Identification of genetic transcriptional factors involved in non-Candida albicans Candida species biofilm development

Candida albicans is the major cause of candidosis, however recently non- C. albicans Candida (NCAC) species have emerged as common pathogens. One of the most important virulence factors is the ability to form biofilms that have important clinical repercussions due to their increased resistant to antifungal therapy. In the case of C. albicans, the transcriptional network of biofilm formation is composed by six transcriptions factors (BCR1, EFG1, TEC1, NDT80, ROB1 and BRG1). However, in the case of NCAC species little is known about the influence of these genes in biofilm formation. Thus, in order to identify targets to be used as biofilm controllers in NCAC species it was characterized the role of BCR1, EFG1 and TEC1 genes on C. parapsilosis and C. glabrata species biofilm formation. After planktonic cells and biofilms grown, the RNAs were extracted and the expression levels of BCR1, EFG1 and TEC1 compared by quantitative real time PCR. CFUs enumeration and crystal violet staining were used to quantify biofilm formation. The results demonstrated that in both Candida species all genes are expressed but in a species and lifestyle dependent manner. Specifically, in opposite to observed in C. glabrata, BCR1 and TEC1 expression levels, are higher in biofilm than in planktonic cells of C. parapsilosis. Interestingly the EFG1 levels of expression was superior to 100% in both conditions for C. parapsilosis, however higher in planktonic cells. Thus, it is possible to assume that BCR1, TEC1 and EFG1 are biofilm regulators in C. parapsilosis, as in C. albicans, but not in C. glabrata and they could be suggested to be used as future targets to control C. parapsilosis biofilm formation

Controlling ETEC colonization on cultures of an intestinal pig cell line with a T4-like phage

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high producing costs. The rise of antibiotic selective pressure together with on-going limitation on their use demands news strategies to tackle this pathology. The pertinence of using phages to tackle this problematic is being explored, and in this work, the efficacy of a T4-like phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC O9:H9 (Sta, F5/F41) was assessed. FJ1 has a 170,053 bp genome, and of the 270 coding sequences none corresponds to identified undesirable proteins, such as integrases or transposases. Envisaging the oral application to piglets, FJ1 was previously encapsulated on CaCO3/alginate. Assays were performed on 15-day cultures of the intestinal pig cell line IPEC-1 seeded in transwell inserts. Phage treatment occurred 2 hours after ETEC infection, when, in average, 5x105 CFU.cm-2 were adhered to cultured cells. Encapsulated phage provided reductions of, approximately, 2.3 Log CFU.cm-2 and 2.8 Log CFU.cm-2 on adhered bacteria, respectively 3 and 6 hours after administration. The repeated exposure of the host to FJ1 led to the emergence of phage-insensitive mutants, phenotype that brought fitness costs to the host strain: they were 70% more vulnerable to the pig complement system and less efficient in adhering to cultured cells (in about 90%). Overall, FJ1 is presented here as promising to fight against ETEC infections through oral administration to piglets.info:eu-repo/semantics/publishedVersio

Assessing the virulence potential of different Listeria monocytogenes clonal complexes with Galleria mellonella larvae model

Listeria monocytogenes is a highly diverse species, exhibiting differential virulence potential within strains from different clonal complexes (CCs). Hypervirulent CCs strains tend to be associated with higher frequency in clinical cases and severe outcomes, while hypovirulent CCs are characterized by a reduced level of virulence and are often associated with food-related contamination. Recently, researchers have employed Galleria mellonella larvae as an in vivo model to characterize these variable virulence patterns among Listeria strains. Although it has only been utilized once to study CC-related virulence thus, there is still uncertainty about its relevance as an in vivo model. Infection studies with G. mellonella larvae were performed to evaluate the virulence potential of 16 Lm strains from CC1, CC2, CC4, CC6, CC388, CC87, CC9 and CC121. Hence, the survival rate and health index scores of larvae were used to quantify the virulence capacity of this pathogen. Results obtained indicate that: the CC2 strain exhibited a hypovirulent phenotype in the larvae with the highest survival rate and health index scores, followed by two strains from CC1 and CC6. In contrast, another CC6 strain exhibited reduced larvae survival rates, followed by the CC4 strain. Furthermore, strains from CC9, which is considered hypovirulent, caused around 47% mortality (Figure 1). Our findings revealed clear variations in virulence patterns that were previously determined with other in vitro and in vivo models. Moreover, it was observed a strain-dependent intra-clonal complex virulence difference in the infection of G. mellonella larvae. Additionally, by eliminating the dependence of L. monocytogenes strains on the inlA gene for host cell invasion, it was observed that hypovirulent clones demonstrated an infection potential equal to or greater than that of hypervirulent strains. Hence, there are still virulence markers that need to be characterized to improve the genotypic distinction of these CCs.This work was supported by National Funds from FCT - Fundação para a Ciência e a Tecnologia through project GenoPhenoTraits4Persitence - Genomic and phenotypic traits contributing to persistence of Listeria monocytogenes in food processing environment (PTDC/BAAAGR/4194/2021).info:eu-repo/semantics/publishedVersio

Aptamers for blocking enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the major cause of enteric infections in swine, resulting in significant costs for the swine industry. Among other virulence factors, fimbriae are essential for the initial adhesion of ETEC to the intestinal epithelial cells. In particular, the F4-type (K88) fimbriae are commonly associated with neonatal infections and most post-weaning diarrhoeal infections. These diseases are traditionally prevented or treated with antibiotics, but the use of antibiotics is being highly restricted due to the growing phenomenon of antimicrobial resistance. Therefore, novel strategies such as aptamers, which are small single-stranded oligonucleotides capable of binding to target molecules, seem to be a promising alternative to block the initial adhesion of F4-ETEC. The present study focuses on two parallel studies, the first in which two pre-selected aptamers (31/37) were tested in an in vivo model, Galleria mellonella, to evaluate their toxicity at three inoculated concentrations (1M, 10M, 20M) and the performance as treatment (Capt=500 nM) with infection (108 CFU/mL) of five strains (F4-ETEC, F18-ETEC, Escherichia coli K12, Klebsiella pneumonia ATCC 43216, Staphylococcus aureus ATCC 25929). Secondly, new specific DNA aptamers were selected through an innovative cell-SELEX approach against F4-ETEC bacteria, which involved four main steps: library incubation, partitioning, elution, and amplification. Both preselected aptamers showed no toxicity in Galleria mellonella after 96 h, regardless of the inoculated concentration. Furthermore, inoculation of aptamer 31 + F4-ETEC in Galleria mellonella increased the larval survival rate and health index when compared to inoculation with F4-ETEC alone. Regarding the new selection of DNA aptamers, 12 rounds of SELEX were successfully carried out and a final pool of potential aptamers against F4-ETEC was obtained, which will now be evaluated for their specificity and affinity. This work demonstrates the potential of aptamers in the treatment of ETEC infections as an alternative to antibiotics.This work was financially supported by: Project PTDC/CVT-CVT/4620/2021, funded by FEDER funds through COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC), and by FCT under the scope of the strategic funding of UIDB/04469/2020 unit (CEB). The authors are also grateful to FCT for the PhD Grant SFRH/BD/2023.04664.BDANA and for the CEEC Individual (2022.06886.CEECIND).info:eu-repo/semantics/publishedVersio

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Aplicação de oligómeros de antisense para prevenir infeções por Candida albicans

Tese de doutoramento em Chemical and Biological EngineeringCandida albicans is the main cause of candidiasis and its pathogenicity is supported by a series of virulence factors, including the switch from yeast to filamentous forms. The transcription factor EFG1 plays a central role in C. albicans regulation morphology. In addition, there is rise of C. albicans multidrug resistance accompanied by the scarcity of new classes of fungal drugs. Antisense oligonucleotides (ASOs) can hold great promise as a therapeutic agent, however yet they are poorly explored for controlling Candida infections. Therefore, the main goal of this work was to develop a therapeutic approach based on antisense therapy (AST) to track C. albicans filamentation. To this end, two major objectives were addressed: I) Exploitation and application of ASOs to control C. albicans filamentation; II) Creation of strategies for ASOs cargo and delivery. The first goal of this research was the exploitation of the second and third generations of ASOs to control EFG1 gene and C. albicans filamentation. To this end, the anti-EFG1 2’OMethylRNA was firstly projected based on second generation of ASOs and was proved to have an impact on the reduction of EFG1 gene expression (by 60 %) and on Efg1p protein translation (by 60 %). Interestingly, the ASO was able to inhibit filamentation and to attenuate the virulence of C. albicans on in vivo Galleria mellonella (increasing its survival on 30 % at 72h with a double-dose administration). Based on the third generation of ASOs, a set of anti-EFG1 LNA (Locked nucleic acids) ASOs were developed, and their performance was tested in vitro and in vivo. All LNA-ASOs were able to reduce EFG1 gene expression (by 40-80 %) and consequently filamentation (by 45-55 %) in vitro. The in vivo results revealed that the LNA-ASO modified with PS-linkages and palmitoyl-2’-amino-LNA as the most favorable for an in vivo application (increasing G. mellonella survival by 40 % only with a single-dose administration). The second goal of this research was the development of strategies for anti-EFG1 ASO cargo and delivery. Two distinct approaches were tested: (i) development of polyplexes based on polyamide 4 and 6, that revealed to be feasible carriers, once the ASO maintains its activity against C. albicans cells; (ii) application of lipid-based formulations, where the DOTAP/DOPC 80/20 ρ=3 revealed to be the most promising when tested in a G. mellonella model (25 % of increase on larvae survival in comparison to ASO-free at 72 h). In summary, this research underlines the therapeutic potential of ASOs for controlling C. albicans virulence determinants, as is the case of EFG1 gene, as well as the importance of developing carriers’ systems for ASOs cargo. Considering the future research into the management of C. albicans infections, this research provides valuable information to the development of a credible and alternative method to control C. albicans infections, based on AST.Candida albicans é uma das principais causas de candidíase e a sua patogenicidade é suportada por um conjunto de fatores de virulência, incluindo a capacidade de filamentar. EFG1 é um fator de transcrição com papel fundamental na filamentação. O aumento da multirresistência de C. albicans é acompanhado pela escassez de novos antifúngicos. Os oligonucleotídeos de antisense (OAS) têm revelado potencial como agentes terapêuticos, contudo ainda pouco explorados no controlo da candidíase. Assim, o principal objetivo deste trabalho foi desenvolver uma abordagem terapêutica baseada na Terapia Antisense (TA) para controlar a filamentação de C. albicans. Para tal, dois grandes objectivos complementares foram definidos: I) Exploração e aplicação de OAS para controlar a filamentação e II) Criação de estratégias para o transporte e entrega dos OASs. Como primeiro objetivo explorou-se a segunda e terceira gerações de OASs para controlar o gene EFG1. O anti-EFG1 2’OMethylRNA foi projetado com base na segunda geração, e provou ter um impacto na redução da expressão do gene EFG1 (em 60 %) e na tradução da proteína Efg1p (em 60 %). O OAS foi capaz de inibir a filamentação e atenuar in vivo a virulência de C. albicans em Galleria mellonella (30 % de aumento de sobrevivência às 72 h após dupla administração). Por outro lado, um conjunto de OAS modificados com LNA (terceira geração) foram desenvolvidos e testados in vitro e in vivo. In vitro, os OAS-LNA inibiram a expressão do gene de EFG1 (em 40-80 %) e a filamentação (em 45-55 %). In vivo, o OAS-LNA com as modificações de PS e palmitoyl-2’amino-LNA revelou ser o mais favorável (40 % de aumento de sobrevivência da G. mellonella com uma administração). Como segundo objetivo exploraram-se poliplexos e lipoplexos como estratégias para o transporte e entrega do anti-EFG1 2’OMe. Como primeira abordagem, desenvolveram-se poliplexos à base de poliamida 4 e 6, que revelaram ser veículos viáveis, uma vez que o OAS manteve a sua atividade contra as células de C. albicans. Em seguida, foram exploradas formulações lipídicas, sendo que o DOTAP/DOPC 80/20 ρ=3 revelou ser a mais promissora quando testada in vivo (25 % de aumento de sobrevivência da G. mellonella em comparação com o OAS livre às 72 h). Em suma, esta investigação destaca o potencial terapêutico de OAS para o controlo de determinantes de virulência de C. albicans, como o caso do gene EFG1, bem como a importância do desenvolvimento de sistemas de veículos para o seu transporte. Considerando futuros trabalhos sobre o controlo de infeções por C. albicans, esta investigação fornece informação valiosa para o desenvolvimento de um método credível e alternativo para o controlo destas infeções, com base na TA.This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/121417/2016 and the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was also supported by the project funding by the “02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893”, designated as Antisen4CandiB - Application of antisense oligomers for controlling Candida species biofilm formation on medical surfaces”

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.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and Daniela Eira Araújo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge the project funding by the “02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893”. This research is also supported by Microfluidic Layer-by-layer Assembly of Cationic Liposome-Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020.info:eu-repo/semantics/publishedVersio

Combined application of antisense oligomers to control transcription factors of Candida albicans biofilm formation

Antisense oligomers (ASOs) have been little exploited to control determinants of Candida albicans virulence. Biofilm formation is an important virulence factor of C. albicans, that is regulated by a complex network of transcription factors (such as EFG1, BRG1 and ROB1). Thus, the main goal of this work was to project ASOs, based on the 2'-OMethyl chemical modification, to target BRG1 and ROB1 mRNA and to validate its application either alone or in combination with the EFG1 mRNA target, to reduce C. albicans biofilm formation. The ability of ASOs to control gene expression was evaluate by qRT-PCR. The effect on biofilm formation was determined by the total biomass quantification, and simultaneously the carbohydrates and proteins reduction on extracellular matrix. It was verified that all the oligomers were able to reduce the levels of gene expression and the ability of C. albicans to form biofilms. Furthermore, the combined application of the cocktail of ASOs enhances the inhibition of C. albicans biofilm formation, minimizing biofilm thickness by reducing the quantity of matrix content (protein and carbohydrate). So, our work confirms that ASOs are useful tools for research and therapeutic development on the control of Candida species biofilm formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020. The authors also acknowledge the project funding by the ‘‘02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnolo´gico (IC&DT) – POCI-01–0145-FEDER-028893’’ and ‘‘04/SAICT/2017—Proteção de Direitos de Propriedade Intelectual- Projectos individuais—POCI-01–0145-FEDER-042779’’.info:eu-repo/semantics/publishedVersio