Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus

With the increasing spread of methicillin-resistant Staphylococcus aureus worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant S. aureus infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX) and mutations of the target enzyme MurA or the membrane transporter proteins UhpT and GlpT. Our previous studies indicated that the fos genes might not the major fosfomycin resistance mechanism in S. aureus, whereas mutations of glpT and uhpT seemed to be more related to fosfomycin resistance. However, the precise role of these two genes in S. aureus fosfomycin resistance remains unclear. The aim of the present study was to investigate the role of glpT and uhpT in S. aureus fosfomycin resistance. Homologous recombination was used to knockout the uhpT and glpT genes in S. aureus Newman. Gene complementation was generated by the plasmid pRB473 carrying these two genes. The fosfomycin minimal inhibitory concentration (MIC) of the strains was measured by the E-test to observe the influence of gene deletion on antibiotic susceptibility. In addition, growth curves were constructed to determine whether the mutations have a significant influence on bacterial growth. Deletion of uhpT, glpT, and both of them led to increased fosfomycin MIC 0.5 μg/ml to 32 μg/ml, 4 μg/ml, and >1024 μg/ml, respectively. By complementing uhpT and glpT into the deletion mutants, the fosfomycin MIC decreased from 32 to 0.5 μg/ml and from 4 to 0.25 μg/ml, respectively. Moreover, the transporter gene-deleted strains showed no obvious difference in growth curves compared to the parental strain. In summary, our study strongly suggests that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that uhpT mutation may play a more important role. The high resistance and low biological fitness cost resulting from uhpT and glpT deletion suggest that these strains might have an evolutionary advantage in a fosfomycin-rich clinical situation, which should be closely monitored.


INTRODUCTION
Staphylococcus aureus is one of the most common bacterial pathogens worldwide in both community and hospital settings. Methicillin-resistant Staphylococcus aureus (MRSA) is an important multi-resistant pathogen. To date, vancomycin has remained the cornerstone drug in the management of invasive MRSA infections. However, the continuous rise in the vancomycin minimum inhibitory concentration (MIC), known as the "vancomycin MIC creep" phenomenon, poses a significant challenge to MRSA therapy; therefore, fosfomycin has recently been used alone or in combination with other antibiotics in treating MRSA infections (del Rio et al., 2016;VanEperen and Segreti, 2016). Nevertheless, this situation has inevitably led to the emergence of fosfomycin-resistant MRSA strains. In a recent review, the susceptibility of S. aureus to fosfomycin ranged between 33.2 and 100% in the nine available studies (frequency = 91.7%, 95% confidence interval 88.7-94.9%); in seven of the studies susceptibility rate was >90% (Vardakas et al., 2016). According to the CHINET surveillance program in China in 2010, 29.5% of the MRSA clinical isolates were resistant to fosfomycin . And Yu et al. (2010) reported a fosfomycin susceptible rate of 33.2%.
The mechanisms of action and resistance of fosfomycin have been studied for decades. Fosfomycin was first discovered in 1969 as an effective bactericidal agent against Grampositive and Gram-negative organisms. The mechanism of action of fosfomycin differs from that of most commonly used antimicrobials. In general, fosfomycin is transported across the bacterial wall primarily with the help of the glycerol-3-phosphate (G-3-P) transport (GlpT) system. In the presence of glucose-6-phosphate (G-6-P), the hexose phosphate uptake transport (UhpT) system is induced, and provides an alternative route to the GlpT system. UhpT are important membrane transporter proteins for small molecules, including fosfomycin (Castaneda- Garcia et al., 2009). When transported into the cytosol of a bacterium, fosfomycin deactivates the target protein UDP-N-acetylglucosamine-3enolpyruvyltransferase (MurA, encoded by the murA gene), thereby preventing the formation of N-acetylmuramic acid from N-acetylglucosamine and phosphoenolpyruvate, which is the initial step in peptidoglycan chain formation of the bacterial wall (Kahan et al., 1974). The key resistance mechanisms to fosfomycin include the loss or reduced production of transporters, reduced affinity to MurA, and production of fosfomycin-modifying enzymes (Sastry and Doi, 2016).
However, to date, the mechanisms contributing to fosfomycin resistance have been mostly studied in Gram-negative bacteria, with few related studies on Gram-positive bacteria. We have conducted several studies to investigate the fosfomycin resistance mechanisms in Gram-positive cocci, including Enterococcus faecium and S. aureus (Xu et al., 2013;Chen et al., 2014;Fu et al., 2016a,b). These previous studies indicated that the fos gene was not the major mechanism of fosfomycin resistance in MRSA isolates from our hospital, whereas mutations of glpT and uhpT seemed to be more closely related to fosfomycin resistance. However, the exact roles of these two genes in S. aureus fosfomycin resistance remain unclear. Thus, we designed the present study to investigate the roles of glpT and uhpT in S. aureus fosfomycin resistance.

Bacterial Strains and Plasmids
The strains and plasmids used in this study are presented in Table 1. The clinical fosfomycin-resistant MRSA strains SA2, SA94, and SA30 were collected from the blood or cerebral spinal fluid of patients at Huashan Hospital and were characterized previously (Fu et al., 2016b). And the strain names are in accordance with that in the Supplementary Table S1 of the previous article (Fu et al., 2016b). Each of the clinical MRSA strains was with a different type of transporter gene mutation ( Table 1). The S. aureus strains Newman and RN4220, and the plasmid pKOR1 were used in the homologous recombination assay (Bae and Schneewind, 2005;Wang et al., 2015). In addition, S. aureus ATCC29213 (American Type Tissue Culture Collection, Manassas, VA, United States) was used for the quality control of susceptibility testing. These strains and plasmids were laboratory collection.

Allelic Gene Deletion by Homologous Recombination
Knockout of the transporter genes glpT and uhpT was conducted as previously described (Bae and Schneewind, 2005;Wang et al., 2015). The plasmids and primers used are listed in Tables 1, 2, respectively. Proper gene deletion was verified by analytical polymerase chain reaction (PCR) and sequencing of the genomic DNA at the borders of the PCR-derived regions. Sequencing was then performed to confirm the nucleotides. The amplified fragments were used to construct the homologous recombinant pKOR1-uhpT/glpT with Gateway R BP Clonase TM II Enzyme mix (Thermo Fisher Scientific, Waltham, MA, United States).
pKOR1-uhpT and pKOR1-glpT were introduced into S. aureus RN4220 by electroporation for modification. The plasmid extracted from strain RN4220 was then introduced into S. aureus Newman. The desired uhpT and glpT deletion mutants were selected as described previously (Bae and Schneewind, 2005).

Construction of the Complemented Strain
Fragments were PCR-amplified from S. aureus Newman using the primers C-uhpT-F/R and C-glpT-F/R. The PCR products and vector pRB473 were double-digested with the designed restriction enzymes BamHI and EcoRI (for uhpT), or BamHI and KpnI (for glpT), and ligation was performed with T4 ligase. The resulting plasmids were transferred into S. aureus RN4220, and then introduced into the deletion and clinical strains with defects on uhpT and/or glpT, SA2, SA94, and SA30.

Antimicrobial Susceptibility Testing
Fosfomycin susceptibility of the knockout and clinical strains with defects on uhpT and/or glpT, and their complemented strains were tested with the E-test (BioMerieux SA, La Balme Les Grotts, France), according to the manufacturer's guidance. Results were interpreted according to European committee on antimicrobial susceptibility testing criteria (European Committee on Antimicrobial Susceptibility Testing [EUCAST], 2017) (susceptible, ≤32 mg/L; resistant, ≥64 mg/L).

Measurement of Growth Curves
To evaluate the influence of deletion of the transporter genes on bacterial growth, we measured the in vitro growth curves of S. aureus Newman, Newman-uhpT, Newman-glpT, Newman-uhpT&glpT, and the clinical strains. The strains were cultivated in tryptic soy broth overnight at 37 • C. The bacterial solution was diluted to an optical density at 600 nm (OD 600 ) of 0.1 and cultivated again. The OD 600 was then measured at 0, 2, 4, 6,8,10,12,14,16,18,20,22, and 24 h for each strain, by

Phenotype Microarray (PM) Analysis
Phenotype Microarray analysis was performed using BIOLOG Phenotype Microarray TM (BIOLOG, Hayward, CA, United States) according to the manufacturer's recommendations. The deletion mutants, namely Newman-uhpT, Newman-glpT, Newman-uhpT&glpT, and the parental strain Newman, were tested with the 96-wells plates PM1 and PM2, containing 190 carbon substrates, including G-6-P (PM1 plate, well C1). To assess the altered phenotypes in carbon metabolism of the deletion mutants, the growth was compared to the parent S. aureus Newman.

RESULTS
The deletion mutants showed considerably increased MIC values to fosfomycin compared to that of the parental strain. The Newman-uhpT&glpT strain, in which both transporter genes were knocked out, showed high-level resistance (MIC > 1024 µg/ml) to fosfomycin, as determined by the E-test (Table 3). When uhpT or glpT was knocked out from S. aureus Newman, the fosfomycin MICs increased from 0.5 to 32 µg/ml or 4 µg/ml, respectively. Complementing uhpT and glpT led to a reduced fosfomycin MIC in the deletion mutants and the clinical fosfomycinresistant S. aureus strains with defects at both sites. By complementing plasmid pRB473-uhpT into Newman-uhpT, the strain's fosfomycin MIC decreased from 32 to 0.5 µg/ml. Similarly, by complementing glpT into Newman-glpT, strain's fosfomycin MIC decreased from 4 to 0.25 µg/ml. S. aureus SA2, SA94, and SA30 were clinical fosfomycin-resistant strains, with mutations of both uhpT and glpT, uhpT only, and glpT only, respectively. When complemented with the functional transporter genes, the fosfomycin MICs decreased considerably, as shown in Table 3.
In vitro bacterial growth curves of the wild-type strain S. aureus Newman and the deletion mutants were compared to evaluate the potential fitness cost of these resistant-conferring mutations. As shown in Figure 1, no significant depression in growth was observed in Newman-uhpT and Newman-glpT compared to the wild-type strain. However, the strain Newman-uhpT&glpT presented slight growth inhibition compared to the wild-type.
Phenotype Microarray analysis was performed using carbon utilization panels, PM1 and PM2, in 190 carbon substrates. The changes in carbon metabolism were listed in Figure 2. S. aureus Newman showed metabolic advantage over Newman-uhpT and Newman-uhpT&glpT in wells containing G-6-P (Figure 2, PM1, well C1). G-3-P was not included in the substrate list, and there was no obvious change found in Newman-glpT.

DISCUSSION
Intravenous fosfomycin is broadly used in the treatment of multidrug-resistant pathogens in Europe and Asia owing to its unique antibiotic mechanism, high permeability, and high susceptibility rate (Falagas et al., 2009). Falagas et al. (2010 performed fosfomycin susceptibility testing in non-urinary MRSA isolates, among which 99.2% (129/130) were found to be susceptible. The same group reviewed the susceptibility data of Gram-positive cocci, and reported a cumulative susceptibility rate of 87.9% (4240/4892) in S. aureus (Falagas et al., 2009).
There is less known of fosfomycin resistance mechanism in Gram-positive cocci. In previous works, we collected MRSA clinical strains, and found that only the minority of the fosfomycin-resistant MRSA strains carried the fos gene or murA mutation, while glpT and uhpT mutations were common (82.1%, 55/67, vs. 77.6%, 52/67, respectively) (Fu et al., 2016a,b). This fact indicated that, fosB or murA mutation is not the major contributor to fosfomycin resistance in MRSA, while mutations within the glpT and/or uhpT genes might play an important FIGURE 1 | The in vitro growth curves of S. aureus strains. The strains were cultivated in tryptic soy broth overnight at 37 • C. The bacterial solution was diluted to an optical density at 600 nm (OD 600 ) of 0.1 and cultivated again. The OD 600 was then measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24  FIGURE 2 | PM analysis for carbon metabolism. The strains were grown in a 96-well plate under 37 • C over two nights. Carbon utilization kinetics was measured with OmniLog instrument. Data were superimposed using OmniLog software. The kinetic results show consensus data comparing S. aureus Newman and its transporter gene deletion mutants. A metabolic advantage by S. aureus Newman is indicated by red, while a metabolic advantage by the deletion mutants is shown by green. The yellow part indicates that both strains have equal metabolism in the well. The white box around PM1 plate, well C1, highlights the G-6-P metabolism. Detailed substrate information of PM1 and PM2 were shown in the Supplementary Table S1. role in S. aureus fosfomycin resistance. In the present study, we established uhpT and/or glpT deletion mutants. Knocking out both genes resulted in high-level fosfomycin resistance (MIC > 1024 µg/ml). Complementing either of the two genes into the deletion mutants and clinical mutated strains resulted in a decreased fosfomycin MIC. Direct comparison of uhpT and glpT according to the level of increase of the MIC suggested that uhpT has a greater effect on the strain's MIC to fosfomycin than glpT.
To evaluate the possible, the in vitro fitness cost of the transporter gene mutation, we compared growth curves between the fosfomycin-sensitive wild-type strain, laboratory deletion mutant strain, and clinical strains with defects on uhpT and/or glpT. Previous reports have shown that mutations of uhpT and glpT can compromise the growth of strains of E. coli, Klebsiella pneumoniae, and Proteus mirabilis (Li Pira et al., 1987;Marchese et al., 2003). A probable mechanism might be that mutations of the glpT and/or uhpT transporting systems prevent carbon source getting into the cytoplasm, and therefore disturb cell metabolism (Venkateswaran and Wu, 1972;Nilsson et al., 2003). But in P. aeruginosa, the glpT mutation was found to lead to fosfomycin resistance with no obvious fitness cost (Castaneda- Garcia et al., 2009). In the present study, there was only a slight reduction of growth observed in the strain Newman-uhpT&glpT compared to the wild type, and no significant growth suppression was observed either in the laboratory deletion mutant strains or in clinical strains with defects on uhpT and/or glpT, which is similar as observed in P. aeruginosa. So S. aureus might also compensate the disadvantage in energy obtainment caused by uhpT and/or glpT mutation through other transporting systems. But further study is still in need for verification.
We observed that G-6-P utilization was defected in both Newman-uhpT and Newman-uhpT&glpT. UhpT is the membrane transporter of this substrate, deletion mutants of uhpT showed defects in G-6-P metabolism is as expected. The G-3-P metabolism in S. aureus seems to be more complicated. G-3-P seems to be an intermediate product in carbon/phosphorus metabolism pathway. As another low G+C Gram-positive bacteria, B. subtilis shares similar carbon metabolism pattern as S. aureus. In B. subtilis, G-3-P is produced from glycerol with glycerol kinase. And G-3-P dehydrogenase can oxidize G-3-P to dihydroxyacetone phosphate, an intermediated in glycolysis (Holmberg et al., 1990). We have not observed significant change in metabolism. This may be because that G-3-P utilization defect is easily compensated by other pathways.
In summary, the results of our study strongly suggest that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that the uhpT mutation may play a more important role. The high resistance and low fitness cost resulting from uhpT and glpT mutations suggest that these mutated strains might have an evolutionary advantage in a fosfomycin-rich clinical situation.
The widely observed uhpT or glpT mutation in S. aureus might be a threat in hospital settings. Further studies are needed to evaluate the frequency of S. aureus fosfomycin mutants, and virulence of these mutants.

AUTHOR CONTRIBUTIONS
Designed and conceived the experiments: YL, XX, and MW. Performed the experiments: SX, ZF, and YZ. Analyzed the data: SX, ZF, and YZ. Wrote and reviewed the manuscript: SX, ZF, XX, and YL.

FUNDING
This study was supported by grants from the National Natural Science Foundation of China (81171613 to XX and 81120108024 to MW).

ACKNOWLEDGMENT
We thank Professor M. Li, Renji Hospital, Shanghai Jiaotong University, for providing strain S. aureus RN4220 and plasmid pKOR1.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00914/full#supplementary-material TABLE S1 | Carbon sources tested in PM1 and PM2 plates.