RESUMO
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.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Ferro , Klebsiella pneumoniae , Lipopolissacarídeos , Regiões Promotoras Genéticas , Proteínas Repressoras , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Lipopolissacarídeos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ferro/metabolismo , Sítios de Ligação , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismoRESUMO
Acinetobacter baumannii is a Gram-negative bacterium considered an emerging multi-drug-resistant pathogen. Furthermore, this bacterium can survive in extreme environmental conditions, which makes it a frequent cause of nosocomial infection outbreaks. Gene expression analyses by Reverse Transcription Quantitative real-time PCR (RT-qPCR) depend on a reference gene, also called an endogenous gene, which is used to normalize the generated data and thus ensure an accurate analysis with minimal errors. Currently, gene expression analyses in A. baumannii are compromised, as there are no reports in the literature describing the identification of validated reference genes for use in RT-qPCR analyses. For this reason, we selected twelve candidate reference genes of A. baumannii and assessed their expression profile under different experimental and culture conditions. The expression stability of the candidate genes was evaluated by using statistical algorithms such as BestKeeper, geNorm, NormFinder, Delta CT, and RefFinder, in order to identify the most suitable candidate reference genes for RT-qPCR analyses. The statistical analyses indicated rpoB, rpoD, and fabD genes as the most adequate to ensure accurate normalization of RT-qPCR data in A. baumannii. The accuracy of the proposed reference genes was validated by using them to normalize the expression of the ompA gene, encoding the outer membrane protein A, in A. baumannii sensible and resistant to the antibiotic polymyxin. The present work provides suitable reference genes for precise RT-qPCR data normalization on future gene expression studies with A. baumannii.