Research Description

Microbial Biofilm Formation: From Molecular Mechanisms to Therapeutics

Molecular Mechanisms

A microbial biofilm is a surface attached community of microbial cells encased in a self-produced polymeric matrix. Biofilms grow on any moist biotic or abiotic surface, and provide a number of environmental advantages such as protecting microbes from the host immune response, and conferring tolerance to antibiotics and detergents. Established biofilms are extremely difficult to eradiate and cost the health care systems in the US and Canada upwards of $25 billion annually to treat. They are responsible for greater than 65-80% of all chronic bacterial infections.

Using a multidisciplinary approach, which combines in vivo microbiological studies with structural biology, biophysical and biochemical techniques, we want to understand at the molecular level how two critical fundamental processes occur.

• How are exopolysaccharides – a major component of the biofilm matrix – synthesized, chemically modified, and exported from the cell; and

• What is required for the assembly and function of type IV pili – key virulence factors used by Pseudomonas aeruginosa to establish infection and for biofilm formation.

Therapeutics

Our research on the biosynthetic machinery of various exopolysaccharides has led to the identification of several glycoside hydrolases that are capable of both preventing biofilm formation and degrading preformed biofilms. These enzymes are effective at low nano-molar concentration and are being developed as combination therapies for the treatment of a range of bacterial and fungal infections, and as disinfectants and coatings for cleaning and protecting surfaces.

In addition, as modification of exopolysaccharides by the removal or addition of acetyl groups is crucial for the ability of many pathogenic organisms to initiate and maintain infection, we are employing high-throughput screening techniques and structure based drug design methods to identify small molecules that modulate the de-N-acetylation state of several different polymers involved in biofilm formation.

Exopolysaccharide polymerization, modification and export.

Micrograph showing surface attached biofilm

Exopolysaccharides are a major component of many microbial biofilms. These long sugar polymers are often found both associated with the bacterial or fungal cell surface and in a secreted form. Exopolysaccharide biosynthesis is a multistep process that requires the polymerization of nucleotide-sugar precursors, and transport across either the cytoplasmic or plasma membrane. In the case of gram-negative bacteria the polymer also needs to transverse the periplasm and be exported from the cell. The properties of the polymer are frequently modulated by chemically modification, which can occur either in the periplasm or extracellular space.

Ongoing projects seek to determine the molecular mechanisms involved in the biosynthesis of:
• alginate;
• Pel polysaccharide;
• Psl polysaccharide;
• poly β-1,6-N-acetyl-D-glucosamine (PNAG); and
• galactosaminogalactan (GAG).

Assembly of Pseudomonas aeruginosa Type IV pili

Micrograph showing lack of surface adherence in a P. aeruginosa pilA mutant.

Type IV pili (T4P) are strong flexible filaments that mediate attachment to both living and artificial surfaces. They are also involved in bacteriophage adsorption, DNA uptake, biofilm initiation and development, and “twitching motility”, a unique form of surface-associated movement whereby the bacteria pull themselves rapidly towards or along a surface by retracting their T4P. Bacteria lacking T4P cannot adhere to surfaces and are therefore avirulent.

Regulation and assembly of the T4P is a complex process involving over 50 proteins. We are interested in understanding how a subset of the proteins in this molecular machine interact with each other to assembly, extend and retract the pilus.

Publications

View all publications on PubMed

Structure of the Pseudomonas aeruginosa Type IVa Pilus Secretin at 7.4 Å.
Koo J, Lamers RP, Rubinstein JL, Burrows LL, Howell PL.
Structure. 2016 Oct 4;24(10):1778-1787. doi: 10.1016/j.str.2016.08.007.  Read

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.
Perrin Baker, Preston J. Hill, Brendan D. Snarr, Noor Alnabelseya, Matthew J. Pestrak, Mark J. Lee, Laura K. Jennings, John Tam, Roman A. Melnyk, Matthew R. Parsek, Donald C. Sheppard, Daniel J. Wozniak andP. Lynne Howell.
Science Advances 20 May 2016: Vol. 2, no. 5, e1501632. DOI: 10.1126/sciadv.1501632  Read

Modification and periplasmic translocation of the biofilm exopolysaccharide Poly-β-1,6-N-acetyl-D-glucosamine.
J. Little, G. Li, C. Ing, B.R. DiFrancesco, N. Bamford, H. Robinson, M. Nitz, R. Pomès, P. L. Howell.
Proc. Nat. Acad. Sci., July 3, 2014.  Read

P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. P. aeruginosa SCNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.
Baker P, Ricer T, Moynihan PJ, Kitova EN, Walvoort MT, Little DJ, Whitney JC, Dawson K, Weadge JT, Robinson H, Ohman DE, Codée JD, Klassen JS, Clarke AJ, Howell PL.
PLoS Pathog. 2014 10(8):e1004334.  Read

Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.
Tammam, P. Sundaram, J. Koo, L. Sampaleanu, M. Ayers, A. Chong, J. Forman-Kay, L.L. Burrows, P. L. Howell.
Mol. Micro. 82(6), 1496-1514, 2011.  Read