Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

Abstract

e Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken Enterococcus faecalis is an intestinal resident of humans and many animals and is a major cause of severe nosocomial infections that are difficult to treat due to intrinsic and acquired resistances to major classes of antibiotics. It is a homofermentative lactic acid bacterium that uses mainly the Embden-MeyerhoffParnas (glycolysis) pathway for the catabolism of glucose or related carbohydrates (1). The dominant end product of fermentation under these conditions is lactate (2, 3). Reduction of the glycolytic end product pyruvate to lactate by lactate dehydrogenases (LDH) allows the regeneration of the NADH formed during glycolysis to NAD ϩ , which keeps glycolysis going. E. faecalis possesses two cytosolic L-(ϩ)-lactate dehydrogenases encoded by the ldh-1 (EF_0255) and ldh-2 (EF_0641) genes. In the ldh-1 promoter region, a potential cre box was identified 114 bp upstream of the ATG start codon (4). cre boxes are cis-acting sequences targeted by CcpA, the major global transcription regulator of carbon catabolite repression in Firmicutes (5). Northern blot analysis showed that ldh-1 is expressed as a monocistronic transcript. The amount of transcript was 2.2-fold lower in a ccpA mutant than in the wild-type strain, suggesting that CcpA activates ldh-1 transcription (4). Most of the activity is associated with LDH-1, and LDH-2 plays only a minor role (2, 3). Recently, ldh deletion mutants of E. faecalis have been constructed and characterized. Deficiency in either LDH-1, LDH-2, or both activities causes a significant increase in the levels of mixed acid fermentation end products in strain V583 (2, 3, 6). All LDH mutants showed growth rates similar to that of the parental strain (2, 6, 7). This demonstrates that the loss of the characteristic metabolic reaction for lactic acid bacteria does not have an obvious impact on fitness under normal growth conditions in E. faecalis. Similar results have been obtained for Lactococcus lactis (8) and Streptococcus pyogenes (7), where little or no difference in the growth rate between the parent and ⌬ldh mutants has been observed. The growth yields were also comparable in E. faecalis when the strains grew under aerobic conditions, but final cell densities were even higher than those for the wild type when the ⌬ldh-1 mutant and the ⌬ldh-1 ⌬ldh-2 double mutant strain grew under anaerobic conditions (2). These combined results demonstrate the impressive metabolic flexibility of E. faecalis. Transcriptomic ...

Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

“…Lactate and acetate were quantified in the NMR sample extract by 1 H-NMR in a Bruker AMX300 (Bruker BioSpin GmbH). The concentration of other metabolites was determined in fully relaxed 13 C spectra of the NMR sample extracts as previously described (21). Due to the fast pulsing conditions used for acquiring in vivo 13 C spectra, correction factors for resonances due to the C-1 and C-6 of fructose 1,6-bisphosphate (FBP) (0.39 Ϯ 0.04), lactate (0.53 Ϯ 0.01), and acetate (0.93 Ϯ 0.09) were determined to convert peak intensities into concentrations.…”

“…The concentration of other metabolites was determined in fully relaxed 13 C spectra of the NMR sample extracts as previously described (21). Due to the fast pulsing conditions used for acquiring in vivo 13 C spectra, correction factors for resonances due to the C-1 and C-6 of fructose 1,6-bisphosphate (FBP) (0.39 Ϯ 0.04), lactate (0.53 Ϯ 0.01), and acetate (0.93 Ϯ 0.09) were determined to convert peak intensities into concentrations. The concentration of labeled lactate (in R6 cell extracts) or acetate (in SPR1078M cell extracts) determined by 1 H-NMR was used as a standard to calculate the concentration of the other metabolites in the sample as described previously (21).…”

“…In vivo NMR experiments were performed using an online system described earlier (21). Glucose (20 mM), specifically labeled with 13 C on carbon one, was added to the cell suspension at time point zero. The time course of glucose consumption, amount of product formation, and changes in the pools of intracellular metabolites were monitored in vivo, which refers to analysis in living cells.…”

“…In the absence of LDH, the bacterium was shown to be susceptible to various stressors, including solvents, oxidative stress, hypochlorite, detergent, acidity, and salt. Furthermore, LDH deficiency led to attenuation of the ability of the microbe to colonize host organs in a murine model of systemic infection (13). In Staphylococcus aureus, nitric oxide-induced LDH activity was shown to be important for metabolic adaptation to nitrosative stress by maintaining redox homeostasis and to be essential for virulence (14).…”

“…Besides its role in fermentative metabolism, accumulating evidence suggests its involvement in microbial virulence and pathogenesis. In En-terococcus faecalis, it has been shown that resistance to different environmental stresses is determined by the ability of the microbe to maintain redox balance via LDH (13). In the absence of LDH, the bacterium was shown to be susceptible to various stressors, including solvents, oxidative stress, hypochlorite, detergent, acidity, and salt.…”