Unpuzzling friunavirus-host interactions one piece at a time: phage recognizes Acinetobacter pittii via a new K38 capsule depolymerase

Acinetobacter pittii is a species that belong to the Acinetobacter calcoaceticus-baumannii complex, increasingly recognized as major nosocomial bacterial pathogens, often associated with multiple drug-resistances. The capsule surrounding the bacteria represents a main virulence factor, helping cells avoid phage predation and host immunity. Accordingly, a better understanding of the phage infection mechanisms is required to efficiently develop phage therapy against Acinetobacter of different capsular types. Here, we report the isolation of the novel A. pittii-infecting Fri1-like phage vB_Api_3043-K38 (3043-K38) of the Podoviridae morphotype, from sewage samples. Its 41,580 bp linear double-stranded DNA genome harbours 53 open reading frames and 302 bp of terminal repeats. We show that all studied Acinetobacter Fri1-like viruses have highly similar genomes, which differentiate only at the genes coding for tailspike, likely to adapt to different host receptors. The isolated phage 3043-K38 specifically recognizes an untapped Acinetobacter K38 capsule type via a novel tailspike with K38 depolymerase activity. The recombinant K38 depolymerase region of the tailspike (center-end region) forms a thermostable trimer, and quickly degrades capsules. When the K38 depolymerase is applied to the cells, it makes them resistant to phage predation. Interestingly, while K38 depolymerase treatments do not synergize with antibiotics, it makes bacterial cells highly susceptible to the host serum complement. In summary, we characterized a novel phage-encoded K38 depolymerase, which not only advances our understanding of phage-host interactions, but could also be further explored as a new antibacterial agent against drug-resistant Acinetobacter.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. This project has been also funded by a Research Grant 2020 of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) to H.O.info:eu-repo/semantics/publishedVersio

Anti-RAS1 and anti-RIM101 oligomers for controlling Candida albicans filamentation under different body fluids

Dissertação de mestrado em Genética MolecularCandida albicans continua a ser a espécie do género Candida responsável por maior número de infeções fúngicas em humanos. A patogenicidade de C. albicans está relacionada com a capacidade de transitar da forma de levedura para a forma filamentosa (dimorfismo). Esta transição de forma tem sido relacionada com alguns genes, entre eles RAS1 e RIM101. Assim, o principal objetivo deste trabalho, com base na terapia antisense (TA), foi desenvolver oligómeros antisense (OAS) capazes de reduzir a expressão genética dos genes RAS1 e RIM101. Pretende-se assim contribuir para o desenvolvimento de um cocktail de OAS capazes de controlar a filamentação de C. albicans em diferentes fluidos corporais humanos. Assim, como primeira tarefa avaliou-se a capacidade de filamentação de C. albicans em fluidos corporais humanos simulados, preparados a diferentes pH. Verificou-se que C. albicans é capaz de filamentar em saliva e urina artificial com diferentes valores de pH (4, 5.8 e 7), evidenciando a elevada capacidade de adaptabilidade e plasticidade de C. albicans em diferentes contextos ambientais. A segunda tarefa consistiu no desenho de dois OAS, o anti-RAS1 e o anti-RIM101, e na validação da sua funcionalidade in vitro. A ausência de citotoxidade de anti-RAS1 2'-OMethylRNA (2'OMe) e anti-RIM101 2'OMe, conjuntamente com a capacidade de reduzir a níveis significativos tanto a expressão dos genes RAS1 e RIM101, como a filamentação de C. albicans em diferentes fluidos corporais simulados, valida a aplicabilidade da TA no controlo da virulência de C. albicans. Assim, este trabalho reforça a possibilidade do desenvolvimento de novos antifúngicos a partir de um cocktail de OAS para o tratamento de candidíases.Candida albicans remains the species of the genus Candida responsible for the largest number of fungal infections in humans. The pathogenicity of C. albicans is related with the ability to switch from yeast to filamentous forms(dimorphism). RAS1 and RIM101 genes are identified as important regulators of this phenomenon. Thus, based on antisense therapy (AST), the main goal of this work was to develop antisense oligomers (ASOs) capable of reducing the genetic expression of the RAS1 and RIM101 genes. It is intended to contribute to the development of an ASOs cocktail capable of controlling the filamentation of C. albicans in different human body fluids. So, as a first task, the filamentation capacity of C. albicans on human body fluids simulated at different pH was evaluated. It was verified that C. albicans is able to develop filaments on saliva and artificial urine under different pH values (4, 5.8 and 7), demonstrating the high plasticity and adaptability of C. albicans on different environmental contexts. The second task was to design two ASOs, anti-RAS1 and anti-RIM101, and to validate its functionality in vitro. The absence of anti-RAS1 2'-OMethylRNA (2'OMe) e anti-RIM101 2'OMe cytotoxicity, jointly with its ability to reduce the expression of RAS1 and RIM101 genes and C. albicans filamentation on different simulated body fluids to significant levels, validates the applicability of AT to control of C. albicans virulence. Thus, this work reinforces the possibility of developing new antifungals from an ASO cocktail to be used in the treatment of candidiasis.Este estudo foi suportado pelo projeto Antisen4CandiB - Application of antisense oligomers for controlling Candida species biofilm formation on medical surfaces, com referência POCI-01-0145- FEDER-028893, financiado pelo programa 02/SAICT/2017 - Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) através do COMPETE 2020, Portugal 2020 e a Fundação para a Ciência e a Tecnologia (FCT)

Anti-EFG1 oligomer able to control Candida albicans filamentation in human body fluids

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Sfl1 is required for Candida albicans biofilm formation under acidic conditions

Candida albicans is a common Candida species, responsible for infections in various anatomical sites under different environmental conditions, aggravated in the presence of its biofilms. As such, this study aimed to reveal the regulation of C. albicans biofilms under acidic conditions by the transcription factor Sfl1, whose role on biofilm formation is unclear. For that, microbiologic and transcriptomic analyses were performed with the knock-out mutant C. albicans sfl1/sfl1 and its parental strain SN76, grown in planktonic and biofilm lifestyles at pH 4 (vaginal pH). The results revealed that despite being a filamentation repressor Sf1 is required for maximal biofilm formation under acidic conditions. Additionally, Sfl1 was found to induce 275 and 126 genes in biofilm and planktonic cells, respectively, with an overlap of 19 genes. The functional distribution of Sfl1 targets was similar in planktonic and biofilm modes but an enrichment of carbohydrate metabolism function was found in biofilm cells, including some genes encoding proteins involved in the biofilm matrix production. Furthermore, this study shows that the regulatory network of Sfl1 in acidic biofilms is complex and include the positive and negative regulation of transcription factors involved in adhesion and biofilm formation, such as AHR1, BRG1, TYE7, TEC1, WOR1, and various of their targets. Overall, this study shows that Sfl1 is a relevant regulator of C. albicans biofilm formation in acidic environments and contributes to a better understanding of C. albicans virulence under acidic conditions.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation [grant NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 e Programa Operacional Regional do Norte. The authors also acknowledge the project funding by the “02/SAICT/2017 e Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT)”grant POCI-01-0145-FEDER-028893] [grant NORTE-01-0145-FEDER-031515].info:eu-repo/semantics/publishedVersio