A Glimpse into Dendrimers Integration in Cancer Imaging and Theranostics.

Cancer is a result of abnormal cell proliferation. This pathology is a serious health problem since it is a leading cause of death worldwide. Current anti-cancer therapies rely on surgery, radiation, and chemotherapy. However, these treatments still present major associated problems, namely the absence of specificity. Thus, it is urgent to develop novel therapeutic strategies. Nanoparticles, particularly dendrimers, have been paving their way to the front line of cancer treatment, mostly for drug and gene delivery, diagnosis, and disease monitoring. This is mainly derived from their high versatility, which results from their ability to undergo distinct surface functionalization, leading to improved performance. In recent years, the anticancer and antimetastatic capacities of dendrimers have been discovered, opening new frontiers to dendrimer-based chemotherapeutics. In the present review, we summarize the intrinsic anticancer activity of different dendrimers as well as their use as nanocarriers in cancer diagnostics and treatment.

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms.

BACKGROUND: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. OBJECTIVES: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. METHODS: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. RESULTS: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. CONCLUSIONS: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Lipid Droplets in Cancer: From Composition and Role to Imaging and Therapeutics.

Cancer is the second most common cause of death worldwide, having its origin in the abnormal growth of cells. Available chemotherapeutics still present major drawbacks, usually associated with high toxicity and poor distribution, with only a small fraction of drugs reaching the tumour sites. Thus, it is urgent to develop novel therapeutic strategies. Cancer cells can reprogram their lipid metabolism to sustain uncontrolled proliferation, and, therefore, accumulate a higher amount of lipid droplets (LDs). LDs are cytoplasmic organelles that store neutral lipids and are hypothesized to sequester anti-cancer drugs, leading to reduced efficacy. Thus, the increased biogenesis of LDs in neoplastic conditions makes them suitable targets for anticancer therapy and for the development of new dyes for cancer cells imaging. In recent years, cancer nanotherapeutics offered some exciting possibilities, including improvement tumour detection and eradication. In this review we summarize LDs biogenesis, structure and composition, and highlight their role in cancer theranostics.

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

Half-Sandwich Cyclopentadienylruthenium(II) Complexes: A New Antimalarial Chemotype.

A small library of "half-sandwich" cyclopentadienylruthenium(II) compounds of the general formula [(η5-C5R5)Ru(PPh3)(N-N)][PF6], a scaffold hitherto absent from the toolbox of antiplasmodials, was screened for activity against the blood stage of CQ-sensitive 3D7-GFP, CQ-resistant Dd2, and artemisinin-resistant IPC5202 Plasmodium falciparum strains and the liver stage of Plasmodium berghei. The best-performing compounds displayed dual-stage activity, with single-digit nanomolar IC50 values against blood-stage malaria parasites, nanomolar activity against liver-stage parasites, and residual cytotoxicity against HepG2 and Huh7 mammalian cells. The parasitic absorption/distribution of 7-nitrobenzoxadiazole-appended fluorescent compounds Ru4 and Ru5 was investigated by confocal fluorescence microscopy, revealing parasite-selective absorption in infected erythrocytes and nuclear accumulation of both compounds. The lead compound Ru2 impaired asexual parasite differentiation, exhibiting fast parasiticidal activity against both ring and trophozoite stages of a synchronized culture of the P. falciparum 3D7 strain. These results point to cyclopentadienylruthenium(II) complexes as a highly promising chemotype for the development of dual-stage antiplasmodials.

A Transcriptomics Approach To Unveiling the Mechanisms of In Vitro Evolution towards Fluconazole Resistance of a Candida glabrata Clinical Isolate.

Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms.

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.

Biophysical study of human induced Pluripotent Stem Cell-Derived cardiomyocyte structural maturation during long-term culture.

Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) have an enormous potential for the development of drug screening and modeling cardiac disease platforms. However, early hiPSC-CMs usually exhibit low structural development, precluding the applicability of these cells. Here, we follow during 120 days the progressive structural maturation of hiPSC-CM microtissues obtained using the Wnt signaling modulation protocol. For this purpose, we designed a user friendly custom-written program to quantify cardiac fiber alignment and sarcomere length. Cardiomyocyte shape, cardiac fiber density and multinucleation were also analyzed. Derived cardiomyocytes showed significant progression in cardiomyocyte fiber density and sarcomere length during the long-term culture, with a peak at day 90 of 40% multinucleated cells. In addition, cardiomyocyte microtissues remained functional with progressive maturation leading to a decrease in the percentage of cTnT positive cells from 59% to 22% at day 120, a value similar to the content present in tissues of the adult left ventricle. These data and the framework that we provide to quantify cardiomyocyte structural features can be important to set new metrics to develop applications for drug screening and disease modeling.

Stress Response Protein BolA Influences Fitness and Promotes Salmonella enterica Serovar Typhimurium Virulence.

The intracellular pathogen Salmonella enterica serovar Typhimurium has emerged as a major cause of foodborne illness, representing a severe clinical and economic concern worldwide. The capacity of this pathogen to efficiently infect and survive inside the host depends on its ability to synchronize a complex network of virulence mechanisms. Therefore, the identification of new virulence determinants has become of paramount importance in the search of new targets for drug development. BolA-like proteins are widely conserved in all kingdoms of life. In Escherichia coli, this transcription factor has a critical regulatory role in several mechanisms that are tightly related to bacterial virulence. Therefore, in the present work we used the well-established infection model Galleria mellonella to evaluate the role of BolA protein in S Typhimurium virulence. We have shown that BolA is an important player in S Typhimurium pathogenesis. Specifically, the absence of BolA leads to a defective virulence capacity that is most likely related to the remarkable effect of this protein on S Typhimurium evasion of the cellular response. Furthermore, it was demonstrated that BolA has a critical role in bacterial survival under harsh conditions since BolA conferred protection against acidic and oxidative stress. Hence, we provide evidence that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence.IMPORTANCE BolA has been described as an important protein for survival in the late stages of bacterial growth and under harsh environmental conditions. High levels of BolA in stationary phase and under stresses have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator. Here, we show that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. This work constitutes a relevant step toward an understanding of the role of BolA protein and may have an important impact on future studies in other organisms. Therefore, this study is of utmost importance for understanding the genetic and molecular bases involved in the regulation of Salmonella virulence and may contribute to future industrial and public health care applications.

Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties.

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

Characterization of BCAM0224, a multifunctional trimeric autotransporter from the human pathogen Burkholderia cenocepacia.

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 µg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 µg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Necroptosis induced by ruthenium (II) complexes as mitochondrial disruptors.

Inducing necroptosis in cancer cells has emerged as an effective strategy to overcome drug resistance. However, while organic small molecules have been extensively studied for this purpose, metal-based compounds have received relatively little attention as triggers of necroptosis. The development of ruthenium (II) hybrid compounds, particularly those containing triazene (Ru-TRZ), highlights a novel avenue for modulating necroptotic cell death. Here we show that incorporating a methyltriazene moiety, a known alkylating warhead, confers superior mitochondrial-targeting properties and enhances cell death compared to amide-containing counterparts. Ru-hybrid TRZ2 exhibits also antitumor efficacy against in vivo drug-resistant cancer cells. Mechanistically, we demonstrate that Ru-TRZ hybrids induce apoptosis. In addition, by activating downstream RIPK3-driven cell death, TRZ2 proficiently restrains normal mitochondrial function and activity, leading to cancer cell necroptosis. Finally, TRZ2 synergizes anti-proliferative activity and cell death effects induced by conventional drugs. In conclusion, Ru-TRZ2 stands as a promising ruthenium-based chemotherapeutic agent inducing necroptosis in drug resistant cancer cells.

Extracellular Vesicles and Infection: From Hijacked Machinery to Therapeutic Tools.

Extracellular vesicles (EVs) comprise a broad range of secreted cell-derived membrane vesicles. Beyond their more well-characterized role in cell communication, in recent years, EVs have also been shown to play important roles during infection. Viruses can hijack the biogenesis of exosomes (which are small EVs) to promote viral spreading. Additionally, these exosomes are also important mediators in inflammation and immune responses during both bacterial and viral infections. This review summarizes these mechanisms while also describing the impact of bacterial EVs in regulating immune responses. Finally, the review also focuses on the potential and challenges of using EVs, in particular, to tackle infectious diseases.

The role of RNA regulators, quorum sensing and c-di-GMP in bacterial biofilm formation.

Biofilms provide an ecological advantage against many environmental stressors, such as pH and temperature, making it the most common life-cycle stage for many bacteria. These protective characteristics make eradication of bacterial biofilms challenging. This is especially true in the health sector where biofilm formation on hospital or patient equipment, such as respirators, or catheters, can quickly become a source of anti-microbial resistant strains. Biofilms are complex structures encased in a self-produced polymeric matrix containing numerous components such as polysaccharides, proteins, signalling molecules, extracellular DNA and extracellular RNA. Biofilm formation is tightly controlled by several regulators, including quorum sensing (QS), cyclic diguanylate (c-di-GMP) and small non-coding RNAs (sRNAs). These three regulators in particular are fundamental in all stages of biofilm formation; in addition, their pathways overlap, and the significance of their role is strain-dependent. Currently, ribonucleases are also of interest for their potential role as biofilm regulators, and their relationships with QS, c-di-GMP and sRNAs have been investigated. This review article will focus on these four biofilm regulators (ribonucleases, QS, c-di-GMP and sRNAs) and the relationships between them.

Acetate modulates the inhibitory effect of Lactobacillus gasseri against the pathogenic yeasts Candida albicans and Candida glabrata.

The exploration of the interference prompted by commensal bacteria over fungal pathogens is an interesting alternative to develop new therapies. In this work we scrutinized how the presence of the poorly studied vaginal species Lactobacillus gasseri affects relevant pathophysiological traits of Candida albicans and Candida glabrata. L. gasseri was found to form mixed biofilms with C. albicans and C. glabrata resulting in pronounced death of the yeast cells, while bacterial viability was not affected. Reduced viability of the two yeasts was also observed upon co-cultivation with L. gasseri under planktonic conditions. Either in planktonic cultures or in biofilms, the anti-Candida effect of L. gasseri was augmented by acetate in a concentration-dependent manner. During planktonic co-cultivation the two Candida species counteracted the acidification prompted by L. gasseri thus impacting the balance between dissociated and undissociated organic acids. This feature couldn't be phenocopied in single-cultures of L. gasseri resulting in a broth enriched in acetic acid, while in the co-culture the non-toxic acetate prevailed. Altogether the results herein described advance the design of new anti-Candida therapies based on probiotics, in particular, those based on vaginal lactobacilli species, helping to reduce the significant burden that infections caused by Candida have today in human health.