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Acid phosphatase activity as a measure of Haemophilus influenzae adherence to mucin
D L Chance1, T J Reilly, A L Smith
1Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, 65212, USA. chanced@health.missouri.edu
Abstract:
Haemophilus influenzae is an important respiratory tract pathogen. Toward understanding the progression of H. influenzae from commensal to pathogen, we need to understand the steps of colonization and infection, processes which must involve overcoming the normal host mucociliary clearance mechanism. A reliable method for the screening and quantitation of mucin-H. influenzae binding to allow for the assessment of the physiological variables significant to H. influenzae-mucin interactions in the normal and diseased conditions, will provide insight on how to intervene to prevent, inhibit, or treat infection. The current methods for enumeration of mucin-bound H. influenzae are labor intensive and rely on viable organisms. In this report, we present a new detection method, which reduces the number of variables, processing steps, and time involved, providing an economical, rapid, and reliable means to screen for and quantitate mucin-bound H. influenzae. Organisms are applied to mucin-coated microtiter wells for a set time; nonadherent organisms are removed with gentle rinses; wells are incubated with the phosphomonoesterase substrate p-nitrophenyl phosphate; and the absorbance, reflecting phosphatase activity of the mucin-bound organisms, is read at 410 nm in a microtiter plate reader against enzymatic activity calibration curves. All nonencapsulated and encapsulated H. influenzae tested exhibited significant acid phosphate activity within 20 min, which provided linear relationships with the numbers of organisms present. H. influenzae mucin binding characteristics obtained by this method were generally comparable to published data, and ranged from 10(3) to 10(6) organisms per well, depending on both strain of organism and type of mucin employed. This convenient, rapid and economical mucin adherence assay, will enable more extensive and comprehensive studies of the interactions of H. influenzae adhesins and specific ligands on mucin macromolecules, as well as the nonspecific means by which mucins function in preventing bacterial infection.
Insights
A new assay quantifies Haemophilus influenzae binding to mucins, aiding understanding of respiratory infections. This rapid, economical method helps study bacterial adherence and develop new treatments.
Area of Science:
- Microbiology
- Infectious Diseases
- Biochemistry
Background:
- Haemophilus influenzae is a significant respiratory pathogen.
- Understanding its transition from commensal to pathogen requires studying colonization and infection mechanisms.
- Overcoming host mucociliary clearance is crucial for H. influenzae survival.
Purpose of the Study:
- To develop a reliable, rapid, and economical method for screening and quantifying mucin-H. influenzae binding.
- To assess physiological variables influencing H. influenzae-mucin interactions.
- To gain insights for preventing, inhibiting, or treating H. influenzae infections.
Main Methods:
- A novel assay using mucin-coated microtiter wells.
- Incubation of H. influenzae with mucin-coated wells, followed by rinsing.
- Measurement of bound organism phosphatase activity using p-nitrophenyl phosphate substrate.
- Quantification via absorbance readings at 410 nm against calibration curves.
Main Results:
- The assay demonstrated significant acid phosphatase activity in both encapsulated and nonencapsulated H. influenzae strains within 20 minutes.
- Linear relationships were observed between enzyme activity and the number of bound organisms.
- Mucin adherence characteristics obtained were comparable to published data, ranging from 10^3 to 10^6 organisms per well.
Conclusions:
- The developed assay is a convenient, rapid, and economical tool for studying H. influenzae mucin adherence.
- It enables comprehensive research into H. influenzae adhesins, mucin ligands, and mucin's role in preventing infection.
- This method facilitates a deeper understanding of H. influenzae pathogenesis and potential therapeutic interventions.