A Virulence Associated Siderophore Importer Reduces Antimicrobial Susceptibility of Klebsiella pneumoniae

Abstract

The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

Keywords: ABC transporter; Klebsiella pneumoniae; antimicrobial efflux; integrative conjugative element; mobile genetic element; siderophore; yersiniabactin.

FIGURE 1

Comparison of ICEKp in the study strain KpRR2 with the first characterized ICEKp in strain NTUH-K2044, and with the yersiniabactin gene cluster in Yersinia pestis. The first characterization of ICEKp in K. pneumoniae was in strain Kp NTUH-K2044 (Lin et al., 2008). Yellow shading indicates the synteny and 99% DNA identity between the yersiniabactin gene cluster in the study strain KpRR2 and that in Yersinia pestis (AF091251) High Pathogenicity Island (HPI). The genes cluster responsible for conjugation are indicated by blue shading region, these share 94% identity.

FIGURE 2

The cargo genes of ICEKp from K. pneumoniae KpRR2 are an iron siderophore biosynthesis cluster (yersiniabactin) that includes an ABC transporter and an MFS family permease. (A) The entire cargo genes of ICEKp are shown, with annotation. The only transmembrane transporters amongst the cargo genes are in the four-gene operon: ybtP-ybtS. This operon is unique to yersiniabactin clusters and conserved with >80% amino acid identity in all organisms. The previously reported deletion strain ΔICE lacks the entire ICEKp (and therefore lacks the entire yersiniabactin cluster). (B) YbtP and YbtQ share 34% amino acid identity with each other, and form a heterodimeric transporter YbtPQ, homologous to ABC transporters of drugs, heme, lipid A and enterobactin. ABC transporters are recognized by their nucleotide binding domain: the C-terminal domain of YbtP and YbtQ (residues 342–556 and 344–558) share 29% and 31% identity with the nucleotide binding domain of MacB. (C) YbtX belongs to the major facilitator superfamily (MFS) and is a homolog of aerobactin and rhizobactin transporters, multidrug efflux transporters and peptidoglycan recycling transporters. Ce, Caenorhabditis elegans; Ec, E. coli; Mt, Mycobacterium tuberculosis; Gv, Gloeobacter violaceus; Ko, Klebsiella oxytoca; Kp, K. pneumoniae; Mb, Methanobrevibacter; Ms, Mycobacterium smegmatis; Mx, Myxococcus xanthus; Pa, Pseudomonas aeruginosa; Pp, Pseudomonas putida; Sa, Staphylococcus aureus; Rc, Rhodobacter capsulatus; Rm, Rhizobium meliloti; St, Salmonella typhimurium; Vo, Vibrio orientalis; Yp, Yersinia pestis. Functional annotations of proteins: AmpG (muropeptide MFS transporter), Bcr (MDR efflux), EmrD (MDR efflux), FebDG (ferric enterobactin transporter), IrtA (iron transporter), MacAB (MDR efflux), MdlAB (MDR efflux), MsbA (lipid ABC transporter), PdtE (inner membrane permease) Pgp-1 (multidrug resistance protein), SetC (sugar efflux system), SmdAb (MDR efflux), RhtX (rhizobactin transporter), Rv0194 (MDR ABC transporter), Rv2886c/Rv2887c (MDR efflux). Phylogenetic tree of amino acids sequence of ybt genes was inferred by maximum-likelihood method using MEGA10 (Kumar et al., 2018). Bootstrap values were calculated with 1000 replications. The bar shows changes observed between the two sequences; 0.20 means 20% amino acid changes.

FIGURE 3

Deletion of the integrative and conjugative element ICEKp from K. pneumoniae rendered it more sensitive to multiple classes of antimicrobial, and the effect could be reversed by reintroduction of the yersiniabactin transporter gene cluster. A deletion mutant lacking the entire ICEKp element, ΔICE, had a significantly larger zone of inhibition than the parent strain KpRR2 for all antimicrobials tested. Reintroduction of the yersiniabactin transporter gene cluster on plasmid pSXPQA restored the zone size to that of the parental strain. Data show the mean and standard deviation of three replicates. The quantity of antimicrobial per disk was: gentamicin 10 μg, trimethoprim 5 μg, rifampicin 5 μg, novobiocin 30 μg, imipenem 10 μg, doxycycline 30 μg, polymyxin B 300 μg, ofloxacin 5 μg. Each strain was compared with the parent strain by Student’s t test, and significant differences in zone diameter were indicated with an asterisk. *p < 0.05.

FIGURE 4

Efflux pump inhibitor reserpine increased the susceptibility of K. pneumoniae, but not the ICEKp knockout, to antimicrobials. Addition of reserpine (RP, 50 μg/ml) significantly increased the diameter of the zone of inhibition of study strain KpRR2 and the plasmid-complemented mutant ΔICE + pSXPQA for trimethoprim (A) and tetracycline (B). By contrast, the presence (+) or absence (-) of reserpine had no significant effect on the zones of inhibition of the knockout strain ΔICE. Trimethoprim was used at 5 μg per disk and tetracycline at 25 μg per disk. Data are the mean and standard deviation of three replicates. *indicates p < 0.05 using Student’s t test. “ns” indicates p > 0.05. The assay was also performed with other antimicrobials giving similar results (Supplementary Figure 1).

FIGURE 5

The yersiniabactin importer YbtPQ reduced antimicrobial susceptibility of K. pneumoniae ICEKp mutant and the effect of YbtPQ was reversed by reserpine. A The enhanced trimethoprim susceptibility (larger zone of inhibition) of mutant ΔICE was fully complemented by plasmid pPQ, encoding transporter YbtPQ. Plasmids pYbtX and pYbtS did not complement the defect of ΔICE (no significant change in zone of inhibition compared to ΔICE). Trimethoprim was used at 5 μg per disk. B The enhanced imipenem susceptibility of mutant ΔICE was fully complemented by plasmid pPQ, encoding transporter YbtPQ, but not by the vector control, pCtrl. The effect of plasmid pPQ on imipenem susceptibility was reversed by the addition of reserpine (RP) 50 μg/disk). Data are the mean and standard deviation of at least three replicates. *indicates p < 0.05 using Student’s t test. The effect of plasmid YbtPQ on antimicrobial susceptibility was also tested with additional antimicrobials (Supplementary Figure 2) and the effect of YbtPQ on antimicrobial susceptibility in the presence/absence of reserpine was additionally tested by MIC assay (Table 2).

FIGURE 6

Antimicrobial susceptibility of E. coli was reduced by ICEKp or by YbtPQ alone. (A) Transconjugant E. coli + ICE carrying the ICEKp was significantly less sensitive than E. coli to trimethoprim and tetracycline in a disk diffusion assay (25 and 10 μg per disk, respectively). Inhibition of efflux using reserpine (50 μg/disk) significantly increased the susceptibility of the transconjugant to each antimicrobial. Reserpine (RP) had no significant effect on the susceptibility of parental E. coli to either antimicrobial. (B) E. coli carrying plasmid pPQ, encoding YbtPQ, were significantly less susceptible to all antimicrobials tested, whereas control plasmid pYbtX had no significant effect on antimicrobial susceptibility. From left to right, disks contained 10 μg, 5 μg, 10 μg, 30 μg, 300 units, 5 μg, 30 μg, or 5 μg of antimicrobial. (C) The effect of YbtPQ on susceptibility to imipenem was reduced by reserpine. pCtrl indicates a control plasmid without ybtPQ (vehicle only control). Data are the mean and standard deviation of six replicates. Strains and conditions were compared by Student’s t test. * indicates p < 0.05. (D) The effect of YbtPQ on the minimum inhibitory concentrations (MIC) of E. coli for tetracycline was determined using E-test assay in the presence or absence of reserpine (RP 50 μg/ml) in triplicate (range tested was 0.016–256 μg/ml).