The Klebsiella pneumoniae tellurium resistance gene terC contributes to both tellurite and zinc resistance

Abstract

Klebsiella pneumoniae is widely recognized as a pathogen responsible for hospital-acquired infections and community-acquired invasive infections. It has rapidly become a significant global public health threat due to the emergence of hypervirulent and multidrug-resistant strains, which have increased the challenges associated with treating life-threatening infections. Tellurium resistance genes are widespread on virulence plasmids in K. pneumoniae isolates. However, the core function of the ter operon (terZABCDEF) in K. pneumoniae remains unclear. In this study, the multidrug-resistant K. pneumoniae P1927 strain was isolated from the sputum of a hospitalized pneumonia patient. The ter operon, along with antimicrobial resistance and virulence genes, was identified on a large hybrid plasmid in K. pneumoniae P1927. We generated a terC deletion mutant and demonstrated that this mutant exhibited reduced virulence in a Galleria mellonella larva infection model. Further physiological functional analysis revealed that terC is not only important for Te(IV) resistance but also for resistance to Zn(II), Mn(II), and phage infection. All genes of the ter operon were highly inducible by Zn(II), which is a stronger inducer than Te(IV), and the terBCDE genes were also induced by Mn(II). Collectively, our study demonstrates novel physiological functions of TerC in Zn(II) resistance and virulence in K. pneumoniae.IMPORTANCEKlebsiella pneumoniae has rapidly become a global threat to public health. Although the ter operon is widely identified in clinical isolates, its physiological function remains unclear. It has been proposed that proteins encoded by the ter operon form a multi-site metal-binding complex, but its exact function is still unknown. TerC, a central component of the tellurium resistance determinant, was previously shown to interact with outer membrane proteins OmpA and KpsD in Escherichia coli, suggesting potential changes in outer membrane structure and properties. Here, we report that TerC confers resistance to Zn(II), Mn(II), and phage infection, and Zn(II) was shown to be a strong inducer of the ter operon. Furthermore, TerC was identified as a novel virulence factor. Taken together, our results expand our understanding of the physiological functions encoded by the ter operon and its role in the virulence of K. pneumoniae, providing deeper insights into the link between heavy metal(loid) resistance determinants and virulence in pathogenic bacteria.

Keywords: Klebsiella pneumoniae; TerC; virulence; zinc detoxification.

Fig 1

Circular genome map of K. pneumoniae P1927. (A) Chromosome circle map. (B) Circle map of plasmid 1. (C) Circle map of plasmid 2. (D) Circle map of plasmid 3. From the outside to the inside, the first two circles represent the coding sequences (CDS) on the forward and reverse strands. The third circle represents the GC content, with the outer part indicating that the GC content in this region was higher than the average GC content of the whole genome. The fourth circle represents the GC skew value. When the value was positive, the positive chain was more likely to transcribe the CDS, and when it was negative, the negative chain was more likely to transcribe the CDS. The circular genome map was generated by CGview (v.1.0). Antimicrobial resistance genes, heavy metal(loid) resistance genes, conjugative transfer genes, and virulence factors were labeled.

Fig 2

Distribution of the ter operon in different bacteria (GenBank accession numbers in parentheses). K. pneumoniae P1927 (CP073378.1), E. coli DETEC-P793 (CP116116.1), R. planticola KpNDM1 (JX515588.1), Superficieibactor sp. HKU1 (CP119754.1), C. koseri 2022LN-00378 (ABJULC000000000.3), S. enterica CFSAN056598 (AACWHP000000000.1), Morganella morganii 2023GN-00017 (ABKLBV000000000.3), P. aeruginosa 22 (JASEZL000000000.1), Veillonella sp. N5_258_062G1 (JAWFEU000000000.1), Kluyvera intermedia CAV6332 (DACSNU000000000.1), Serratia marcescens CAV1492 (CP011641.1).

Fig 3

Growth phenotypes of the WT and terC mutant grow on the LB agar plate supplemented with a range of Te(IV) (A), Zn(II) (B), and Mn(II) (C) at 37°C. Data are five (n = 5) independent technical replicates.

Fig 4

Growth phenotypes of the WT and terC mutant grow in LB liquid medium supplemented with a range of Te(IV) (A), Zn(II), (B) and Mn(II) (C) at 37°C. Data are mean OD_{600 nm} values (± SD) from three (n = 3) independent biological experiments. Statistical significance of the differences determined by two-way ANOVA with Sidak posttest: **** (P < 0.0001), *** (P < 0.001), ** (P < 0.01), and * (P < 0.05), using GraphPad Prism 9.5.1.

Fig 5

RT-qPCR analysis of tellurium resistance genes (terZABCDEF), manganese resistance genes (mntPRS), and zinc resistance genes (fieF, zupT, and znuBCA) from K. pneumoniae P1927. Strain was grown in LB liquid medium containing 10 µM and 100 µM Te(IV) (A), 1 mM and 2 mM Zn(II) (B), and 5 mM and 10 mM Mn(II) (C) for 1.5 h, and RNA was isolated and analyzed by RT-qPCR. (D) Expression of ter operon in LB liquid medium supplemented with 100 µM Te(IV), 2 mM Zn(II), and 10 mM Mn(II). Log₂ fold change in the expression for treated K. pneumoniae P1927 WT cultures, by comparison with untreated. Data are mean values (± SD) from three (n = 3) independent biological experiments. Statistical significance of the differences determined by two-way ANOVA with Sidak posttest: **** (P < 0.0001), *** (P < 0.001), ** (P < 0.01), and * (P < 0.05), using GraphPad Prism 9.5.1.

Fig 6

Phage plaques of phage 55–2, 2113–2, 2134–2, 1596–2, 2102–2, 2095–2, 2157–2, and 2093–2 on the LB agar plates. Figures are the same scales (250 × 250 pixels).

Fig 7

Growth of the WT and terC mutant in the presence of phage 55–2 (A), 2134–2 (B), 2102–2 (C), and 2095–2 (D) over 24 h in LB liquid medium. Data are mean OD_{600 nm} values (± SD) from three (n = 3) independent biological experiments. Statistical significance of the differences determined by two-way ANOVA with Sidak posttest: **** (P < 0.0001), *** (P < 0.001), ** (P < 0.01), and * (P < 0.05), using GraphPad Prism 9.5.1.

Fig 8

Kaplan–Meier plots showing the percent survival of G. mellonella larvae over 48 h post-infection with 10⁹ colony-forming units (CFU) (A), 10⁸ CFU (B), 10⁷ CFU (C), and 10⁶ CFU (D) of the WT or terC mutant. The experiments were controlled by PBS-injected larvae. Survival curves were plotted using the Kaplan–Meier method (GraphPad Prism 9.5.1 software). Each experiment was performed in triplicate with 10 animals per treatment per replicate, and shaded areas show 95% confidence intervals in survival probability. Statistical significance of the differences determined by the Log-rank test using GraphPad Prism 9.5.1.