Phenazine-1 carboxylic acid of Pseudomonas aeruginosa induces the expression of Staphylococcus aureus Tet38 MDR efflux pump and mediates resistance to phenazines and antibiotics.

In this study, we showed that phenazine-1 carboxylic acid (PCA) of Pseudomonas aeruginosa induced the expression of Tet38 efflux pump triggering Staphylococcus aureus resistance to tetracycline and phenazines. Exposure of S. aureus RN6390 to supernatants of P. aeruginosa PA14 and its pyocyanin (PYO)-deficient mutants showed that P. aeruginosa non-PYO phenazines could induce the expression of Tet38 efflux pump. Direct exposure of RN6390 to PCA compound at 0.25× MIC led to a five-fold increase in tet38 transcripts. Expression of Tet38 protein was identified through confocal microscopy using RN6390(pRN-tet38p-yfp) that expressed YFP under control of the tet38 promoter by PCA at 0.25× MIC. The MICs of PCA of a Tet38-overexpressor and a Δtet38 mutant showed a three-fold increase and a two-fold decrease, respectively, compared with that of wild-type. Pre-exposure of RN6390 to PCA (0.25× MIC) for 1 hour prior to addition of tetracycline (1× or 10× MIC) improved bacteria viability of 1.5-fold and 2.6-fold, respectively, but addition of NaCl 7% together with tetracycline at 10× MIC reduced the number of viable PCA-exposed RN6390 of a 2.0-log10 CFU/mL. The transcript levels of tetR21, a repressor of tet38, decreased and increased two-fold in the presence of PCA and NaCl, respectively, suggesting that the effects of PCA and NaCl on tet38 production occurred through TetR21 expression. These data suggest that PCA-induced Tet38 protects S. aureus against tetracycline during coinfection with P. aeruginosa; however, induced tet38-mediated S. aureus resistance to tetracycline is reversed by NaCl 7%, a nebulized treatment used to enhance sputum mobilization in CF patients.

Role of Staphylococcus aureus Tet38 in Transport of Tetracycline and Its Regulation in a Salt Stress Environment.

Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. In this study, we evaluated the effects of sodium and potassium on tet38 expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters. tet38 transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator. tet38 transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a ΔkdpD mutant. KdpE bound specifically to the tet38 promoter. Under extreme salt stress, the survival of S. aureus with intact tet38 was reduced compared to that of a Δtet38 mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing tet38 and tetK and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of tet38 through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined. IMPORTANCE This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of tet38 through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na+/K+. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Molecular Mechanisms and Epidemiology of Fosfomycin Resistance in Staphylococcus aureus Isolated From Patients at a Teaching Hospital in China.

Staphylococcus aureus is a major cause of hospital- and community-acquired infections placing a significant burden on the healthcare system. With the widespread of multidrug-resistant bacteria and the lack of effective antibacterial drugs, fosfomycin has gradually attracted attention as an "old drug." Thus, investigating the resistance mechanisms and epidemiology of fosfomycin-resistant S. aureus is an urgent requirement. In order to investigate the mechanisms of resistance, 11 fosfomycin-resistant S. aureus isolates were analyzed by PCR and sequencing. The genes, including fosA, fosB, fosC, fosD, fosX, and tet38, as well as mutations in murA, glpT, and uhpT were identified. Quantitative real-time PCR (qRT-PCR) was conducted to evaluate the expression of the target enzyme gene murA and the efflux pump gene tet38 under the selection pressure of fosfomycin. Furthermore, multilocus sequence typing (MLST) identified a novel sequence type (ST 5708) of S. aureus strains. However, none of the resistant strains carried fosA, fosB, fosC, fosD, and fosX genes in the current study, and 12 distinct mutations were detected in the uhpT (3), glpT (4), and murA (5) genes. qRT-PCR revealed an elevated expression of the tet38 gene when exposed to increasing concentration of fosfomycin among 8 fosfomycin-resistant S. aureus strains and reference strain ATCC 29213. MLST analysis categorized the 11 strains into 9 STs. Thus, the mutations in the uhpT, glpT, and murA genes might be the primary mechanisms underlying fosfomycin resistance, and the overexpression of efflux pump gene tet38 may play a major role in the fosfomycin resistance in these isolates.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Tet38 Efflux Pump Contributes to Fosfomycin Resistance in Staphylococcus aureus.

Fosfomycin inhibits MurA following uptake by the GlpT transporter of glycerol-3-phosphate in Escherichia coli In Staphylococcus aureus, plasmid overexpression of the Tet38 efflux pump and a glpT mutant resulted in increased MICs and decreased accumulation of fosfomycin, with MICs affected by glycerol-3-phosphate. In contrast, a tet38 mutant had a lower MIC and increased accumulation of fosfomycin, suggesting that Tet38 acts as an efflux transporter of fosfomycin.

Native efflux pumps contribute resistance to antimicrobials of skin and the ability of Staphylococcus aureus to colonize skin.

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.