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Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

ABSTRACT: Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.

SUBMITTER: Kuan CS

PROVIDER: S-EPMC3155361 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

Kuan Chee Sian CS Wong Mun Teng MT Choi Sy Bing SB Chang Ching Ching CC Yee Yoke Hiang YH Wahab Habibah A HA Normi Yahaya Mohd YM Too Wei Cun See WC Few Ling Ling LL

International journal of molecular sciences 20110707 7

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease ...[more]

PMID: 21845088

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Project description:Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Essential to the colonization and infection by K. pneumoniae is the acquisition of nutrients, such as the transition metal ion zinc. Zinc has crucial structural and catalytic roles in the proteome of all organisms. Nevertheless, in excess, it has the potential to mediate significant toxicity by dysregulating the homeostasis of other transition elements, disrupting enzymatic processes, and perturbing metalloprotein cofactor acquisition. Here, we sought to elucidate the zinc detoxification mechanisms of K. pneumoniae, which remain poorly defined. Using the representative K. pneumoniae AJ218 strain, we showed that the P-type ATPase, ZntA, which is upregulated in response to cellular zinc stress, was the primary zinc efflux pathway. Deletion of zntA rendered K. pneumoniae AJ218 highly susceptible to exogenous zinc stress and manifested as an impaired growth phenotype and increased cellular accumulation of the metal. Loss of zntA also increased sensitivity to cadmium stress, indicating a role for this efflux pathway in cadmium resistance. Disruption of zinc homeostasis in the K. pneumoniae AJ218 ΔzntA strain also impacted manganese and iron homeostasis and was associated with increased production of biofilm. Collectively, this work showed the critical role of ZntA in K. pneumoniae zinc tolerance and provided a foundation for further studies on zinc homeostasis and the future development of novel antimicrobials to target this pathway. IMPORTANCE Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Treatment of K. pneumoniae infections is becoming increasingly challenging due to high levels of antibiotic resistance and the rising prevalence of carbapenem-resistant, extended-spectrum β-lactamases producing strains. Zinc is essential to the colonization and infection by many bacterial pathogens but toxic in excess. This work described the first dissection of the pathways associated with resisting extracellular zinc stress in K. pneumoniae. This study revealed that the P-type ATPase ZntA was highly upregulated in response to exogenous zinc stress and played a major role in maintaining bacterial metal homeostasis. Knowledge of how this major bacterial pathogen resists zinc stress provided a foundation for antimicrobial development studies to target and abrogate their essential function.

Project description:IntroductionZinc exhibits potent antimicrobial properties due to its ability to compromise bacterial structure and protein functionality, effectively inhibiting and eradicating bacteria. However, bacteria have evolved mechanisms to expel excess zinc ions from their cells, enabling them to thrive in environments rich in metal ions at high concentrations. This evolutionary advancement limits the clinical application of metal ions as antimicrobial agents. In this study, we aimed to investigate the potential of zinc ionophores to overcome bacterial resistance by elevating intracellular zinc ion levels.MethodsWe employed the zinc ionophore PBT2 to elevate intracellular zinc ion levels in Klebsiella pneumoniae, a bacterium known for its resistance to various antibiotics. By treating K. pneumoniae with PBT2, we aimed to assess its impact on bacterial resistance to tigecycline, an antibiotic commonly used in clinical settings. The changes in intracellular zinc ion levels, superoxide dismutase activity, reactive oxygen species concentration, and cell wall synthesis pathway were monitored to evaluate the mechanism of action of PBT2.ResultsOur results revealed that PBT2 successfully reversed the resistance of K. pneumoniae to tigecycline. Specifically, PBT2 increased the concentration of intracellular zinc ions in K. pneumoniae, leading to a suppression of superoxide dismutase activity within the cell and an elevation of reactive oxygen species concentration. These changes impaired the oxidative stress response of the bacteria. Additionally, the disruption of zinc homeostasis significantly inhibited the cell wall synthesis pathway in K. pneumoniae, potentially restricting the efflux pump mechanism that predominantly drives tigecycline resistance.DiscussionThe findings of this study pave the way for innovative strategies and approaches in the clinical development of novel antimicrobial agents. By using zinc ionophores such as PBT2 to elevate intracellular zinc ion levels, we can overcome bacterial resistance to antibiotics like tigecycline. The suppression of superoxide dismutase activity and elevation of reactive oxygen species concentration suggest that PBT2 impairs the oxidative stress response of K. pneumoniae, further enhancing its susceptibility to antibiotics. Furthermore, the inhibition of the cell wall synthesis pathway and restriction of the efflux pump mechanism provide additional mechanisms by which PBT2 reverses antibiotic resistance. These results highlight the potential of zinc ionophores as a novel class of antimicrobial agents and warrant further investigation into their clinical applications.

Dataset Information

Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

Publications

Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

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