The twin-arginine translocation system is vital for cell adhesion and uptake of iron in the cystic fibrosis pathogen Achromobacter xylosoxidans

Abstract

Achromobacter xylosoxidans is an emerging pathogen that causes airway infections in patients with cystic fibrosis. Knowledge of virulence factors and protein secretion systems in this bacterium is limited. Twin arginine translocation (Tat) is a protein secretion system that transports folded proteins across the inner cell membranes of gram-negative bacteria. Tat has been shown to be important for virulence and cellular processes in many different bacterial species. This study aimed to investigate the role of Tat in iron metabolism and host cell adhesion in A. xylosoxidans. Putative Tat substrates in A. xylosoxidans were identified using the TatFind, TatP, and PRED-Tat prediction tools. An isogenic tatC deletion mutant (ΔtatC) was generated and phenotypically characterized. The wild-type and ΔtatC A. xylosoxidans were fractionated into cytosolic, membrane, and periplasmic fractions, and the expressed proteome of the different fractions was analysed using liquid chromatography-mass spectrometry (LC-MS/MS). A total of 128 putative Tat substrates were identified in the A. xylosoxidans proteome. The ΔtatC mutant showed attenuated host cell adhesion, growth rate, and iron acquisition. Twenty predicted Tat substrates were identified as expressed proteins in the periplasmic compartment, nine of which were associated with the wild type. The data indicate that Tat secretion is important for iron acquisition and host cell adhesion in A. xylosoxidans.

Keywords: Twin-arginine translocation; achromobacter xylosoxidans; bacterial protein secretion; cystic fibrosis; infection; iron metabolism.

Figure 1.

VENN diagram illustrating the results from three Tat substrate prediction algorithms; PRED-Tat (red), TatFind (green) and TatP (blue). The numbers inside the circles represent the number of putative Tat substrates identified by one, two, or three of the prediction tools.

Figure 2.

Percentage of remaining bacteria adhered to BEAS-2B lung epithelial cells after incubation and washing steps. Wild-type (WT) A. xylosoxidans adheres significantly better to epithelial cells than the ΔtatC mutant. The box plots illustrate the mean value of 3 repeats and the error bars the standard deviation. The dots represent individual values. Statistical comparison was performed using Student’s T-test. * = p < 0.05.

Figure 3.

Growth rates of wild-type A. xylosoxidans and the ΔtatC mutant. The wild type and isogenic ΔtatC mutant were grown in minimal medium supplemented with different concentrations of iron (0, 10 or 20 uM, respectively). Optical density at 600 nm was monitored continuously to measure growth, and the doubling times were calculated during the exponential growth phase. The figure shows the mean growth rate expressed as doubling times (min) for the wild type (WT; black bars) and ΔtatC mutant (ΔtatC; white bars). Error bars represent SD. Statistical comparisons were made with Student’s T-test. * = p < 0.05, ns = non-significant.

Figure 4.

Expression of predicted Tat substrates in the periplasm of A. xylosoxidans during normal and iron limited conditions. The wild type (WT) and ΔtatC mutant were grown to mid-exponential phase in the absence or presence of iron. The cells were then separated into cytosolic, membrane and periplasmic fractions and the protein contents of the fractions were analysed with LC-MS/MS. The figure shows log2-transformed sum-normalized intensities for the putative Tat substrates expressed in the periplasmic fractions of the wild type and the ΔtatC mutant from three independent experiments (Supplementary table S3). The box plots show mean values and the error bars show standard deviation. The dots represent individual values. Comparisons between groups were made with Student’s T-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 5.

Expression of the predicted Tat substrate ferripyoverdine receptor. The wild type (WT) and the ΔtatC mutant were grown to mid-exponential phase in the absence or presence of iron. The cells were then fractionated and the protein contents of the periplasmic fractions were analysed with LC-MS/MS. The figure shows log2-transformed sum-normalized intensities for the putative Tat substrate ferripyoverdine receptor in the wild type and ΔtatC mutant from three independent experiments. The box plot shows the mean value and the error bars the standard deviation. The dots represent individual values. Comparisons between groups were made with Student’s T-test. ** = p < 0.01.