Serologic reactivity to the emerging pathogen Granulibacter bethesdensis

Abstract

Background: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown.

Methods: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA).

Results: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH.

Conclusions: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.

Figure 1.

Immunoblots performed using serum from the 4 infected patients with chronic granulomatous disease against pooled Granulibacter extracts. The stars on the left of the blots denote the 11 bands subsequently used to determine seropositivity. A, Sera from patients 1 and 3 and negative control sera were used at a 1:250 dilution. Serum from patient 4 was used at 1:1000 dilution. Serum from patient 2 was used at a 1:5000 dilution. B, Titration of serum from patient 2.

Figure 2.

Immunoblots comparing Granulibacter organisms to other Acetobacteraceae species. Serum from patient 1 immunoblotted against pooled whole bacterial extracts of Granulibacter bethesdensis isolates (National Institutes of Health [NIH] strains NIH1.1, NIH 2.1, NIH3.1, and NIH4.1), along with similar extracts from 5 closely related Acetobacteraceae species. Each lane contains 2 μg total protein. Patient serum was used at a 1:250 dilution.

Figure 3.

Human seropositivity determined by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G against Granulibacter bethesdensis methanol dehydrogenase (MDH). A, Sera from 5 culture-confirmed cases and 7 healthy donors were titered. For patients, a 4-parameter nonlinear regression analysis (plotted as solid lines) was performed to model the titer curves based on 73–129 data points per donor (symbols at mean ± SD). For the healthy donors, the mean and range are plotted. B, ELISA screening of human sera for antibodies against MDH at a dilution factor of 1024 (greatest ratio of patients with culture-confirmed infection to healthy donors). Individual absorbances are shown for 79 healthy donors (+), 153 patients with chronic granulomatous disease (o), and 5 patients with culture-confirmed infection (•). For the cases, the mean ± SD is shown, and the black dotted line indicates the cutoff used for determining seropositivity (ie, 3 SDs below the culture-confirmed sample mean).

Figure 4.

Chronic granulomatous disease mouse seropositivity determined by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G against Granulibacter bethesdensis methanol dehydrogenase. p47^phox−/− mice were infected with G. bethesdensis strains NIH1.1, NIH2.2, NIH3.1, and NIH4.1 (2 mice per strain), as described in Methods, and serum was collected at the indicated times and analyzed by ELISA. High-titer antibody was present by 5 weeks after infection and remained positive >200 days later.

Figure 5.

Methanol dehydrogenase antibodies in culture-confirmed Granulibacter bethesdensis infections over time. A, Enzyme-linked immunosorbent assay (ELISA) data for patient 5 over a 20-year period prior to diagnosis of G. bethesdensis infection in May 2009. B, ELISA data for patient 3 over an approximately 10-year period prior to diagnosis in January 2006. The onset of the patient's clinical illness ascribed to Granulibacter infection is shown.