The deficiency of poly-β-1,6-N-acetyl-glucosamine deacetylase trigger A. baumannii to convert to biofilm-independent colistin-tolerant cells

Abstract

Acinetobacter baumannii is a nosocomial pathogen that can be resistant to antibiotics by rapidly modulating its anti-drug mechanisms. The multidrug-resistant A. baumannii has been considered one of the most threatening pathogens to our society. Biofilm formation and persistent cells within the biofilm matrix are recognized as intractable problems, especially in hospital-acquired infections. Poly-β-1,6-N-acetyl-glucosamine (PNAG) is one of the important building blocks in A. baumannii's biofilm. Here, we discover a protein phosphoryl-regulation on PNAG deacetylase, AbPgaB1, in which residue Ser411 was phosphorylated. The phosphoryl-regulation on AbPgaB1 modulates the product turnover rate in which deacetylated PNAG is produced and reflected in biofilm production. We further uncovered the PgaB deficient A. baumannii strain shows the lowest level of biofilm production but has a high minimal inhibition concentration to antibiotic colistin and tetracycline. Based on bactericidal post-antibiotic effects and time-dependent killing assays with antibacterial drugs, we claim that the PgaB-deficient A. baumannii converts to colistin-tolerant cells. This study utilizes a biofilm-independent colistin-tolerant model of A. baumannii to further investigate its characteristics and mechanisms to better understand clinical outcomes.

Figure 1

Schematic illustration of two PNAG synthesis operons in A. baumannii. (a) The coding genes A1S-0938 to A1S-0940 and A1S-2162 to A1S-2159 were annotated as PNAG synthesis operons in A. baumannii. PgaC (brown) and PgaD (orange) Protein complexes were located on the cytoplasmic membrane to polymerize N-acetylglucosamine. PgaB is a PNAG deacetylase that anchors on the outer membrane and is located at the periplasm. Outer membrane transporter protein PgaA contributed to the transport of partially deacetylated PNAG as an exopolysaccharide. (b) Phylogenetic analysis of polysaccharide deacetylases in CE4 family of CAZy database from bacteria based on Neighbor Joining clustering method.

Figure 2

The identified phosphorylated sites on AbPgaB1 marked in secondary structure and tertiary structure. (a) The secondary structure of AbPgaB1 was predicted by Jpred4. Alpha helix and beta sheet structures were marked in purple and yellow, respectively. Identified p-sites were marked “p” (red) on AbPgaB1 sequences. (b) Tetrasaccharide of PNAG, 4-NAG, which the main chain showed in sticks (green) were docked into the AbPgaB1 modeled structure (cartoon, predicted based on template PDB: 4P7R²⁶ by SWISS-MODEL) to display the location of phosphorylated residues (yellow) within the 3D structure. The modeled structure was edited using PyMOL. (c) MS/MS spectra of p-peptide “⁴⁰⁷TDPVSKDLVVTEQAK⁴²¹” on AbPgaB1. Each peak reveals the m/z of fragments after tandem mass spectrometry separation and is processed by MaxQuant. The N-terminal b-ion and C-terminal y-ion fragments were highlighted in blue and red, respectively.

Figure 3

Observation and quantification of biofilm production in A. baumannii ATCC15151 and the AbpgaB1-mediated mutant strains. (a) Observation of biofilm formation in Ab15151 and its derivatives via SEM. The upper panel showed the view with 7,500 × magnification and the lower panel showed with 10,000 × magnification. The bar indicated a scale of 1 µm. (b) Biofilm was quantified by crystal violet staining and determined the absorbance at 595 nm. The OD₅₉₅ values of Ab15151 were defined as 100% to calculate the relative biofilm amounts from its derivative mutant strains. Each data point was averaged from at least 6 repeats. *Indicated a significant difference from Ab15151 whose p-value of the t-test was less than 0.001.

Figure 4

Time-dependent post-antibiotic effects of colistin and tetracycline to A. baumannii strains in this study. The overnight cultures were diluted to OD₆₀₀ 0.1 for colistin or 0.01 for tetracycline administration. After a 1 h treatment with colistin (a) or tetracycline (c), the cultures were tenfold serially diluted to spot on the LB agar plate. With the administration of 32 µg/mL colistin (b) or 8 µg/mL tetracycline (d) within 4 h, all tested strains were tenfold-diluted and incubated overnight for evaluating their survival rate.

Figure 5

Time-dependent killing assays of WT and PgaB-mediated A. baumannii mutant strains demonstrated its colistin tolerance. In this study, the overnight cultures of A. baumannii strains were diluted to OD₆₀₀ 0.05 for growth curve determination. The antibiotic colistin (red) and tetracycline (blue) were administrated with the final concentration of 16.0 µg/mL after 2 h incubation. During 27 h incubation, the optical density of each culture was determined at 600 nm by a microplate reader. The growth curve drawn in black was the condition without antibiotic administration as a control.