Although the involvement of the TCA and urea cycles in biofilm formation has been previously described as above 7, 15, 16, it is not known how exogenous glutamate, a major amino acid in central metabolism, is involved in biofilm formation in conjunction with the TCA and urea cycles. Because the gltS mutant was the only one that demonstrated alteration in biofilm formation among all the sodium ion transporters, we tested our hypothesis that glutamate-induced metabolic changes would affect MRSA biofilm formation by utilizing chemically defined media (CDM) depleted of glutamate, mass spectrometry-based metabolomic analyses, and RNA transcriptomic analyses. We found significantly increased biofilm formation in a major sodium-glutamate symporter gltS transposon mutant strain ( gltS::Tn) 13 compared to the parental strain. With the hypothesis that a subset of MRSA ion transporters would be involved in biofilm formation, we screened biofilm formation for ion transposon (Tn) mutants in the Nebraska library 14. However, their role in biofilm has not been extensively studied. For example, alsT functions as an efficient glutamine transporter 13 and gltT is an aspartate transporter 11. The biological roles of ion transporters in MRSA were recently studied by several investigators 10, 11, 12, 13. We have previously shown that the a subset of ion transporters was involved in biofilm formation in Escherichia coli 9. Ion transporters play a critical role in sensing environments and regulating ion trafficking. The involvement of the urea cycle in biofilm formation is also described 7. During biofilm formation, the TCA cycle may be initially activated 5, but the inactivation of the TCA cycle generally ensues 6, contributing to an increase in the percentage of protein components in the USA300 biofilm matrix 7, 8. The tricarboxylic acid (TCA) cycle is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. An investigation into the underlying molecular mechanisms of biofilm formation in MRSA is critical to developing a therapeutic approach. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading nosocomial pathogen and commonly associated with significant morbidity, hospital mortality, length of stay, and economic burden, with an estimated attributable healthcare cost of $1.7 billion in 2017 in the United States 4. The transition of bacterial cells from planktonic to biofilm form is induced by the regulation of gene expression in response to sensed physicochemical environmental signals such as ions, pH, and nutrients, which in turn affects various signaling pathways 3. Biofilm infections are highly resistant to antibiotic therapy and host defense, making biofilms difficult to be eradicated 2. Biofilm-related infections occupy a large percentage of nosocomial infections 1, including device and indwelling catheter infections. An intervention to target glutamate intake may be a potential useful approach against biofilm.īiofilms are aggregates of bacteria that are adherent to a material surface and encased in a self-synthetic extracellular polymer matrix. In conclusion, we showed that gltS was a critical regulator of biofilm formation by controlling the intake of exogenous glutamate. We also showed that urea cycle activation was critical for biofilm formation. The deficiency of exogenous glutamate intake accelerated endogenous glutamate/glutamine production, which led to the activation of the urea cycle. The lack of exogenous glutamate also enhanced biofilm formation in JE2 strain. This was shown by gltS transposon mutant as well as its complementation. Here we examined the role of ion transporters in biofilm formation and found that the sodium-glutamate transporter gltS played an important role in biofilm formation in MRSA. Although biofilm is induced by a number of environmental signals, the molecule responsible for environmental sensing is not well delineated. Methicillin-resistant Staphylococcus aureus (MRSA) is one of major organisms responsible for biofilm infection. Biofilm-based infection is a major healthcare burden.
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