Antibody-guided identification of Achromobacter xylosoxidans protein antigens in cystic fibrosis

Abstract

Persistent bacterial airway infection is a hallmark feature of cystic fibrosis (CF). Achromobacter spp. are gram-negative rods that can cause persistent airway infection in people with CF (pwCF), but the knowledge of host immune responses to these bacteria is limited. The aim of this study was to investigate if patients develop antibodies against Achromobacter xylosoxidans, the most common Achromobacter species, and to identify the bacterial antigens that induce specific IgG responses. Seven serum samples from pwCF with Achromobacter infection were screened for antibodies against bacteria in an ELISA coated with A. xylosoxidans, A. insuavis, or Pseudomonas aeruginosa. Sera from pwCF with or without P. aeruginosa infection (n = 22 and 20, respectively) and healthy donors (n = 4) were included for comparison. Serum with high titers to A. xylosoxidans was selected for affinity purification of bacterial antigens using serum IgGs bound to protein G beads. The resulting IgG-antigen complexes were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selected antigens of interest were produced in recombinant form and used in an ELISA to confirm the results. Four of the seven patients with Achromobacter infection had serum antibodies against Achromobacter. Using patient serum-IgG for affinity purification of A. xylosoxidans proteins, we identified eight antigens. Three of these, which were not targeted by anti-P. aeruginosa antibodies, were expressed recombinantly for further validation: dihydrolipoyl dehydrogenase (DLD), type I secretion C-terminal target domain-containing protein, and domain of uncharacterized function 336 (DUF336). While specific IgG against all three recombinant antigens was confirmed in the patient serum with high titers against Achromobacter, DLD and DUF336 showed the least binding to serum IgG from pwCF without Achromobacter spp. infection. Using serum IgG affinity purification in combination with LC-MS/MS and confirming the results using ELISA against recombinant proteins, we have identified bacterial antigens from A. xylosoxidans.IMPORTANCEAchromobacter species are opportunistic pathogens that can cause airway infections in people with cystic fibrosis. In this patient population, persistent Achromobacter infection is associated with low lung function, but the knowledge about bacterial interactions with the host is currently limited. In this study, we identify protein antigens that induce specific antibody responses in the host. The identified antigens may potentially be useful in serological assays, serving as a complement to culturing methods for the diagnosis and surveillance of Achromobacter infection.

Keywords: Achromobacter xylosoxidans; IgG antigens; cystic fibrosis; systems antigenomics.

Fig 1

Antibody responses of donor serum against whole bacteria and Achromobacter proteins. Serum samples from pwCF and healthy controls were analyzed for IgG titers against Achromobacter xylosoxidans (A), A. insuavis (B), and Pseudomonas aeruginosa (C) by direct ELISA using plates coated with whole bacteria. Bars represent the mean OD₄₂₀ value per group. Each dot is the average value of three technical replicates. Serum from individuals infected with both Achromobacter and P. aeruginosa (gray triangles) was included in the Achromobacter group when screening for those antibodies (A, B), and vice versa for P. aeruginosa (C). The dotted lines represent the average of the healthy control group +3 SD, which was considered the cutoff value for positive titers. Groups were compared using the Mann-Whitney test. (D, E) Western blot assay of serum IgG binding to A. xylosoxidans proteins. Secreted proteins (D) and trypsinated surface fractions (E) were separated on SDS-PAGE followed by Western blotting using CF_Achro and healthy control serum.

Fig 2

Identification of antigens in Achromobacter xylosoxidans. (A) Brief description of the systems antigenomics workflow used to identify antigenic proteins. (B) Enrichment analysis of bacterial proteins associated with serum from pwCF infected with Achromobacter compared to omalizumab, two-tailed t-test, FDR 0.05. Panel A created with BioRender.com.

Fig 3

Specificity of Achromobacter antigens compared to anti-P. aeruginosa IgG. (A) Enrichment analysis of bacterial proteins associated with CF_Achro serum compared to CF_PsA serum, two-tailed t-test, FDR 0.05. (B) LFQ intensities of the Achromobacter antigens DUF336, T1S-DCP, DLD, and ABC-TSBP in pulldowns using CF_Achro and CF_PsA serum are compared using the Mann-Whitney U-test. n = 6, ** = P < 0.01. (C) Abundance of significant Achromobacter antigens in the total bacterial protein fraction. LFQ intensities (log10) of all 935 identified Achromobacter proteins in the pooled bacterial protein fraction are plotted in descending order. The four CF_Achro-associated bacterial antigens are marked with red triangles. (D) ELISA plates were coated with recombinantly expressed antigens, and IgG titers were tested in the same sera used in the IgG pulldown assay.

Fig 4

Serum IgG binding to recombinantly expressed A. xylosoxidans antigens. ELISA plates were coated with recombinantly expressed dihydrolipoyl dehydrogenase (A), domain of unknown function (DUF336) (B), and type I secretion C-terminal target domain-containing protein (C), and IgG titers in sera from pwCF and healthy controls were measured by direct ELISA. Each dot represents the average value of three technical replicates, and bars represent the mean value. The dotted lines represent the average of the healthy control group + 3 SD, which was considered the cutoff for positivity.