Final Report Summary - PSL (Intercellular signalling functions of bacterial biofilm extracellular matrix)
Biofilm is a community of microbial growth attached to a surface. Biofilm bacteria are encapsulated by extracellular polymeric substances, including polysaccharides. The overall function of these polysaccharides are to provide adhesive means for bacteria to aggregate and develop three dimensional ordered structures, but there are reports of antimicrobial barrier functions. In this project, further uncharacterised or unknown functions of the extracellular sugars were investigated mainly using opportunistic pathogen Pseudomonas aeruginosa as the model organism.
Work performed during the project:
Inspection of the social evolutionary outcome of PSL expression in P. aeruginosa biofilm
Genetic screen to attempt elucidating the signal transduction system of PSL signalling
Investigation of extracellular matrix signalling by other species than P. aeruginosa
The phenotypic effects of antibiotics tolerance by P. aeruginosa biofilm
Novel genetic regulatory cascade for P. aeruginosa PEL polysaccharides
Discovery of novel extracellular polysaccharides in respiratory pathogen Bordetella bronchiseptica
Results and conclusion summary:
• P. aeruginosa polysaccharide PSL and PEL play very different social evolutionary roles in biofilms.
o PSL appears to be shared goods, but do not follow a typical trend of public goods and cheating phenomenon.
o PEL appears to be private goods, and the mutants are rapidly outcompeted by PEL producers.
o PSL polysaccharides provides protection against antibiotics, and non-producers in the biofilm community can take some advantage.
o PEL provides no common protection against external antimicrobial killing.
• Micro-CT scanners can be a useful tool of 3D macroscopic imaging and substratum analyses for microbial growth on a semi-solid or solid surface.
o Semi-quantitative imaging and analyses are possible for metallic molecules, including known antimicrobial agents such as silver and gallium as the new generation anti-biofilm therapeutic drugs are promising but not well studied.
• Type IV pilus, traditionally characterised as surface motility organelle of P. aeruginosa, appears to be important for cellular post-adhesion organisation, but are not PSL receptors for signalling. However, as revealed by micro-CT scans, there may be direct interactions between PSL and Type IV that mediates surface motility.
• The main polysaccharide components of B. bronchiseptica and Staphylococcus aureus do not appear to affect biofilm development or growth in vitro. However, previously described polysaccharides from B. bronchiseptica were not identified in our most recent GC/MS analyses, and instead novel oligosaccharides were revealed.
• Although PEL and PSL polysaccharides are co-regulated in P. aeruginosa, less is known regarding the precise regulatory mechanisms of these polysaccharide expressions. RsmA was previously described as a post-transcriptional regulator of psl, and RsmA has been suspected of regulating pel, but no direct data supporting this is present in the literature. My work has revealed that RsmA is not a direct regulator of PEL, but two intermediate transcriptional regulators fall between the cascade of genetic events involving RsmA and pel. I have genetic and phenotypic evidence to support this, and further biochemical tests will need to be done to complete this finding.
Potential impact:
• Studies on social evolution of biofilm bacteria is a current hot topic in microbiology. The findings in this field are revolutionising our basic understanding of fundamental bacteriology to not treat these organisms as ‘stand alone entities’ but rather tackle our research strategies as a collective community of organisms, almost akin to tissues of higher organisms in order to comprehend the biology of microbes that typically grow as biofilm in their natural habitat. This includes pathogenic bacteria such as P. aeruginosa, which is posing significant damage to the medical field for their intrinsic capability of forming biofilm on innate hospital surfaces, medical devices, and on human tissues. Pathogenic biofilms generally exhibit higher capacity for negating antimicrobial therapies current in existence, and there is an immediate emergency need to re-think strategies for combating these infection. Part of this stems from our lack of biological understanding of bacteriology as community organismal studies that we are only recently taking steps towards describing.
• Novel strategies for combating biofilms will have wide societal impact, but in the biomedical field, CT scan-based approach of testing antimicrobials against microbial biofilms can add an additional use for understanding pathogenesis.
• An effective anti-biofilm strategy is to interfere with biofilm polysaccharide production. Further characterisation of the genetic and biochemical pathways will reveal novel drug targets that are not designed to kill bacteria, which increases the selective pressure for genetic resistance, but a more targeted approach non-lethal approach to deal with biofilm-forming species.
Work performed during the project:
Inspection of the social evolutionary outcome of PSL expression in P. aeruginosa biofilm
Genetic screen to attempt elucidating the signal transduction system of PSL signalling
Investigation of extracellular matrix signalling by other species than P. aeruginosa
The phenotypic effects of antibiotics tolerance by P. aeruginosa biofilm
Novel genetic regulatory cascade for P. aeruginosa PEL polysaccharides
Discovery of novel extracellular polysaccharides in respiratory pathogen Bordetella bronchiseptica
Results and conclusion summary:
• P. aeruginosa polysaccharide PSL and PEL play very different social evolutionary roles in biofilms.
o PSL appears to be shared goods, but do not follow a typical trend of public goods and cheating phenomenon.
o PEL appears to be private goods, and the mutants are rapidly outcompeted by PEL producers.
o PSL polysaccharides provides protection against antibiotics, and non-producers in the biofilm community can take some advantage.
o PEL provides no common protection against external antimicrobial killing.
• Micro-CT scanners can be a useful tool of 3D macroscopic imaging and substratum analyses for microbial growth on a semi-solid or solid surface.
o Semi-quantitative imaging and analyses are possible for metallic molecules, including known antimicrobial agents such as silver and gallium as the new generation anti-biofilm therapeutic drugs are promising but not well studied.
• Type IV pilus, traditionally characterised as surface motility organelle of P. aeruginosa, appears to be important for cellular post-adhesion organisation, but are not PSL receptors for signalling. However, as revealed by micro-CT scans, there may be direct interactions between PSL and Type IV that mediates surface motility.
• The main polysaccharide components of B. bronchiseptica and Staphylococcus aureus do not appear to affect biofilm development or growth in vitro. However, previously described polysaccharides from B. bronchiseptica were not identified in our most recent GC/MS analyses, and instead novel oligosaccharides were revealed.
• Although PEL and PSL polysaccharides are co-regulated in P. aeruginosa, less is known regarding the precise regulatory mechanisms of these polysaccharide expressions. RsmA was previously described as a post-transcriptional regulator of psl, and RsmA has been suspected of regulating pel, but no direct data supporting this is present in the literature. My work has revealed that RsmA is not a direct regulator of PEL, but two intermediate transcriptional regulators fall between the cascade of genetic events involving RsmA and pel. I have genetic and phenotypic evidence to support this, and further biochemical tests will need to be done to complete this finding.
Potential impact:
• Studies on social evolution of biofilm bacteria is a current hot topic in microbiology. The findings in this field are revolutionising our basic understanding of fundamental bacteriology to not treat these organisms as ‘stand alone entities’ but rather tackle our research strategies as a collective community of organisms, almost akin to tissues of higher organisms in order to comprehend the biology of microbes that typically grow as biofilm in their natural habitat. This includes pathogenic bacteria such as P. aeruginosa, which is posing significant damage to the medical field for their intrinsic capability of forming biofilm on innate hospital surfaces, medical devices, and on human tissues. Pathogenic biofilms generally exhibit higher capacity for negating antimicrobial therapies current in existence, and there is an immediate emergency need to re-think strategies for combating these infection. Part of this stems from our lack of biological understanding of bacteriology as community organismal studies that we are only recently taking steps towards describing.
• Novel strategies for combating biofilms will have wide societal impact, but in the biomedical field, CT scan-based approach of testing antimicrobials against microbial biofilms can add an additional use for understanding pathogenesis.
• An effective anti-biofilm strategy is to interfere with biofilm polysaccharide production. Further characterisation of the genetic and biochemical pathways will reveal novel drug targets that are not designed to kill bacteria, which increases the selective pressure for genetic resistance, but a more targeted approach non-lethal approach to deal with biofilm-forming species.