Yes / Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 C) and an arbitrarily-selected temperature of 46 C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. / University of Malaya High Impact Research (HIR) UM-MOHE HIR Grants (UM.C/625/1/HIR/MOHE/CHAN/14/1, No. H-50001-A000027; UM.C/625/1/HIR/MOHE/CHAN/01, No. A000001-50001); PPP Grant (PG081-2015B)

Pseudomonas aeruginosa and Candida albicans have shown antagonistic relationships, both in vivo and in vitro. The interaction between P. aeruginosa and C. albicans is one of the many prokaryotic-eukaryotic interactions existing in nature and needs more research due to their complex pathogenicity in humans. In this work, we have studied growth dynamics of Candida in a mixed population of Pseudomonas and Candida, and their receptor and ligand specificities, both from an ecological and a molecular point of view. Initially, growth, viability, and morphogenesis of Candida were studied in the presence of Pseudomonas and the conditioned medium of Pseudomonas using two Candida strains, namely CAF2 and tup1 mutant. The killing effect of Pseudomonas was more pronounced on the hyphal form of Candida. However, growth of Candida was inhibited by Pseudomonas irrespective of its morphological form. The conditioned medium had no effect on the growth rate of Candida. Nevertheless, it completely inhibited the germination of Candida. The attachment of Pseudomonas to Candida was studied using different strains of both, and with Pseudomonas cells harvested at different stages of its growth. The percent attachment varied with the age of the textit Pseudomonas culture. A lecB mutant of Pseudomonas showed a two-fold reduction in attachment compared to the wild type PAK strain. Carbohydrate inhibition studies confirmed that LecB is not directly involved in the attachment mechanism, but indirectly involves through regulating the expression of other proteins required for attachment. A genomic DNA library of Pseudomonas PAO1 was screened for clones that had acquired the ability to attach to C. albicans. A clone was isolated with a small increase in attachment and was subjected to genetic analysis. It contained nucleotides 458565 to 475917 of the Pseudomonas genome including some genes of the Pil-Chp gene cluster. Seven transposon mutants that represent mutations in ChpA,B,C,D,E operon and three other ORFs were selected, and their attachment ability was tested. All seven mutants showed a reduction in attachment indicating that this was a non specific effect, which could be attributed to the in vitro manipulation of the bacteria. We conclude that the attachment of Pseudomonas to Candida is multi-factorial.

Le développement de salissures marines (ou biofouling) est à l’origine de nombreux problèmes économiques et écologiques. Ces salissures marines sont constituées de microorganismes (bactéries, microalgues…) formant le microfouling sur lequel va se développer le macrofouling constitué de macroorganismes tels que les algues, coquillages et éponges. La formation de ces salissures va induire un ralentissement des navires provoquant une surconsommation de carburant. De plus, l’utilisation de revêtements antisalissures ou peintures antifouling à base de métaux lourds et de biocides dans le passé a conduit à des problèmes environnementaux. L’objectif de la thèse est de rechercher une alternative aux composés actuellement utilisés (cuivre) en respectant le milieu marin. L’environnement est une source d’inspiration, une approche biomimétique pourrait être une stratégie de lutte efficace contre le biofouling. Dans une première partie, un criblage d’une centaine de composés est réalisé contre des souches bactériennes. Huit composés issus des deux familles : les batatasins et les hemibastadins sont étudiées en vue de comprendre leurs modes d’action. Parmi les composés sélectionnés, la famille des hemibastadins comprenant le DiBromoHemiBastadin-1 (DBHB) a montré des propriétés antifouling prometteuses. En effet, cette molécule est capable d’inhiber 50 % la formation du biofilm avec une IC50= 6,44 µg/mL pour la bactérie Pseudomonas aeruginosa PAO1 et une IC50 = 12,8 µg/mL pour la bactérie marine Paracoccus sp. 4M6. Afin de comprendre le mode d’action de cette molécule, son impact sur la communication bactérienne, le quorum sensing est étudié et le composé DBHB est capable de l’inhiber. Dans une seconde partie, un autre groupe d’organismes participant au microfouling est étudié : les microalgues. Afin d’évaluer l’impact de composés de la famille des hemibastadins et notamment du DBHB, l’adhésion et la formation de biofilm de microalgues sont étudiées. Le DBHB montre des inhibitions de l’adhésion et de la formation du biofilm uniquement envers la diatomée Cylindrotheca closterium. Une dernière partie, s’est intéressée à l’évaluation de revêtements contenant six composés de la famille des batatasins et des hemibastadins sur le microfouling naturel. Une méthode d’immersion de revêtements en conditions contrôlées (photobioréacteur) est mise au point afin de pallier aux contraintes environnementales. L’ensemble de cette étude a permis de mettre en évidence les propriétés antifouling du DBHB et de caractériser son mode d’action. Ce composé offre d’intéressantes voies d’étude dans la lutte contre le biofouling. De plus, l’approfondissement des connaissances sur les procédés d’adhésion et de formation de biofilm de microalgues permet de définir de nouvelles stratégies de lutte. / Biofouling induces important economic and ecological problems. This phenomenon includes microorganisms (bacteria, microalgae…) giving the microfouling which allows the macrofouling development with algae, invertebrates and sponges. These organisms colonize every immersed surface as boat hull. The colonization induces reduced speed of ships and fuel overconsumption. In the past, the utilization of AF coatings with heavy metals or pesticides caused environmental problems. The purpose of the study is to find an alternative to AF compounds (copper) respecting the marine medium. The marine environment is an inspiration; a biomimetic approach could be an interesting strategy to inhibit biofouling. In a first part, a screening of one hundred compounds is realized against marine and terrestrial bacteria. Eight molecules from two families (batatasins and hemibastadins) are studied to understand the way of action. Among selected compounds, Dibromohemibastadin-1 (DBHB) from hemibastadin family shows promising AF activities. This molecule is able to inhibit the biofilm formation with an IC50 of 6,44 µg/mL against the bacterium Pseudomonas aeruginosa PAO1 and 12,8 µg/mL for the marine bacterium Paracoccus sp. 4M6. To identify the way of action of DBHB, the impact on the bacterial communication named quorum sensing is investigated. The molecule shows an anti-quorum sensing property. In a second part, another group participating at microfouling is studied: microalgae. In order to evaluate the impact of hemibastadin family in particular DBHB, microalgae adhesion and biofilm formation are characterized. DBHB induces inhibition only on the adhesion and the biofilm formation of the diatom Cylindrotheca closterium. The last part presents the formulation of coatings containing six compounds from the batatasin and hemibastadin families. These coatings have been immersed in a harbor to evaluate the impact on natural microfouling. Furthermore, a new method for the evaluation of AF coatings is developed in controlled conditions, in a photobioreactor. This method allows the evaluation of coatings on the formation of a mixed biofilm (bacteria and microalgae). This method has been established to avoid environmental constraints by immersion in natural condition. This study allows the characterization of the AF property of DBHB. This compound provides promising research path to limit biofouling. Moreover, the development of a test allowing adhesion and microalgae biofilm formation in dynamic condition improves the characterization of compounds activities.

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