## 5 Carbon Catabolite Regression Systems Coordinate Antibiotic Persistence and Tolerance  
Pathogenic microbes are heterotrophic and rely on a variety of carbon sources for growth [68]. The ability to sense and efficiently utilize a diverse pool of carbon sources, which increases nutritional fitness, is contingent upon highly coordinated metabolite sensing coupled with rapid and appropriate responses. Bacterial growth and metabolism are intricately linked to the availability of carbon sources and cellular responses to this availability. Thus, persister formation is also closely linked to carbon flux within the cell.  
Perhaps one of the best described and most conserved metabolite response systems is the carbon catabolite repression (CCR) system. CCR is a global regulatory mechanism by which utilization of secondary carbon sources is dampened in the presence of preferred carbon sources such as glucose [69]. In Gram-negative species, CCR is activated by transcriptional repression of a pro-catabolic cyclic-AMP-CRP protein complex. In Gram-positive species, CCR is negatively regulated. Environmental glucose triggers phosphorylation of the histidine protein (HPr), which complexes with a pleiotropic transcription factor, carbon catabolite protein A (CcpA). This heterotrophic complex binds to responsive DNA elements, thereby repressing catabolic gene expression [69]. CcpA has been demonstrated to regulate a massive proportion of glucose-responsive genes, almost 80 percent in _Bacillus subtilis_, and carbon sources have been implicated in _E. coli_ persister formation, hinting at a possible connection between CCR and persisters [70].  
Nutrient transitions, starvation, and the CCR response have been recently implicated as important triggers of antibiotic tolerance [1; 71]. Experimental inactivation of _ccpA_ has been shown to decrease tolerance in various clinically relevant species [57]. In _E. coli_, CCR knockout increases sensitivity to penicillin due to ablation of metabolic flux [71]. In _Streptococcus gordonii_, _ccpA_ knockout ablates tolerance to multiple drug classes, both in vitro and in a rat endocarditis model [70]. Complementation with a functional _ccpA_ copy is experimentally sufficient to restore tolerance in both clinical and laboratory strains [70]. _Streptococcus suis_, a zoonotic pig pathogen, and _Streptococcus pneumoniae_ lose tolerance to \(\beta\)-lactam antibiotics when _ccpA_ is mutated or deleted [70]. In methicillin-resistant _S. aureus_ (MRSA), _ccpA_ deletion results in severe reductions in \(\beta\)-lactam and glycopeptide resistance amongst highly resistant strains despite the presence of genetically-encoded resistance determinants [70; 72].  
_Staphylococcus epidermidis_ growth and tolerance is enhanced in vitro through _ccpA_. Recently, TCA cycle activity and CCR linkage have been identified as the connecting mechanisms [73]. Interestingly, this linkage seems conserved across _Staphylococci_. In _S. aureus_, _ccpA_ represses TCA cycle genes, removing inhibition of intercellular adhesion and biofilm formation, which themselves have been implicated in increased antibiotic tolerance [73]. In a clinical context, many _Staphylococcal_ infections cause abscess formation, where preferred carbon sources are limited [74]. Thus, catabolism of secondary carbon sources must be highly regulated in order to adopt an antibiotic-tolerant biofilm lifestyle. As previously discussed, the formation of these tolerant biofilms has the potential to increase persister formation. Clinically, the connection between CCR, tolerance, and persistence has many implications, particularly for hosts with metabolic disorders. In hyperglycmic patients, for example, it is possible that increased glucose bioavailability triggers CCR activity, protecting pathogenic microbes from therapeutic regimens while increasing virulence and infection burden.