scholarly journals Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells

2015 ◽  
Vol 83 (11) ◽  
pp. 4362-4372 ◽  
Author(s):  
Q. C. Truong-Bolduc ◽  
G. R. Bolduc ◽  
H. Medeiros ◽  
J. M. Vyas ◽  
Y. Wang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Unsaturated Fatty Acids ◽  
Efflux Pump ◽  
Fold Increase ◽  
Growth Patterns ◽  
Heart Cell ◽  
Content Type

ABSTRACTWe previously identified the protein Tet38 as a chromosomally encoded efflux pump ofStaphylococcus aureusthat confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator oftet38, named tetracycline regulator 21 (TetR21), that bound specifically to thetet38promoter and repressed pump expression. A ΔtetR21mutant showed a 5-fold increase intet38transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to thetetR21promoter and indirectly repressed the expression oftet38. To further assess the full role of Tet38 inS. aureusadaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We usedS. aureusRN6390, its Δtet38, ΔtetR21, and ΔmgrAmutants, and a Δtet38 ΔtetR21double mutant. After 2 h of contact, the Δtet38mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrAmutant. The growth patterns of RN6390 and the ΔmgrAand ΔtetR21mutants within A549 cells were similar, while no growth was observed for the Δtet38mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability ofS. aureusto internalize and replicate within epithelial cells.

scholarly journals DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus

2018 ◽  
Vol 201 (3) ◽  
Author(s):  
Heba Alnaseri ◽  
Robert C. Kuiack ◽  
Katherine A. Ferguson ◽  
James E. T. Schneider ◽  
David E. Heinrichs ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Dna Binding ◽  
Unsaturated Fatty Acids ◽  
Efflux Pump ◽  
Ectopic Expression ◽  
Exogenous Fatty Acid ◽  
Content Type ◽  
Tetr Family Regulator

ABSTRACT Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER. Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE. In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the −10 element of the PfarR promoter, (ii) in palindrome PAL1 immediately in the 3′ direction of PfarR, or (iii) within PAL2 upstream of the predicted PfarE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in PfarR. Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded. IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus. Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.

scholarly journals Transcriptional Profiling Analysis of the Global Regulator NorG, a GntR-Like Protein of Staphylococcus aureus

2011 ◽  
Vol 193 (22) ◽  
pp. 6207-6214 ◽  
Author(s):  
Q. C. Truong-Bolduc ◽  
P. M. Dunman ◽  
T. Eidem ◽  
D. C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Target Genes ◽  
Efflux Pump ◽  
Transcriptional Profiling ◽  
Fold Increase ◽  
Regulatory Function ◽  
Drug Transporter ◽  
Content Type ◽  
Global Regulators ◽  
Inorganic Ion

The GntR-like protein NorG has been shown to affectStaphylococcus aureusgenes involved in resistance to quinolones and β-lactams, such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional-profiling assays usingS. aureusRN6390 and its isogenicnorG::catmutant. Our data showed that NorG positively affected the transcription of global regulatorsmgrA,arlS, andsarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold) genes. TheS. aureuspredicted MmpL protein showed 53% homology with the MmpL lipid transporter ofMycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA ofStaphylococcus hominis. Two pump genes most negatively affected by NorG were the NorC (4-fold) and AbcA (6-fold) genes. Other categories of genes, such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time reverse transcription (RT)-PCR assays formgrA,arlS,sarZ,norB,norC,abcA,mmpL, andbcrA-like were carried out to verify microarray data and showed the same level of up- or downregulation by NorG. ThenorGmutant showed a 2-fold increase in resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression ofnorCon a plasmid. These data indicate that NorG has broad regulatory function inS. aureus.

scholarly journals Transcriptional Regulator TetR21 Controls the Expression of the Staphylococcus aureus LmrS Efflux Pump

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Q. C. Truong-Bolduc ◽  
Y. Wang ◽  
C. Chen ◽  
D. C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Parental Strain ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Fold Increase ◽  
Efflux Pumps ◽  
Transcription Level ◽  
Content Type

ABSTRACT TetR21 controls the expression of Tet38 and LmrS efflux pumps. A tetR21 mutant, QT21, exhibited a 4-fold increase in the transcription level of lmrS. Staphylococcus aureus lmrS overexpressor showed increases of 4-fold and 2-fold, respectively, in the MICs of chloramphenicol and erythromycin, while the MICs of lmrS mutant QT18 and lmrS-tetR21 mutant QT1821 remained similar to those of parental strain RN6390. TetR21 does not bind to the promoter of lmrS, suggesting indirect regulation of lmrS.

scholarly journals Oleate Hydratase (OhyA) Is a Virulence Determinant in Staphylococcus aureus

2021 ◽  
Author(s):  
Christopher D. Radka ◽  
Justin L. Batte ◽  
Matthew W. Frank ◽  
Jason W. Rosch ◽  
Charles O. Rock
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Unsaturated Fatty Acids ◽  
Commensal Bacteria ◽  
Hydroxy Fatty Acids ◽  
Infection Model ◽  
Infection Site ◽  
Human Pathogen ◽  
Content Type ◽  
Oleate Hydratase

The oleate hydratase protein family was discovered in commensal bacteria that utilize host unsaturated fatty acids as the substrates to produce a spectrum of hydroxylated products. These hydroxy fatty acids are thought to act as signaling molecules that suppress the inflammatory response to create a more tolerant environment for the microbiome. S. aureus is a significant human pathogen, and defining the mechanisms used to evade the immune response is critical to understanding pathogenesis. S. aureus expresses an OhyA that produces at least three 10-hydroxy fatty acids from host unsaturated fatty acids at the infection site, and an S. aureus strain lacking the ohyA gene has compromised virulence in an immunocompetent infection model.

scholarly journals Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

2015 ◽  
Vol 197 (11) ◽  
pp. 1893-1905 ◽  
Author(s):  
Heba Alnaseri ◽  
Benjamin Arsic ◽  
James E. T. Schneider ◽  
Julienne C. Kaiser ◽  
Zachariah C. Scinocca ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Linoleic Acid ◽  
Defense Mechanisms ◽  
Efflux Pump ◽  
Content Type ◽  
Inducible Resistance ◽  
Resistance Nodulation Division ◽  
Y Substitution ◽  
Arachidonic Acids

ABSTRACTAlthoughStaphylococcus aureusis exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome ofS. aureusUSA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H121Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genesfarRandfarE, forregulator andeffector offattyacidresistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression offarEwas strongly induced by arachidonic and linoleic acids in anfarR-dependent manner. Second, an H121Y substitution in FarR resulted in increased expression offarEand was alone sufficient to promote increased resistance ofS. aureusto linoleic acid. Third, inactivation offarEresulted in a significant reduction in the inducible resistance ofS. aureusto the bactericidal activity of 100 μM linoleic acid, increased accumulation of [14C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen.IMPORTANCEStaphylococcus aureuscolonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts,S. aureusmust have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes,farRandfarE, that constitute a dedicated regulator and effector ofS. aureusresistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression offarE, which encodes an efflux pump, is induced in anfarR-dependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess.

scholarly journals Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Q. C. Truong-Bolduc ◽  
N. S. Khan ◽  
J. M. Vyas ◽  
D. C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Efflux Pump ◽  
Scavenger Receptor ◽  
A549 Cells ◽  
Cell Receptor ◽  
Lung Epithelial Cells ◽  
Microscopy Imaging ◽  
Content Type ◽  
Bacterial Uptake

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

scholarly journals Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Zachary DeMars ◽  
Vineet K. Singh ◽  
Jeffrey L. Bose
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Lipid Composition ◽  
Unsaturated Fatty Acids ◽  
Mouse Skin ◽  
Membrane Composition ◽  
Wild Type ◽  
Content Type

ABSTRACT Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA. We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology. IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus. Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

scholarly journals Skin-Specific Unsaturated Fatty Acids Boost the Staphylococcus aureus Innate Immune Response

2015 ◽  
Vol 84 (1) ◽  
pp. 205-215 ◽  
Author(s):  
Minh Thu Nguyen ◽  
Dennis Hanzelmann ◽  
Thomas Härtner ◽  
Andreas Peschel ◽  
Friedrich Götz
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Human Skin ◽  
Immune Activation ◽  
Pathogenic Bacteria ◽  
Unsaturated Fatty Acids ◽  
Innate Immune ◽  
Immune Stimulation ◽  
Content Type ◽  
Lipid Moiety

Antimicrobial fatty acids (AFAs) protect the human epidermis against invasion by pathogenic bacteria. In this study, we questioned whether human skin fatty acids (FAs) can be incorporated into the lipid moiety of lipoproteins and whether such incorporation would have an impact on innate immune stimulation in the model organismStaphylococcus aureusUSA300 JE2. This organism synthesized only saturated FAs. However, when feeding USA300 with unsaturated FAs present on human skin (C16:1, C18:1, or C18:2), those were taken up, elongated stepwise by two carbon units, and finally found in the bacterial (phospho)lipid fraction. They were also observed in the lipid moiety of lipoproteins. When USA300 JE2 was fed with the unsaturated FAs, the cells and cell lysates showed an increased innate immune activation with various immune cells and peripheral blood mononuclear cells (PBMCs). Immune activation was highest with linoleic acid (C18:2). There are several pieces of evidence that the enhanced immune stimulating effect was due to the incorporation of unsaturated FAs in lipoproteins. First, the enhanced stimulation was dependent on Toll-like receptor 2 (TLR2). Second, anlgtmutant, unable to carry out lipidation of prolipoproteins, was unable to carry out immune stimulation when fed with unsaturated FAs. Third, the supplied FAs did not significantly affect growth, protein release, or expression of the model lipoprotein Lpl1. AlthoughS. aureusis unable to synthesize unsaturated FAs, it incorporates long-chain unsaturated FAs into its lipoproteins, with the effect that the cells are better recognized by the innate immune system. This is an additional mechanism how our skin controls bacterial colonization and infection.

scholarly journals Implication of the NorB Efflux Pump in the Adaptation of Staphylococcus aureus to Growth at Acid pH and in Resistance to Moxifloxacin

2011 ◽  
Vol 55 (7) ◽  
pp. 3214-3219 ◽  
Author(s):  
Que Chi Truong-Bolduc ◽  
Gilles R. Bolduc ◽  
Ryo Okumura ◽  
Brian Celino ◽  
Jennifer Bevis ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Fold Increase ◽  
Low Ph ◽  
Bacterial Killing ◽  
Content Type ◽  
Phosphorylated Form ◽  
Fold Reduction ◽  
Genes Encoding

ABSTRACTStaphylococcus aureusis an important pathogen that adapts and survives in low-pH environments. One component of this adaptation involves the regulation of genes encoding bacterial transporters that could affect response to antibiotics under these conditions. We previously demonstrated that the transcriptional regulator MgrA in its phosphorylated form (MgrA-P) represses the expression ofnorB, encoding the NorB multidrug resistance efflux pump. In this study, we focused on changes in the expression ofmgrAat the transcriptional and posttranslational levels, following a shift from pH 7.0 to pH 4.5. We then correlated those changes with modifications in transcript levels ofnorBand to resistance to moxifloxacin, a substrate of NorB. At pH 4.5,S. aureusMgrA increased 2-fold and MgrA-P decreased 4-fold, associated with an 8-fold increase innorBtranscripts and a 6-fold reduction in bacterial killing by moxifloxacin, and the phenomenon was dependent on intactmgrA. Taken together, these new data showed that phosphoregulation of MgrA at low pH reverses its repression ofnorBexpression, conferring resistance to moxifloxacin.

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