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Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae

Wu, Siva and Baum, Marc M. and Kerwin, James and Guerrero, Debbie and Webster, Simon and Schaudinn, Christoph and VanderVelde, David and Webster, Paul (2014) Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae. Pathogens and Disease, 72 (3). pp. 143-160. ISSN 2049-632X. PMCID PMC4262604.

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Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

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Additional Information:© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. Received 30 April 2014; revised 7 June 2014; accepted 11 June 2014. Final version published online 10 July 2014. Article first published online: 10 Jul 2014. The authors are especially grateful to financial support provided by their respective organizations, and the generosity of the Ahmanson Foundation for their support in equipping the Imaging Center at the House Research Institute. Rapid freezing and freeze substitution was carried out using equipment purchased with funds from the National Science Foundation (NSF #0722354). The study was also supported in part by The Fritz Burns Foundation, The Hope for Hearing Foundation, the Deafness Research Foundation, the Hearst Foundation, and the Capita Foundation. A grant from the NIDCD (5 P-30 DC006276-03) supported the Ahmanson Imaging Core where a substantial part of this work was performed. Prof. T. F. Murphy, University at Buffalo, State University of New York provided polyclonal antibodies to the OMP P2 protein. Dr. Gabriel B. Gugiu of the Mass Spectrometry and Proteomics Core Facility at the Beckman Research Institute of the City of Hope performed the proteomic analysis of protein preparations from the 6.5-h, 12-h, 24-h, and 96-h filters after biofilm removal. Tim Gallaher and Richard Johnson provided expert bioinformatics assistance and instruction. Tyler Woolsey and Nandini Girish assisted with early microscopy and bacterial culturing (NG: immunolabeling, TW viable bacteria and biomass estimates). The authors have stated no conflict of interest.
Funding AgencyGrant Number
Fritz Burns FoundationUNSPECIFIED
Hope for Hearing FoundationUNSPECIFIED
Deafness Research FoundationUNSPECIFIED
Hearst FoundationUNSPECIFIED
Capita FoundationUNSPECIFIED
NIDCD5 P-30 DC006276-03
Subject Keywords:nuclear magnetic resonance; electron microscopy; proteomics; immunocytochemistry; diagnostics
Issue or Number:3
PubMed Central ID:PMC4262604
Record Number:CaltechAUTHORS:20150112-113032020
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Official Citation:Wu, S., Baum, M. M., Kerwin, J., Guerrero, D., Webster, S., Schaudinn, C., VanderVelde, D. and Webster, P. (2014), Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae. Pathogens and Disease, 72: 143–160. doi: 10.1111/2049-632X.12195
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53574
Deposited By: Ruth Sustaita
Deposited On:12 Jan 2015 22:48
Last Modified:03 Oct 2019 07:50

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