BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii

Abstract

Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P < 0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii.

Fig 1. Autoagglutination of Bordetella holmesii isolate BH7.

(A) The clinical isolates BH2, BH6, BH7, and BH8 were suspended in casamino acid solution and incubated for 4 h at 36°C under static conditions. (B) Time course analysis of the turbidity (OD₆₅₀) of the bacterial suspensions. Data are presented as means ± standard deviations of results obtained from 3 separate experiments performed in triplicate. (C) Microscopic analysis of the morphology of the bacterial suspensions. The suspensions were incubated for 4 h at 36°C on glass slides, and observed by phase-contrast microscopy (objective magnification, 100×).

Fig 2. Lack of biofilm formation by Bordetella holmesii isolate BH7.

The clinical isolates BH2 and BH7 were cultured statically on vertically submerged glass slides in mSS broth. After 5 and 72 h, the cell adherence and the biofilm growth at the air-liquid interface were visualized by SEM. Scale bars, 1 μm

Fig 3. Lack of BipA production in Bordetella holmesii isolate BH7.

(A) Comparison of the protein expression profiles of the clinical isolates BH2, BH6, BH7, and BH8. Total protein (20 μg) was subjected to SDS-PAGE followed by CBB staining (left panel). The dashed-boxed area is enlarged (right panel). The arrows indicate the bands that were identified as BipA by nano-LC-MS/MS analysis. (B) Analysis of BipA production in B. holmesii isolates. Total protein (2 μg) was subjected to immunoblot analysis with anti-BipA antisera. (C) Comparison of BipA sequences among the isolates. The bipA gene of BH7 encodes a deletion of a guanine (G) at nucleotide position 2039 (gray box). The deletion mutation generates a premature stop codon at nucleotide position 2066. The translation stop site (amino acid 689) is shown in the deduced amino acid sequence (BH7, gray box).

Fig 4. Autoagglutination of BipA mutant.

(A) BipA mutant BH2Sm^r-ΔBipA and its parental strain, BH2Sm^r, were suspended in casamino acid solution and incubated for 4 h at 36°C under static conditions. (B) Time course analysis of the turbidity (OD₆₅₀) of the bacterial suspensions. Data are presented as the means ± standard deviations of results obtained from 3 separate experiments performed in triplicate. (C) Microscopic analysis of the morphology of the bacterial suspensions. The suspensions were incubated for 4 h at 36°C on glass slides, and observed by phase-contrast microscopy (objective magnification, 100×).

Fig 5. Lack of biofilm formation by BipA mutant.

(A) BipA mutant strain BH2Sm^r-ΔBipA and its parental strain, BH2Sm^r, were cultured in mSS broth for 72 h under static conditions, and biofilm-forming bacteria were stained with crystal violet. (B) Time course analysis of the levels of biofilm biomass production. The biofilm-associated crystal violet was solubilized and quantified after 24, 48, and 72 h incubation. Data are presented as means ± standard deviations of the results obtained from 3 separate experiments performed in triplicate. Statistical significance was determined using Student’s t-test. (C) SEM analysis of biofilm formation. BH2Sm^r-ΔBipA and BH2Sm^r were cultured statically on vertically submerged glass slides in mSS broth. After 5 and 72 h, cell adherence and biofilm growth at the air-liquid interface were visualized by SEM. Scale bars, 1 μm

Fig 6. Initial colonization of BipA mutant in murine lungs.

Groups of three BALB/c mice were intranasally infected with the BipA mutant BH2Sm^r-ΔBipA or its parental strain BH2Sm^r. The colonization levels were assessed after 3, 6, and 24 h. Each symbol represents one mouse, and horizontal lines represent the mean number of CFU recovered. Statistical significance was determined using Student’s t-test. The dashed line indicates the lower limit of detection (100 CFU/lung). The lung CFUs in two mice infected with BH2Sm^r-ΔBipA were below the limit of detection at 24 h post-infection.

Fig 7. Lack of BipA protein and transcript expression in Bordetella holmesii ATCC strains.

(A) B. holmesii BH2, ATCC51541, and ATCC700053 were cultured for 4 days on BG agar. Total protein (2 μg) was subjected to immunoblot analysis with anti-BipA antisera. (B) After culturing for 3 days on BG agar, total RNA was isolated, reverse transcribed into cDNA, and then analyzed by qRT-PCR analysis. Relative bipA transcript levels were calculated using the ΔΔCt method and were normalized to those of recA. The recA transcript was used as an internal control for each sample. Data are presented as fold-changes in expression compared to those observed in BH2. The means ± standard deviations of results obtained from 3 separate experiments are shown.