Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

Siva Wu,David Vandervelde,James Kerwin,Paul Webster,Debbie Guerrero,Christoph Schaudinn,Simon Webster,Marc M Baum

doi:10.1111/2049-632x.12195

Abstract

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.

Highlights

Biofilms are aggregations of sessile bacteria cells embedded in an extracellular matrix, one function of which is to attach bacteria to one another and to biotic and abiotic substrates (Stoodley et al, 2002)
We have previously examined biofilms formed by non-typeable Haemophilus influenzae (NTHi) and shown the extracellular matrix (ECM) to contain a large number of bacterial proteins (Gallaher et al, 2006), including a high molecular weight protein (HMW1), a known adhesin with a role in bacterial adhesion, in an extracellular location (Webster et al, 2006)
Other studies of biofilm ECM has revealed it to be composed of varying amounts of polysaccharide, protein, nucleic acid and lipid, the actual proportions dependent on many different factors, two of which being the species of biofilm-forming bacteria and the developmental stage of the biofilm (Oosthuizen et al, 2002, Sauer et al, 2002, Sauer, 2003, Fux et al, 2005, Cao et al, 2011, Mueller et al, 2011)

Summary

Introduction

Biofilms are aggregations of sessile bacteria cells embedded in an extracellular matrix, one function of which is to attach bacteria to one another and to biotic and abiotic substrates (Stoodley et al, 2002). Proteins within the ECM may assist in forming the complex structures reported previously within biofilms (Schaudinn et al, 2006, Baum et al, 2009, Pelzer et al, 2012, Schaudinn et al, 2014). Extracellular proteins can be firmly bound to the cell surface, associated with the ECM, or be freely diffusing throughout the biofilm matrix (Hoffman & Decho, 1999). The presence and function of extracellular proteins has been reported for a wide range of Gram-positive and Gram-negative bacteria, both in cultures of free-swimming (planktonic) cells and in biofilms (Wingender et al, 1999).

Methods

Results

Conclusion