Phenotypic and Enzymatic Comparative Analysis of the KPC Variants, KPC-2 and Its Recently Discovered Variant KPC-15

<div><p>Sixteen different variants (KPC-2 to KPC-17) in the KPC family have been reported, and most current studies are focusing on KPC-2 and KPC-3. The KPC-15 variant, which isolated from <i>Klebsiella pneumoniae</i> in a Chinese hospital, was a recently discovered KPC enzyme. To compare the characteristics of KPC-15 and KPC-2, the variants were determined by susceptibility testing, PCR amplification and sequencing, and study of kinetic parameters. The strain harboring the KPC-15 showed resistance to 18 conventional antimicrobial agents, especially to cabapenem antibiotics, and the strain involving the KPC-2 also indicated resistance to cabapenem antibiotics, but both strains were susceptible to polymyxin B and colistin. The conjugation experiments showed that the changes of MIC values to the antibiotics were due to the transferred plasmids. The differences of amino acids were characterised at sites of 119 leucine and 146 lysine with KPC-15 and KPC-2. The minimum evolution tree indicated the KPC alleles evolution, and showed that the KPC-15 appeared to be homogenous with KPC-4 closely. Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study. The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2. Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.</p></div

Kinetic parameters for KPC-15 and KPC-2 enzymes.

<p>Kinetic parameters for KPC-15 and KPC-2 enzymes.</p

Promoter elements of the <i>bla</i>_KPC-15 and <i>bla</i>_TEM genes in 8.997 kb-length nucleotide sequence.

<p>The sequence provided the upstream and downstream regions of <i>bla</i>_KPC-15 structural genes. The <i>tnpA</i> gene, which was upstream of the <i>bla</i>_KPC-15 (3,020 bp) gene, was homologous to a putative IS<i>Kpn8</i> transposon, and the downstream region of the <i>bla</i>_KPC-15 gene (1,320 bp) was homologous to a putative IS<i>Kpn6</i>-like transposon. The nucleotides upstream of the <i>bla</i>_KPC-15 and <i>bla</i>_TEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the <i>bla</i>_KPC-15 gene were shown as <i>gattaa</i>, labeled as −10 below, and there were no obvious −35 promoter elements to be discovered in the promoter region. The putative −10 and −35 promoter elements of the <i>bla</i>_TEM-12 gene were shown as <i>tataac</i> and <i>ttattg</i>, labeled as −10 and −35 below the promoter region. The start sites of transcription were indicated as G by +1 residue. RBS was the abbreviation of the ribosome binding site.</p

Comparison of amino acid sequence alignments of KPC carbapenemases.

<p>KPC-15 was recently identified as a carbapenemase in <i>Klebsiella pneumonae</i> from the Taizhou Municipal Hospital of China. The data within parentheses are GenBank accession numbers.</p

Minimum Evolution tree of amino acid sequences for KPC-2 to KPC-17.

<p>KPC-15 carbapenemase was our newly discovered and appeared to be homogenous with KPC-4 closely. The amino acid sequences of KPCs based on the data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111491#pone-0111491-g001" target="_blank">Figure 1</a>. This comparison was designed and analysed using MEGA 5.05 software.</p

Sequences of primers utilised to determine the <i>bla</i>_KPC genetic environment in this study.

<p>Sequences of primers utilised to determine the <i>bla</i>_KPC genetic environment in this study.</p

The susceptibility for strains of <i>E. coli</i> J53Az^R, Kp1241, Kp1769, and transconjugants (J53Az^R-Kp1241 and J53Az^R-Kp1769).

<p>Clinical breakpoints of MICs for the antimicrobial agents see the reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111491#pone.0111491-CLSI1" target="_blank">[25]</a>.</p><p>The susceptibility for strains of <i>E. coli</i> J53Az^R, Kp1241, Kp1769, and transconjugants (J53Az^R-Kp1241 and J53Az^R-Kp1769).</p

Live-cell imaging of both mutants GFP-CTD^{Δ723 −741} and GFP-CTD^Δ714–722.

<p>COS-1 cells co-expressing either GFP-CTD^{Δ723 −741} (<b>A</b>) or GFP-CTD^Δ714–722 (<b>B</b>), along with the ER/DsRed marker, were subjected to live-cell imaging combined with the <i>in vivo</i> membrane protease protection assay, as described above in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109159#pone-0109159-g003" target="_blank">Figure 3</a>. The images shown herein are a representative of at least three independent experiments undertaken on separate occasions that were each performed in triplicate (n = 9).</p

Both AD2 and NST domains positively regulates chimaeric Gal4-Nrf1 and Gal4-Nrf1β factors.

<p>(<b>A</b>) Schematic representation of expression constructs for Gal4D (Gal4 DNA-binding domain) fusion proteins containing various portions of Nrf1 or Nrf1β (<i>left panel</i>). They were created by ligation of their encoding cDNA fragments into the <i>BamHI/EcoRI</i> sites of the pcDNA3/Gal4-V5 vector. The <i>left panel</i> shows Gal4D-directed reporter activity that was measured from COS-1 cells had been cotransfected with each of indicated expression constructs for the various Gal4D/Nrf1 fusion proteins (1.2 µg), together with <i>P_TKUAS</i>×4<i>-</i>Luc (0.6 µg) and b-gal (0.2 µg) plasmids. The data are shown graphically as fold changes (mean ± S.D.) of transactivation by indicated Gal4-fusion factors when compared with the background (value of 1.0). Significant increases ($, p<0.05 and$$, p<0.001, n = 9) and decreases (*p<0.05, **p<0.001, n = 9) in activity relatively to the referenced activity are indicated (<i>arrows</i>). (<b>B</b>) The above-prepared cell lysates were resolved using 4–12% LDS/NuPAGE and examined by western blotting with V5 antibody. The electrophoretic bands representing free Gal4D and Gal4-Nrf1 fusion proteins are indicated. Samples loaded on each well were calculated to contain equal amounts of β-gal activity.</p

Imaging of fixed and live cells expressing GFP fusion protein with CTD of Nrf1 or its mutants.

<p>(<b>A</b>) Schematic of Six expression constructs for the GFP-CTD fusion protein and its mutants; these fusion proteins have been created by attachment of various lengths of CTD of Nrf1 to the C-terminus of GFP. (<b>B</b>) These indicated expression constructs each were transfected into COS-1 cells for 6 h. The cells were then allowed to recover from transfection in fresh medium for 18 h before being fixed by 4% paraformaldehyde and stained for the nuclear DNA by DAPI. The green signals from GFP were observed under confocal microscope and merged with the DNA-staining images. (<b>C</b> and <b>D</b>) Live-cell imaging of GFP-CTD and its mutant GFP-CTD^Δ731–741(lacking its basic c-tail). COS-1 cells had been transfected with expression constructs for either GFP-CTD (<b><i>C</i></b>) or GFP-CTD^Δ731–741 (<b><i>D</i></b>), together with the ER/DsRed marker, before being subjected to real-time live-cell imaging combined with the <i>in vivo</i> membrane protease protection assay. The cells were permeabilized by digitonin 20 µg/ml) for 10 min, before being co-incubated with PK (50 µg/ml) for 30 min. In the time course, real-time images were acquired using the Leica DMI-6000 microscopy system. The merged images of GFP with ER/DsRed are placed (on <i>the third raw of panels</i>), whereas changes in the intensity of their signals are shown graphically (<i>bottom</i>). Overall, the images shown herein are a representative of at least three independent experiments undertaken on separate occasions that were each performed in triplicate (n = 9). The <i>arrow</i> indicates a ‘hernia-like’ vesicle protruded from the cytoplasm.</p