Design of a lipid nano-delivery system containing recombinant Candida albicans chitinase 3 as a potential vaccine against fungal infections.

Opportunistic fungi cause lethal systemic infections and impose high medical costs to health systems. The World Health Organization has recognized the importance of fungal infections, including them in its global priority list guiding research, development, and discovery of new therapeutic approaches. Fungal vaccine development has been proposed as one of the treatment and prevention strategies in the last decade. In this study, we present the design of a lipid antigen delivery system based on Dioctadecyldimethylammonium bromide: Monoolein (DODAB: MO) containing recombinant Candida albicans Chitinase 3 (Cht3) for modulation the immune response against fungal infections. Several DODAB:MO liposomes containing Cht3 were prepared and those prepared by the incubation method and containing 5 µg/mL Cht3 were selected due to their favorable size, ζ-potential and stability, suited for antigen delivery applications. The encapsulation of Cht3 in these liposomes resulted in a significant increase in cellular uptake compared to empty liposomes, demonstrating their efficacy in delivering the antigen. Moreover, the liposomes proved to be safe for use in immunization procedures. Subcutaneous administration of Cht3 liposomes elicited a Th1/Th17 immune response profile, associated with the production of high levels of antibodies against Cht3. These antibodies recognized both the native and the recombinant forms of the protein, opsonizing mother-yeast at the cell scars, which has the potential to disrupt cell separation and hinder yeast growth. The findings suggest that the designed lipid antigen delivery system shows promise as a potential candidate for enhancing immune responses against fungal infections, offering a valuable strategy for future fungal vaccine development.

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis.

UNLABELLED: We evaluated the potential of a liposomal antigen delivery system (ADS) containing Candida albicans cell wall surface proteins (CWSP) in mediating protection against systemic candidiasis. Treatment of bone-marrow-derived dendritic cells with CWSP-loaded dioctadecyldimethylammonium bromide:monoolein (DODAB:MO) liposomes enhanced and prolonged their activation comparatively to free antigen, indicating that liposome-entrapped CWSP were released more sustainable. Therefore, we immunized mice with CWSP either in a free form or loaded into two different DODAB:MO liposome formulations, respectively designated as ADS1 and ADS2, prior to intravenous C. albicans infection. Immunization with ADS1, but not with ADS2, conferred significant protection to infected mice, comparatively to immunization with CWSP or empty liposomes as control. ADS1-immunized mice presented significantly higher serum levels of C. albicans-specific antibodies that enhanced phagocytosis of this fungus. In these mice, a mixed cytokine production profile was observed encompassing IFN-γ, IL-4, IL-17A and IL-10. Nevertheless, only production of IL-4, IL-17 and IL-10 was higher than in controls. In this study we demonstrated that DODAB:MO liposomes enhance the immunogenicity of C. albicans antigens and host protection in a murine model of systemic candidiasis. Therefore, this liposomal adjuvant could be a promising candidate to assess in vaccination against this pathogenic fungus. STATEMENT OF SIGNIFICANCE: This work describes the immunomodulation capacity of the previously validated antigen delivery system (ADS) composed by dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) lipids incorporating the cell wall surface proteins (CWSP) from C. albicans. Here, we not only present the ability of this system in facilitating antigen uptake by DCs in vitro, but also that this system induces higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously. We show that the ADS are efficient nanocarrier and modulate the immune response against intravenous C. albicans infection favoring mouse protection. In sum, we show that the incorporation of C. albicans antigens in DODAB:MO nanocarries are a promising vaccine strategy against C. albicans fungal infection.

Candida glabrata susceptibility to antifungals and phagocytosis is modulated by acetate.

Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.

Genetic variability of Candida albicans Sap8 propeptide in isolates from different types of infection.

The secreted aspartic proteases (Saps) are among the most studied virulence determinants in Candida albicans. These proteins are translated as pre-pro-enzymes consisting of a signal sequence followed by a propeptide and the mature enzyme. The propeptides of secreted proteinases are important for the correct processing, folding/secretion of the mature enzyme. In this study, the DNA sequences of C. albicans Saps were screened and a microsatellite was identified in SAP8 propeptide region. The genetic variability of the repetitive region of Sap8 propeptide was determined in 108 C. albicans independent strains isolated from different types of infection: oral infection (OI), oral commensal (OC), vulvovaginal candidiasis (VVC), and bloodstream infections (BSI). Nine different propeptides for Sap8 processing were identified whose frequencies varied with the type of infection. OC strains presented the highest gene diversity while OI isolated the lowest. The contribution of the Saps to mucosal and systemic infections has been demonstrated and recently Sap8 has been implicated in the cleavage of a signalling glycoprotein that leads to Cek1-MAPK pathway activation. This work is the first to identify a variable microsatellite in the propeptide of a secreted aspartic protease and brings new insights into the variability of Sap8.

DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: a novel adjuvant and delivery system.

We describe the preparation and characterization of DODAB:MO-based liposomes and demonstrate their adjuvant potential and use in antigen delivery. Liposomes loaded with Candida albicans proteins assembled as stable negatively charged spherical nanoparticles with a mean size of 280 nm. High adsorption efficiency (91.0 ± 9.0%) is attained with high lipid concentrations. The nanoparticles were non-toxic, avidly taken up by macrophage cells and accumulated in membrane rich regions with an internalization time of 20 min. Immunized mice displayed strong humoral and cell-mediated immune responses, producing antibodies (IgGs) against specific cell wall proteins, Cht3p and Xog1p. DODAB:MO-based liposomes loaded with C. albicans proteins have an excellent immunogenic potential and can be explored for the development of an immunoprotective strategy against Candida infections.

A new method for yeast phagocytosis analysis by flow cytometry.

Herein we developed a method based on the quenching effect of propidium iodide over Sytox-Green fluorescence to assess yeast phagocytosis by flow cytometry. It allows accurate quantification of living from dead phagocytes; internalized from non-internalized cells, maintaining yeast fluorescence within phagocytes; and the different associations between phagocytes and fungal cells.

Participation of Candida albicans transcription factor RLM1 in cell wall biogenesis and virulence.

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.

Isolates from hospital environments are the most virulent of the Candida parapsilosis complex.

BACKGROUND: Candida parapsilosis is frequently isolated from hospital environments, like air and surfaces, and causes serious nosocomial infections. Molecular studies provided evidence of great genetic diversity within the C. parapsilosis species complex but, despite their growing importance as pathogens, little is known about their potential to cause disease, particularly their interactions with phagocytes. In this study, clinical and environmental C. parapsilosis isolates, and strains of the related species C. orthopsilosis and C. metapsilosis were assayed for their ability to induce macrophage cytotocixity and secretion of the pro-inflammatory cytokine TNF-α, to produce pseudo-hyphae and to secrete hydrolytic enzymes. RESULTS: Environmental C. parapsilosis isolates caused a statistically significant (p = 0.0002) higher cell damage compared with the clinical strains, while C. orthopsilosis and C. metapsilosis were less cytotoxic. On the other hand, clinical isolates induced a higher TNF-α production compared with environmental strains (p < 0.0001). Whereas the amount of TNF-α produced in response to C. orthopsilosis strains was similar to the obtained with C. parapsilosis environmental isolates, it was lower for C. metapsilosis strains. No correlation between pseudo-hyphae formation or proteolytic enzymes secretion and macrophage death was detected (p > 0.05). However, a positive correlation between pseudo-hyphae formation and TNF-α secretion was observed (p = 0.0119). CONCLUSIONS: We show that environmental C. parapsilosis strains are more resistant to phagocytic host defences than bloodstream isolates, being potentially more deleterious in the course of infection than strains from a clinical source. Thus, active environmental surveillance and application of strict cleaning procedures should be implemented in order to prevent cross-infection and hospital outbreaks.

Genetic detection and multilocus sequence typing of vanA-containing Enterococcus strains from mullets fish (Liza ramada).

Enterococci have emerged as important nosocomial and community-acquired pathogens in humans. The presence of vanA-enterococci was investigated in 103 fecal samples recovered from mullets fish (Liza ramada). All fecal samples were inoculated in Slanetz-Bartley agar plates supplemented with 4 mg/L of vancomycin for vancomycin-resistant enterococci (VRE) recovery and two isolates/sample were characterized. Antibiotic susceptibility was tested for 11 antibiotics by disk diffusion and agar dilution methods. VRE identification was performed by biochemical and molecular methods. Additionally, the mechanisms of resistance to glycopeptides (vanA, vanB, vanC1, vanC2, and vanD) and other antibiotics [erm(A), erm(B), tet(L), tet(M), aph(2'')-aac(6'), aph(3')-IIIa, ant(6'), vat(D), vat(E)] as well as the presence of enterococcal surface protein (esp) and hyl virulence factors were investigated. vanA-Enterococcus faecium isolates were recovered from 4 of 103 tested samples, and they showed glycopeptide and erythromycin resistances. Three of them were also ampicillin resistant, two showed resistance to tetracycline, ciprofloxacin, and kanamycin, and one showed resistance to gentamicin. The tet(M) and erm(B) genes were found in all tetracycline- and erythromycin-resistant strains, respectively. The aph(3')-III and aph(2'')-aac(6') genes were identified in the kanamycin- and gentamicin-resistant isolates, respectively. The IS1216 element was identified within vanX-vanY region of Tn1546 in two vanA isolates. The hyl and esp virulence genes were found in four and two isolates, respectively. vanA-strains were ascribed to sequence types ST280 (two isolates) and ST273 (two isolates), including both lineages into the clonal complex CC17. Mullets fish can excrete VRE in their feces and may be a reservoir for such resistant bacteria that can be transmitted to other animals including humans.

Vancomycin-resistant enterococci from Portuguese wastewater treatment plants.

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry-slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA -containing Enterococcus faecium and nine vanC1/vanC2 -containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry-slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A-containing isolates, in all seven vanC-1 isolates and in one of two vanC-2 isolates. The ermB gene in seven erythromycin-resistant isolates; and the aac6 '-aph2 ″ gene in the two high-level-gentamicin-resistant isolates. Moreover, two vanA -containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK-1 allele was detected in our urban vanA -containing E. faecium isolate, and we found as well the purK-6 allele in one poultry-slaughterhouse vanA -containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic-resistant enterococci in Portugal.

Genetic characterization of extended-spectrum beta-lactamases in Escherichia coli isolates of pigs from a Portuguese intensive swine farm.

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coli isolates. We intended to determinate the carriage level and type of ESBLs in E. coli obtained from fecal samples from pigs raised on an intensive pig farm in Portugal; further to characterize other associated resistance genes and their plasmid content, the phylogenetic groups, and the clonal relationship of ESBL-positive isolates. Sixty-five fecal samples were seeded in Levine media supplemented with cefotaxime for E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion agar. ESBL-phenotypic detection was carried out by double-disk test; and the presence of the genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. Other mechanisms of antimicrobial resistance and phylogenetic groups were also determined. Clonal relationship was performed by pulsed-field gel electrophoresis. ESBL-producing E. coli isolates were detected in 16 fecal samples, and one isolate per sample was studied. The CTX-M-1 type ESBL was detected in the 16 isolates. The gene encoding TEM-1 was identified to be associated with eight CTX-M-1-positive isolates. The tet(A) gene was found in 12 of 14 tetracycline-resistant isolates, and the aadA or strA-strB genes were found in the streptomycin-resistant isolates. Fourteen and two ESBL-containing isolates belonged to A and B1 phylogenetic groups, respectively. Clonal relationship of ESBL-containing isolates identified seven unrelated patterns. Swine represent an important reservoir of ESBL-containing E. coli isolates, especially of the CTX-M-1 type.

Detection of CTX-M-14 and TEM-52 extended-spectrum beta-lactamases in fecal Escherichia coli isolates of captive ostrich in Portugal.

The main aim of this study was to determine the frequency of antibiotic resistance among Escherichia coli isolates recovered in Levine agar plates from 54 fecal samples of captive ostriches from a farm in the South of Portugal. Fifty-four nonselected E. coli isolates were obtained (one/sample) and the phenotypes and genotypes of antibiotic resistance were characterized. The following numbers of isolates showed antibiotic resistance: ampicillin (nine), tetracycline (seven), streptomycin (three), amoxicillin-clavulanic acid, cefoxitin, or gentamicin (one), and cefotaxime, ceftazidime, azthreonam, imipenem, nalidixic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole (zero). The bla(TEM) gene was identified in six out of nine ampicillin-resistant isolates, and the tet(A) or tet(B) genes in five out of seven tetracycline-resistant isolates. Mutations at positions -42, -18, -1, and +58 of ampC promoter region were identified in one cefoxitin-resistant isolate. Further, the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates was estimated in the 54 fecal samples of ostriches using cefotaxime-supplemented Levine agar plates for ESBL-positive E. coli recovery. Three samples contained ESBL-positive E. coli isolates of which one isolate/sample was characterized, leading to the detection of the following beta-lactamases: bla(CTX-M-14a) + bla(TEM-1b) (two isolates) and bla(TEM-52c) (one isolate). The three ESBL-positive isolates were classified into the phylogroup B1, and contained class 1 integrons with the gene cassettes dfrA17 + aadA5 (one isolate) and aadA1 (two isolates). This study adds to our knowledge about the wide dissemination of ESBL-producing E. coli isolates in different ecosystems, including captive ostriches, that could be transferred to humans through the food chain.

Effects of adrenomedullin on systolic and diastolic myocardial function.

Adrenomedullin (AM) effects were studied in rabbit papillary muscles by adding increasing concentrations (10(-10) to 10(-6)M) either alone or after pre-treatment with l-NNA, indomethacin, AM22-52 (AM receptor antagonist), CGRP(8-37) (CGRP receptors antagonist), KT5720 (PKA inhibitor), as well as after endocardial endothelium (EE) removal. Passive length-tension relations were constructed before and after a single concentration of AM (10(-6)M). AM concentration-dependently induced negative inotropic and lusitropic effects, and increased resting muscle length (RL). At 10(-6)M, AT, dT/dt(max) and dT/dt(min) decreased 20.9+/-4.9%, 18.3+/-7.3% and 16.7+/-7.8%, respectively, and RL increased to 1.010+/-0.004L/L(max). Correcting RL to its initial value resulted in a 26.6+/-6.4% decrease of resting tension, indicating decreased muscle stiffness, also patent in the down and rightward shift of the passive length-tension relation. The negative inotropic effect of AM was dependent on its receptor, CGRP receptor, PKA, the EE and NO, while the effects of AM on myocardial stiffness were abolished by EE damage and NO inhibition. This latter effect represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that AM is an important regulator of cardiac filling.

Echocardiographic evaluation including tissue Doppler imaging in New Zealand white rabbits sedated with ketamine and midazolam.

Limited data are available on the use of more recent echocardiographic parameters in the rabbit. Echocardiographic examination, including conventional echocardiography and tissue Doppler imaging (TDI), was performed on 26 male New Zealand white rabbits under ketamine-midazolam sedation. Particular emphasis was placed on the more recent systolic and diastolic parameters, such as myocardial performance index (Tei index) and mitral annular motion (from septal and lateral sides of the left ventricle) obtained using pulsed TDI. Parameters that assessed systolic and diastolic function (fractional shortening, Tei index, and maximal mitral E- and A-wave velocities) were comparable to those reported in the literature for rabbits in the awake state. The less cardiodepressive anaesthetic protocol could offer a good alternative in performing echocardiographic evaluation whenever such caution is necessary. TDI is feasible in healthy rabbits and potentially suitable for the investigation of left ventricle systolic and diastolic function.

A pharmacological differentiation between postjunctional (AT1A) and prejunctional (AT1B) angiotensin II receptors in the rabbit aorta.

The effects of angiotensin II and angiotensin III were compared at prejunctional and postjunctional AT(1) receptors of the rabbit thoracic aorta. Furthermore, the influence of PD123319, losartan and eprosartan on these effects was also compared. To study prejunctional effects, the tissues were preincubated with ((3)H)-noradrenaline, superfused and electrically stimulated (1 Hz, 2 ms, 50 mA, 5 min). To study postjunctional effects, non-cumulative concentration-response curves were determined. Both angiotensin II and angiotensin III were more potent prejunctionally than postjunctionally. In the case of angiotensin II, the EC(50) was 12 times lower at the prejunctional than at the postjunctional level, while that of angiotensin III was 30 times lower prejunctionally. Furthermore, whereas angiotensin II was about 33 times more potent than angiotensin III postjunctionally, it was only 12 times more potent than angiotensin III prejunctionally. Eprosartan did not differentiate between prejunctional and postjunctional effects of both angiotensins. In contrast, PD123319 and losartan did differentiate; however, whereas PD123319 concentration-dependently antagonised the facilitation of tritium release caused by angiotensin II and angiotensin III and had no influence on the contraction of the aortic rings elicited by the peptides, losartan did the opposite: it concentration-dependently antagonised the contractions caused by the peptides on the aortic rings and exerted no influence on the facilitatory effect of angiotensin II and angiotensin III. These results show that prejunctional and postjunctional receptors for angiotensin II and angiotensin III are different and underline the hypothesis that postjunctional AT(1) receptors belong to the AT(1A) subtype, while prejunctional AT(1) receptors belong to the AT(1B) subtype.