ABSTRACT
UNLABELLED: Staphylococcus aureus is a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome of S. aureus and used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important for S. aureus fitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important for S. aureus proliferation at sites of infection, which may represent new therapeutic targets. IMPORTANCE: Staphylococcus aureus continues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium's acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identified S. aureus traits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.
Subject(s)
Abscess/microbiology , Genome, Bacterial , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Computational Biology , DNA Transposable Elements/genetics , Drug Resistance, Bacterial , Gene Library , Male , Mice , RNA, Antisense/genetics , Sequence Analysis, DNA , Virulence Factors/geneticsABSTRACT
The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic ß-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Gene Expression Regulation, Bacterial , Staphylococcus aureus/physiology , Stress, Physiological , Abscess/microbiology , Animals , Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Bambermycins/metabolism , Base Sequence , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Gene Expression Profiling , Hydrogen-Ion Concentration , Iron/metabolism , Mice , Molecular Sequence Data , Oxidative Stress , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/metabolism , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
BACKGROUND: Staphylococcus aureus colonizes skin in the presence of antimicrobial fatty acids and polyamines. The chromosomally encoded Tet38 efflux transporter confers resistance to tetracycline and fitness in abscesses, but its natural substrates and those of the Nor quinolone efflux pumps are unknown. METHODS: Susceptibility of tet38 and other pump mutants to and pump gene induction by fatty acids and polyamines were compared. Transport of fatty acids by Tet38 was determined in membrane vesicles. Survival on skin was tested in an adapted mouse skin infection model. RESULTS: The tet38 expression caused a 5- to 8-fold increase in resistance to palmitoleic and undecanoic acids but not polyamines. Subinhibitory concentrations of these fatty acids induced 4-fold increases in tet38 transcripts and competitively inhibited transport of Hoechst 33 342 dye in Tet38 membrane vesicles. Colonization of skin in BALB/c mice was decreased 5-fold in a Δtet38 mutant, which was complemented by plasmid-encoded tet38. Although polyamine minimum inhibitory concentrations (MICs) decreased 4-fold in a norC::cat mutant and increased 8-fold with norC overexpression, spermidine did not induce expression of norC and other pump genes, and norC::cat exhibited wild-type colonization. CONCLUSION: Antibacterial fatty acids may be natural substrates of Tet38, which contributes to resistance and the ability of S. aureus to colonize skin.
