Functional Identification of Hypothetical Proteins in Pseudomonas Aeruginosa PAO1


Student thesis: Doctoral Thesis

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Award date13 Dec 2023


Pseudomonas aeruginosa is a Gram-negative bacterium characterized by its rod-shaped morphology. It is an opportunistic pathogen known for its multidrug resistance, which makes it a frequent cause of nosocomial infections in immunocompromised patients. In particular, P. aeruginosa is a major pathogen responsible for lung infections in individuals with cystic fibrosis (CF), often leading to high mortality rates. One of the remarkable features of P. aeruginosa is its high adaptability to diverse environmental conditions, which can be attributed to its large genome. The bacterium possesses a genome of approximately 6.3 Mbp, encompassing 5570 open reading frames (ORFs). Within this gene repertoire, more than 2000 ORFs correspond to hypothetical proteins (HPs), which are predicted proteins lacking known functions. Surprisingly, these uncharacterized proteins are highly expressed in P. aeruginosa and play a critical role in its ability to withstand environmental stresses and evade host immune responses. Unraveling the functions of these HPs holds immense value, as it can provide crucial insights into the metabolic and regulatory mechanisms of P. aeruginosa. The identification and characterization of these unknown genes are essential for understanding the bacterium's pathogenicity and developing effective treatments against P. aeruginosa infections. Moreover, a systematic functional annotation of HPs can establish relationships between different HPs and classify them, leading to the characterization of novel virulence-associated genes. This comprehensive functional annotation approach can facilitate the discovery of new virulence-associated genes within P. aeruginosa, contributing to a deeper understanding of its pathogenesis and aiding in the development of targeted therapeutic strategies.

According to the Pseudomonas Genome Database, Pseudomonas aeruginosa PAO1 harbors 2194 hypothetical proteins (HPs) distributed throughout its genome. RNA-seq analysis conducted under normal conditions, specifically using LB medium, revealed high expression levels of many of these HPs in wild-type PAO1, indicating their potential importance in the bacterium's survival. To elucidate the functions of these HPs, we employed gene deletion techniques in Pseudomonas aeruginosa PAO1, resulting in the generation of 605 HP mutants encompassing 700 uncharacterized open reading frames (ORFs). RNA-seq analysis of these mutants was performed to assess changes in gene expression compared to wild-type PAO1. By subjecting the RNA-seq data to gene ontology (GO) enrichment analysis, we were able to identify the functional roles of each HP. The top 10 mutants represented a broad range of GO terms, including amide metabolic processes, lipid biosynthesis and metabolism, cellular macromolecule metabolic processes, nucleotide binding, and intracellular organelles. This indicates that HPs have diverse and significant effects on various cellular processes. To further explore the functional relationships among HPs, we employed dimensionality reduction methods on the RNA-seq data from 206 mutants. This analysis revealed distinct clusters of HPs, suggesting conserved functional roles among these uncharacterized proteins. Additionally, we employed weighted correlation network analysis (WGCNA) to group genes into different modules based on their expression levels. These modules encompassed both genome-wide genes and differentially expressed genes (DEGs). By performing GO analysis on the genes within each module, we could characterize the functional attributes of the modules and annotate the HPs based on their regulation within specific modules. This computational approach enabled us to cluster and characterize the functional roles of HPs based on their regulation within specific modules, providing a novel method for understanding these uncharacterized proteins. This approach offers valuable insights into the potential functions and regulatory relationships of these uncharacterized proteins.

Pseudomonas aeruginosa is a versatile pathogen capable of infecting humans, animals, and plants, and it exhibits resilience in various environments due to the presence of numerous virulence factors. These factors include quorum sensing (QS) systems, flagella, type 4 pili (T4P), type III secretion system (T3SS), type VI secretion system (T6SS), type II secretion system (T2SS), and compounds secreted into the extracellular environment. Through screening 605 mutants of hypothetical proteins (HPs) in P. aeruginosa PAO1, we identified 14 HPs that are essential for the early growth in LB medium. Notably, the deletion mutant ΔPA2229-30 exhibited a significant decrease in the production of two QS system signals, namely N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and Pseudomonas quinolone signal, both of which are associated with pathogenicity. These findings suggest that targeting PA2229-30 could be a promising strategy to inhibit the QS system and potentially mitigate P. aeruginosa virulence. Furthermore, four other HP mutants, including ΔPA1550, ΔPA4691-92, PA3016, and ΔPA1788, were found to reduce the production of Pseudomonas quinolone signal, and RNA-seq analysis revealed their positive regulation of the pqs system, which is involved in the virulence of P. aeruginosa. Among the HP mutants, five exhibited a loss of swarming motility, namely ΔPA0307, ΔPA3350, ΔPA4463-65, ΔPA3352, and ΔPA1005. These HPs play a role in modulating the cilium of flagellum-dependent cell motility in PAO1. Additionally, four HP mutants, including PA0392, PA0446, PA0862, and PA3352, inhibited the production of extracellular polymeric substances (EPS), while PA2229-30 positively regulated EPS production. Moreover, the deletion mutant ΔPA0862 displayed enhanced biofilm formation by regulating the macromolecule biosynthetic process, while ΔPA3350, ΔPA3352, and ΔPA4128 exhibited reduced biofilm biomass. Eight HP mutants, including ΔPA4128, ΔPA3674, and ΔPA2854-55, had an impact on the production of pyocyanin, a bacterial pigment that interferes with various biological processes in host cells. These phenotypic screening results highlight the significance of HPs in the regulation of virulence factors during infection. Pseudomonas aeruginosa is an opportunistic pathogen known for its resistance to multiple antibiotics. Determination of the minimal inhibitory concentration (MIC) revealed that many HP mutants displayed increased sensitivity to antibiotics, offering potential new targets for therapies against chronic infections. Subsequent animal experiments demonstrated that 11 deletions, including mutants such as ΔPA3016, ΔPA3674, ΔPA0862, and ΔPA2229-30, exhibited attenuated virulence in mice. These findings further emphasize the role of HPs in P. aeruginosa pathogenicity and provide insights into potential targets for therapeutic interventions.

In conclusion, this thesis provided valuable insights into the functional roles of hypothetical proteins (HPs) in Pseudomonas aeruginosa. By employing computational methods and analyzing transcriptomic data, we elucidated the relationships between different HPs and categorized them based on their regulatory genes. This approach facilitated the functional annotation of the HPs. Phenotypic screening experiments uncovered numerous HPs that modulated virulence factors and played crucial roles in infection. The findings of this thesis underscored the significance of HPs in the context of P. aeruginosa. Moreover, the application of computational methods offered a powerful tool for characterizing and understanding unknown proteins. The identification of virulence-associated HPs also presented potential opportunities for developing novel therapeutic strategies and verified the accuracy of computational methods. By focusing on HPs as drug targets, researchers could explore innovative approaches to combat P. aeruginosa infections. Overall, this thesis contributed to the growing body of knowledge on HPs in P. aeruginosa, shedding light on their functional relevance and showcasing the utility of computational methods in protein characterization. The identification and characterization of virulence-associated HPs provided a foundation for further research and the development of targeted interventions against Pseudomonas aeruginosa infections.

    Research areas

  • Pseudomonas aeruginosa PAO1, hypothetical protein, virulence factor, functional role