The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae

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Muner, Jose Julio ; de Oliveira, Paloma Aparecida Alves ; Baboghlian, Juliana ; Moura, Stefany Casarin ; de Andrade, Abissair Gabriel ; de Oliveira, Michelly Macedo ; Campos, Yasmin Ferreira de ; Mancano, Alquiandra Stefani Ferreira ; Siqueira, Nathalia Maria Goncalves ; Pacheco, Thaisy ; Ferraz, Lucio Fabio Caldas

• Document type: Journal article
• Source: BMC Microbiology; v. 24, n. 1, p. 9-pg., 2024-07-27.

Abstract

Background Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium. Results Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels. Conclusions Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism. (AU)

• FAPESP's process: 08/11365-1 - Study of the mechanism of gene expression regulation by Fur regulator in Klebsiella pneumoniae: consequences on the expression of virulence factors mediated by quorum-sensing
• Grantee: Lúcio Fábio Caldas Ferraz
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/13949-9 - Genotypic and phenotypic characterization of Klebsiella pneumoniae isolated from urinary tract infections and in vitro analysis of the processes of host cells adhesion and invasion
• Grantee: Thaisy Eliza Pacheco dos Santos
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 13/06042-7 - In vivo and in vitro studies of the adhesion and invasion of host cells by a fimbriae-deficient mutant strain of Klebsiella pneumoniae
• Grantee: Lúcio Fábio Caldas Ferraz
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/26203-9 - Study of the contribution of polyamine biosynthesis and transport pathways to the pathogenicity of Klebsiella pneumoniae
• Grantee: Lúcio Fábio Caldas Ferraz
• Support Opportunities: Regular Research Grants