 h (by 90%-100%) and prolonged its efficacy until 72 h of infection (by 30%). Undoubtedly, this work validates the in vivo therapeutic potential of anti-EFG1 2'OMe oligomer for controlling C. albicans infections.

Antisense locked nucleic acid gapmers to control Candida albicans filamentation.

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.

Marine Sponge and Octocoral-Associated Bacteria Show Versatile Secondary Metabolite Biosynthesis Potential and Antimicrobial Activities against Human Pathogens.

Marine microbiomes are prolific sources of bioactive natural products of potential pharmaceutical value. This study inspected two culture collections comprising 919 host-associated marine bacteria belonging to 55 genera and several thus-far unclassified lineages to identify isolates with potentially rich secondary metabolism and antimicrobial activities. Seventy representative isolates had their genomes mined for secondary metabolite biosynthetic gene clusters (SM-BGCs) and were screened for antimicrobial activities against four pathogenic bacteria and five pathogenic Candida strains. In total, 466 SM-BGCs were identified, with antimicrobial peptide- and polyketide synthase-related SM-BGCs being frequently detected. Only 38 SM-BGCs had similarities greater than 70% to SM-BGCs encoding known compounds, highlighting the potential biosynthetic novelty encoded by these genomes. Cross-streak assays showed that 33 of the 70 genome-sequenced isolates were active against at least one Candida species, while 44 isolates showed activity against at least one bacterial pathogen. Taxon-specific differences in antimicrobial activity among isolates suggested distinct molecules involved in antagonism against bacterial versus Candida pathogens. The here reported culture collections and genome-sequenced isolates constitute a valuable resource of understudied marine bacteria displaying antimicrobial activities and potential for the biosynthesis of novel secondary metabolites, holding promise for a future sustainable production of marine drug leads.

Burkholderia cenocepacia BCAM2418-induced antibody inhibits bacterial adhesion, confers protection to infection and enables identification of host glycans as adhesin targets.

Trimeric Autotransporter Adhesins (TAA) found in Gram-negative bacteria play a key role in virulence. This is the case of Burkholderia cepacia complex (Bcc), a group of related bacteria able to cause infections in patients with cystic fibrosis. These bacteria use TAAs, among other virulence factors, to bind to host protein receptors and their carbohydrate ligands. Blocking such contacts is an attractive approach to inhibit Bcc infections. In this study, using an antibody produced against the TAA BCAM2418 from the epidemic strain Burkholderia cenocepacia K56-2, we were able to uncover its roles as an adhesin and the type of host glycan structures that serve as recognition targets. The neutralisation of BCAM2418 was found to cause a reduction in the adhesion of the bacteria to bronchial cells and mucins. Moreover, in vivo studies have shown that the anti-BCAM2418 antibody exerted an inhibitory effect during infection in Galleria mellonella. Finally, inferred by glycan arrays, we were able to predict for the first time, host glycan epitopes for a TAA. We show that BCAM2418 favoured binding to 3'sialyl-3-fucosyllactose, histo-blood group A, α-(1,2)-linked Fuc-containing structures, Lewis structures and GM1 gangliosides. In addition, the glycan microarrays demonstrated similar specificities of Burkholderia species for their most intensely binding carbohydrates.

Burkholderia cenocepacia transcriptome during the early contacts with giant plasma membrane vesicles derived from live bronchial epithelial cells.

Burkholderia cenocepacia is known for its capacity of adherence and interaction with the host, causing severe opportunistic lung infections in cystic fibrosis patients. In this work we produced Giant Plasma Membrane Vesicles (GPMVs) from a bronchial epithelial cell line and validated their use as a cell-like alternative to investigate the steps involved in the adhesion process of B. cenocepacia. RNA-sequencing was performed and the analysis of the B. cenocepacia K56-2 transcriptome after the first contacts with the surface of host cells allowed the recognition of genes implicated in bacterial adaptation and virulence-associated functions. The sensing of host membranes led to a transcriptional shift that caused a cascade of metabolic and physiological adaptations to the host specific environment. Many of the differentially expressed genes encode proteins related with central metabolic pathways, transport systems, cellular processes, and virulence traits. The understanding of the changes in gene expression that occur in the early steps of infection can uncover new proteins implicated in B. cenocepacia-host cell adhesion, against which new blocking agents could be designed to control the progression of the infectious process.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates: In Vivo Virulence Assessment in Galleria mellonella and Potential Therapeutics by Polycationic Oligoethyleneimine.

Klebsiella pneumoniae, one of the most common pathogens found in hospital-acquired infections, is often resistant to multiple antibiotics. In fact, multidrug-resistant (MDR) K. pneumoniae producing KPC or OXA-48-like carbapenemases are recognized as a serious global health threat. In this sense, we evaluated the virulence of K. pneumoniae KPC(+) or OXA-48(+) aiming at potential antimicrobial therapeutics. K. pneumoniae carbapenemase (KPC) and the expanded-spectrum oxacillinase OXA-48 isolates were obtained from patients treated in medical care units in Lisbon, Portugal. The virulence potential of the K. pneumonia clinical isolates was tested using the Galleria mellonella model. For that, G. mellonella larvae were inoculated using patients KPC(+) and OXA-48(+) isolates. Using this in vivo model, the KPC(+) K. pneumoniae isolates showed to be, on average, more virulent than OXA-48(+). Virulence was found attenuated when a low bacterial inoculum (one magnitude lower) was tested. In addition, we also report the use of a synthetic polycationic oligomer (L-OEI-h) as a potential antimicrobial agent to fight infectious diseases caused by MDR bacteria. L-OEI-h has a broad-spectrum antibacterial activity and exerts a significantly bactericidal activity within the first 5-30 min treatment, causing lysis of the cytoplasmic membrane. Importantly, the polycationic oligomer showed low toxicity against in vitro models and no visible cytotoxicity (measured by survival and health index) was noted on the in vivo model (G. mellonella), thus L-OEI-h is foreseen as a promising polymer therapeutic for the treatment of MDR K. pneumoniae infections.

Designing P. aeruginosa synthetic phages with reduced genomes.

In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

A new regulator in the crossroads of oxidative stress resistance and virulence in Candida glabrata: The transcription factor CgTog1.

Candida glabrata is a prominent pathogenic yeast which exhibits a unique ability to survive the harsh environment of host immune cells. In this study, we describe the role of the transcription factor encoded by the gene CAGL0F09229g, here named CgTog1 after its Saccharomyces cerevisiae ortholog, as a new determinant of C. glabrata virulence. Interestingly, Tog1 is absent in the other clinically relevant Candida species (C. albicans, C. parapsilosis, C. tropicalis, C. auris), being exclusive to C. glabrata. CgTog1 was found to be required for oxidative stress resistance and for the modulation of reactive oxygen species inside C. glabrata cells. Also, CgTog1 was observed to be a nuclear protein, whose activity up-regulates the expression of 147 genes and represses 112 genes in C. glabrata cells exposed to H2O2, as revealed through RNA-seq-based transcriptomics analysis. Given the importance of oxidative stress response in the resistance to host immune cells, the effect of CgTOG1 expression in yeast survival upon phagocytosis by Galleria mellonella hemocytes was evaluated, leading to the identification of CgTog1 as a determinant of yeast survival upon phagocytosis. Interestingly, CgTog1 targets include many whose expression changes in C. glabrata cells after engulfment by macrophages, including those involved in reprogrammed carbon metabolism, glyoxylate cycle and fatty acid degradation. In summary, CgTog1 is a new and specific regulator of virulence in C. glabrata, contributing to oxidative stress resistance and survival upon phagocytosis by host immune cells.