A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications

Abstract

Primary Klebsiella pneumoniae liver abscess complicated with metastatic meningitis or endophthalmitis is a globally emerging infectious disease. Its pathogenic mechanism remains unclear. The bacterial virulence factors were explored by comparing clinical isolates. Differences in mucoviscosity were observed between strains that caused primary liver abscess (invasive) and those that did not (noninvasive). Hypermucoviscosity correlated with a high serum resistance and was more prevalent in invasive strains (52/53 vs. 9/52; P < 0.0001). Transposon mutagenesis identified candidate virulence genes. A novel 1.2-kb locus, magA, which encoded a 43-kD outer membrane protein, was significantly more prevalent in invasive strains (52/53 vs. 14/52; P < 0.0001). The wild-type strain produced a mucoviscous exopolysaccharide web, actively proliferated in nonimmune human serum, resisted phagocytosis, and caused liver microabscess and meningitis in mice. However, magA- mutants lost the exopolysaccharide web and became extremely serum sensitive, phagocytosis susceptible, and avirulent to mice. Virulence was restored by complementation using a magA-containing plasmid. We conclude that magA fits molecular Koch's postulates as a virulence gene. Thus, this locus can be used as a marker for the rapid diagnosis and for tracing the source of this emerging infectious disease.

Figure 1.

HV phenotype. (A) Demonstration of a positive string test; formation of a viscous string >0.5 cm in length stretched by the inoculation loop. (B) Under light microscopy, wild-type strain NTUH-K2044 produces an exopolysaccharide web attached to the capsule (Periodic Acid Schiff stain; original magnification, 1,000). (C) magA ⁻ mutant loses the capability of producing an exopolysaccharide web.

Figure 2.

Serum and phagocytosis resistance assays. (A) Difference in serum resistance between NTUH-K2044, MGH-78578, NTUH-K9, and mutants 1–20. The assays were performed sequentially three times. A high degree of consistency was observed between experiments. (B) Restoration of serum resistance by magA complementation in a magA ⁻ mutant, in comparison with magA ⁻ mutant, magA ⁻ mutant–carrying vector only, wild-type–carrying vector only, and wild type. (C) Western blot analysis of complement C3 deposition showing that magA ⁻ mutant (lanes 2 and 5) and cps ⁻ mutant (lanes 3 and 6) have much higher levels of complement C3 deposition than the wild type (lanes 1 and 4). 1-min exposure to serum (lanes 1–3). 5-min exposure to serum (lanes 4–6). (D) Phagocytosis assay showing wild-type NTUH-K2044 is less associated with mouse macrophages than magA ⁻ mutant or cps ⁻ mutant. Complementation with magA-carrying plasmid partially restores the phenotype. (E and F) Fluorescence microscopy images (original magnification, 400). Wild-type NTUH-K2044 (E) is less likely to be ingested or attached by human neutrophils than a magA ⁻ mutant (F). (G) Confocal fluorescence microscopy showing that magA ⁻ mutants were ingested and disintegrated by human neutrophils.

Figure 2.

Serum and phagocytosis resistance assays. (A) Difference in serum resistance between NTUH-K2044, MGH-78578, NTUH-K9, and mutants 1–20. The assays were performed sequentially three times. A high degree of consistency was observed between experiments. (B) Restoration of serum resistance by magA complementation in a magA ⁻ mutant, in comparison with magA ⁻ mutant, magA ⁻ mutant–carrying vector only, wild-type–carrying vector only, and wild type. (C) Western blot analysis of complement C3 deposition showing that magA ⁻ mutant (lanes 2 and 5) and cps ⁻ mutant (lanes 3 and 6) have much higher levels of complement C3 deposition than the wild type (lanes 1 and 4). 1-min exposure to serum (lanes 1–3). 5-min exposure to serum (lanes 4–6). (D) Phagocytosis assay showing wild-type NTUH-K2044 is less associated with mouse macrophages than magA ⁻ mutant or cps ⁻ mutant. Complementation with magA-carrying plasmid partially restores the phenotype. (E and F) Fluorescence microscopy images (original magnification, 400). Wild-type NTUH-K2044 (E) is less likely to be ingested or attached by human neutrophils than a magA ⁻ mutant (F). (G) Confocal fluorescence microscopy showing that magA ⁻ mutants were ingested and disintegrated by human neutrophils.

Figure 3.

(A and B) Histopathology in the liver and brain of a mouse inoculated with 10⁵ CFU of K. pneumoniae NTUH-K2044, examined on day 9 after inoculation (hematoxylin and eosin stain; original magnification, 200). (C and D) Histopathology in the liver and brain of a mouse inoculated with 10⁵ CFU of K. pneumoniae MGH-78578, examined on day 9 after inoculation (hematoxylin and eosin stain; original magnification, 200). A and B show severe meningitis and liver microabscess. C and D show normal histology.

Figure 4.

Dot-blot hybridization of genomic DNA extracted from 40 invasive strains (lanes B–F) and 40 noninvasive strains (lanes G–K). Dot A1 is NTUH-K2044 as the positive control. Dot L1 is the PCR product of cepA (reference 29) as the negative control. (A) Hybridization with magA probe. All the invasive strains shown here were positive. The noninvasive strains at dots 3G, 3H, 4H, 6G, 6H, 7G, 7H, 7I, and 8H were positive. (B) Hybridization with 23S rRNA gene probe as internal positive control. (C) PCR for magA. (lane 1) Marker. (lanes 2–6) 5 positive strains. (lanes 7–11) 5 negative strains. (D) PCR for 23S rRNA gene as control.

Figure 5.

Western blot of magA (lanes 1–7) and NlpB (lanes 8–10). (lanes 1–4) Cytoplasmic membrane, outer membrane, total proteins, and spheroplast of NTUH-K2044. (lanes 5–7) Cytoplasmic membrane, outer membrane, and spheroplast proteins of a magA ⁻ mutant. (lanes 8–10) Outer membrane, cytoplasmic membrane, and periplasmic proteins of NTUH-K2044. (arrow) 43 kD. (arrowhead) 37 kD.