Document text
Principal Investigator: Cindy Menjivar
Organization: UNIVERSITY OF KANSAS MEDICAL CENTER
Fiscal Year: 2024
Award: $37,460
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT
Bacteria require fatty acids for a variety of biological functions, including the construction of phospholipids.
Staphylococcus aureus, like other bacteria, synthesize fatty acids using a fatty acid biosynthesis pathway
referred to as FASII, but can also scavenge fatty acids from the environment using the recently described fatty
acid kinase (Fak) pathway. In addition to fatty acid biosynthesis and acquisition, it is common for bacteria to
degrade fatty acids using fatty acid degradation (Fad) enzymes. Bacteria can degrade fatty acids for energy
production (known as b-oxidation) but can also degrade fatty acids to alter the length of the acyl chain. S. aureus
has been thought to not possess the capacity to degrade or perform b-oxidation of fatty acids. This is due, in
part, to the absence of a key crotonase enzyme. This is surprising considering that S. aureus is annotated to
encode all the other necessary Fad enzymes, though their function has not been confirmed. During RNAseq
studies of the Fak pathway, we observed an ~17-fold increased expression of the group of genes annotated to
encode Fad proteins when the Fak pathway was inactivated. Indeed, no crotonase enzyme was apparent,
though all the other Fad functionalities were annotated. Using bioinformatics, we identified a putative crotonase
enzyme in S. aureus that we now call FadB based on nomenclature in other systems. When co-expressed with
S. aureus FadA, FadB can functionally complement an E. coli fadA and fadB mutants on minimal media with
fatty acid as a sole carbon source. This demonstrates that 1) S. aureus FadB can substitute for the E. coli
crotonase activity-containing enzyme, and 2) S. aureus does possess a complete Fad pathway and likely can
degrade fatty acids. We seek to characterize the regulation of this pathway, demonstrate the saFadA and saFadB
proteins form a complex like the E. coli proteins, and demonstrate the functionality of the S. aureus Fad pathway
in three Specific Aims.
Aim 1 will identify regulatory proteins of the Fad operon. Aim 2 determines if the S. aureus FadA and FadB
proteins form a complex. In addition, Aim 3 examines the ability for S. aureus to degrade fatty acids and use
them as alternative carbon sources using controlled expression of the Fad genes using a combination of 13C-
labeled fatty acids with mass spectrometry and growth assays.
The Fad-encoding genes have been identified in a variety of transcriptomic studies but have remained
unstudied. This is likely due to the known dogma in the field that S. aureus does not possess a complete Fad
pathway and cannot degrade fatty acids. We anticipate that the completion of this application will redefine fatty
acid metabolism in S. aureus and determine for the first time that S. aureus can degrade fatty acids. This will
change how the field understands S. aureus metabolism and will set the stage for future applications examining
the role of this pathway in cell physiology and pathogenesis.
Terms: <3-Hydroxyacyl CoA Hydrolyases><3-Hydroxyacyl Dehydratases><9-Octadecenoic Acid><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Assay><Awareness><Bacteria><Bio-Informatics><Bioassay><Bioinformatics><Biological Assay><Biological Function><Biological Process><Carbon><Cell Function><Cell Physiology><Cell Process><Cellular Function><Cellular Physiology><Cellular Process><Communities><Complement><Complement Proteins><Complex><Coupled><Crotonase><Culture Media><DNA><Data><Death Rate><Degradation Pathway><Degradative Pathway><Deoxyribonucleic Acid><Disease><Disorder><E coli><E coli Proteins><E. coli><E. coli Proteins><Enoyl Hydrase><Enoyl-CoA Hydratase><Environment><Enzyme Gene><Enzymes><Escherichia coli><Escherichia coli Proteins><Essential Fatty Acids><Esters><FADK><FAK><FAK1><Fatty Acid Metabolism Pathway><Fatty Acids><Future><Generalized Growth><Genes><Gram-Positive Bacteria><Growth><High Throughput Assay><Human><Human Figure><Human body><Hybrids><In Vitro><Individual><Interferometry><Intermediary Metabolism><Kinases><Label><Length><Life><Lipopolysaccharides><Lipoproteins><MRSA><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Metabolic><Metabolic Processes><Metabolic Protein Degradation><Metabolism><Methicillin Resistant S. Aureus><Miscellaneous Antibiotic><Modern Man><Morbidity><Morbidity - disease rate><Nomenclature><Oleic Acids><Operon><PTK2><PTK2 gene><Pathogenesis><Pathogenicity Factors><Pathway interactions><Phosphatides><Phospholipids><Phosphotransferase Gene><Phosphotransferases><Photometry><Process><Production><Protein Turnover><Proteins><Pseudomonas fragi><RNA Seq><RNA sequencing><RNAseq><Regulation><Regulatory Protein><Regulatory Protein Degradation><Reporter><Resistance><Role><S aureus><S. aureus><Source><Staph aureus><Staphylococcus aureus><Subcellular Process><System><Testing><Time><Tissue Growth><Transphosphorylases><Virulence Factors><beta-D-Galactosidase><beta-D-Galactoside galactohydrolase><beta-Galactosidase><beta-Hydroxyacyl Dehydratases><beta-Hydroxyacyl-CoA Dehydrases><cis-9-Octadecenoic Acid><complementation><fatty acid biosynthesis><fatty acid metabolism><fatty acid oxidation><genetic regulatory protein><global health><growth media><high throughput screening><human disease><in vivo><lac Z Protein><lipo-teichoic acid><lipoteichoic acid><macromolecule><methicillin resistance Staphylococcus aureus><methicillin resistant Staphylococcus aureus><methicillin resistant strains of Staphylococcus aureus><mortality rate><mortality ratio><mutant><novel><ontogeny><oxidation><pathogen><pathway><pp125FAK><promoter><promotor><protein degradation><protein purification><quorum sensing><regulatory gene product><resistant><social role><transcriptome sequencing><transcriptomic sequencing><transcriptomics><uptake><β-D-Galactosidase><β-D-Galactoside galactohydrolase><β-Galactosidase>