D-alanine modification of a protease-susceptible outer membrane component by the Bordetella pertussis dra locus promotes resistance to antimicrobial peptides and polymorphonuclear leukocyte-mediated killing

Abstract

Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dlt operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of d-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens.

Fig 1

Comparison of dra and dlt loci in Bordetella and other Gram-positive and Gram-negative bacteria. Shown are the organizations of the dra and related operons in different bacterial species. The species shown in the figure (in order of relatedness to the B. pertussis dra locus) and used in sequence comparisons are B. pertussis strain Tohama I, B. bronchiseptica strain RB50, B. parapertussis strain 12822, Achromobacter xylosoxidans A8, B. avium 197N, Acidovorax avenae subsp. avenae ATCC 19860, Delftia acidovorans SPH-1, Dickeya dadantii Ech586, Enterobacter cloacae SCF1, Pectobacterium wasabiae WPP163, Bacillus cereus ATCC 14579, Clostridium difficile 630, and Vibrio cholerae ATCC 39315. Homologous genes use the same patterns across species. The black arrow for V. cholerae represents a lipid A transacylase. The region of the dra operon deleted in the Δdra strain is shown at the top.

Fig 2

Deletion of the dra locus has no effect on cell morphology of B. pertussis. Phase-contrast (A) or electron (B) micrographs of bacterial cells after 72 h of growth are shown.

Fig 3

The dra locus promotes resistance to antimicrobial peptides. The susceptibilities of the WT, Δdra, Δdra^vec, and Δdra^comp strains to various antimicrobial peptides were assessed. Bacteria (1 × 10⁶ CFU) were incubated in 10 mM sodium phosphate buffer with the indicated concentrations of AMPs for 2 h. Bacterial numbers were determined by plate counts on BG agar. Each data point represents the mean and standard deviation of triplicates from one of three independent experiments. *, P < 0.05 compared with the untreated control based on Student's t test. The number 0 on top of the bars in some panels indicates the lack of any detectable bacterial growth. PmB, polymyxin B.

Fig 4

Sensitivities of the WT and the Δdra strains to LL-37 under physiologically relevant conditions. Bacteria (1 × 10⁶ CFU) were incubated in either DMEM (A) or SS medium (B) with the indicated concentrations of LL-37 for 2 h. Bacterial numbers were determined by plate counts on BG agar. Data represent the mean and standard deviation of triplicates from one of three independent experiments. *, P < 0.05.

Fig 5

Interaction of B. pertussis with positively charged protein and peptides. The B. pertussis strains were treated with cytochrome c (A), fluorescein-labeled LL-37 (B), or FITC-labeled poly-l-lysine (C) and washed twice with PBS, and then the cells were resuspended in PBS. The amount of peptide or protein associated with each B. pertussis cell suspension was measured by determination of the absorbance or fluorescence, as described in Materials and Methods. Data are expressed as the percentage of the signal from the B. pertussis suspension compared to the signal from the input protein or peptide. **, P < 0.01.

Fig 6

The dra locus promotes the resistance of B. pertussis to killing by human PMNs. B. pertussis WT, Δdra, Δdra^vec, and Δdra^comp strains were incubated separately with PMNs at multiplicities of infection of 1:1 and 1:5 for 1.5 h. Bacterial survival was calculated by the number of CFU recovered after incubation with PMNs divided by the number of CFU recovered from untreated controls. Each data point represents the mean and standard deviation of triplicate samples from one of three independent experiments. **, P < 0.01.

Fig 7

Dendrogram illustrating the relatedness of DraA homologues from various bacterial species. A rooted dendrogram of homologous DltA/DraA/AlmE protein sequences from the various species described in the legend to Fig. 1 was generated by the Clustal W program and Phylip's Drawtree program using the Biology Workbench at the San Diego Supercomputing Center. Shown in parentheses are the amino acid identities after pairwise alignments (at uniprot.org) of B. pertussis DraA and the corresponding homologue from each species.