Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis

Abstract

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

Fig 1. PIA/PNAG production and adherence of S. aureus to nasal epithelial cells.

(A) PIA/PNAG was extracted from S. aureus strains and detected using WGA-biotin. Following incubation with HRP-streptavidin, PIA/PNAG was visualized by chemiluminescence detection. (B) The adherence of bacteria to RPMI 2650 cells was determined using an adherence assay. The number of bacteria that adhered to the cells was determined by CFU enumeration, and the average number of bacteria adhered to each RPMI 2650 cell was calculated. S. carnosus TM300 was used as a control. Significant differences are denoted with ***p-value < 0.001.

Fig 2. SEM images of adhesion of S. aureus to RPMI 2650 cells.

(A) RPMI 2650 cells were incubated with S. aureus SA113 (a, d), SA113Δica (b, e) and SA113Δica(pCica) (c, f). The images were captured at a magnification of 1000x (a, b, c) and 3000x (d, e, f). The number of bacteria that adhered to 400 cells was enumerated, and the average number of bacteria on each cell was calculated (B). Significant differences are denoted with ***p-value < 0.001.

Fig 3. Effects of glucose on PIA/PNAG production and adherence of S. aureus to RPMI 2650 cells.

S. aureus SA113 was cultured in glucose-containing TSB for 24 h. PIA/PNAG production (A) was determined using WGA-biotin. The average number of S. aureus SA113 adhered to each RPMI 2650 cell (B) was also determined using adherence assay. Significant differences are denoted with ***p-value < 0.001.

Fig 4. Effects of PIA/PNAG on the adherence of S. carnosus TM300 to epithelial cells.

PIA/PNAG production (A), biofilm formation (B) and adherence of S. carnosus TM300 and S. carnosus TM300(pTXicaADBC) to RPMI 2650 cells (C) were determined. (D) EPS that was purified from S. aureus SA113 at various concentrations was mixed with S. carnosus TM300 and incubated with RPMI 2650 cells. The average number of S. carnosus TM300 that adhered to each RPMI2650 cell was determined. EPS (2.4 μg/ml) from S. aureus SA113, SA113Δica, S. epidermidis O-47 and S. epidermidis O-47ΔicaB was treated or untreated with 0.5 mg/ml dispersin B. The amounts of PIA/PNAG were determined by WGA-biotin (E). The average number of S. carnosus TM300 adhered to each RPMI2650 cell was determined by CFU enumeration (F). Significant differences are denoted with ***p-value < 0.001.

Fig 5. Effects of PIA/PNAG on lung infections in C57BL/6 mice.

Mice were challenged with S. aureus SA113 or SA113Δica by intratracheal injection and were sacrificed three days after. (A) Lungs were removed from infected mice and photographed. Arrowheads indicated abscesses formed on SA113-infected lungs (a, c). (B) Lung tissues were fixed, embedded in paraffin, thin-sectioned and stained with H&E. The boxed areas in panels a and b are magnified in panels c and d. The images shown are representative of each group (n = 5 per group). (C) The number of bacteria recovered from the lung tissues of the infected mice was determined (n = 19 per group). The median values are indicated with horizontal lines. Significant differences are denoted with ***p-value < 0.001.