Implications of the expression of Enterococcus faecalis citrate fermentation genes during infection

Abstract

Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system. Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior.

Fig 1. Citrate gene clusters and metabolic pathway.

(A) and (B) Scheme of citrate metabolic pathway gene organization. citM, soluble oxaloacetate decarboxylase; citI, transcriptional regulator (deoR family); citC, citDEF, citX, and citG, citrate lyase subunits and accessory proteins; citP, citrate transporter (2-HCT family); ɸcitO, pseudo gen; citO, transcriptional regulator (gntR family); citH, citrate transporter (citMHS family); H (oadH), G (oadG), oadD, oadB and oadA, subunits of the membrane-bound oxaloacetate decarboxylase; citT, citrate transporter (2-HCT family); citAB, two-component signal transduction system. (C) Citrate and pyruvate pathways and their regulation in E. faecalis. Enzymes involved in citrate metabolism: 1, citrate lyase; 2, oxaloacetate decarboxylase. Enzymes involved in pyruvate metabolism: 3, lactate dehydrogenase; 4, pyruvate formate lyase; 5, pyruvate dehydrogenase; 6, phosphotransacetylase; 7, acetate kinase; 8, alcohol dehydrogenase; 9, non-enzymic oxidative decarboxylation, 10, α-acetolactate synthase and 11, α-acetolactate decarboxylase. Green and red arrows indicate induced or repressed steps (respectively) during growth in blood or urine [3, 4]. O1 and O2 binding sites of the activator CitO; c1, c2 and c3 binding sites of CcpA (cre sites).

Fig 2. Induction of cit promoters in G. mellonella.

(A) Scheme of citrate metabolism genes and fluorescent reporter plasmid pTLGR-Pcit used for microscopy. Fluorescence microscopy at different time points of E. faecalis JH2-2 cells harboring pTLGR-Pcit plasmid grown in LB and LB with 0.5% citrate (LBC), scale bar 5 μm (B); or G. mellonella hemolymph extracted at different time points after E. faecalis JH2-2 pTLGR-Pcit infection, scale bar 50 μm (C). Representative individuals of inoculated larvae are shown in (C). Two independent experiments were carried out and representative images acquired are shown.

Fig 3. Survival of G. mellonella inoculated with different strains of E. faecalis.

(A) Kaplan-Meier survival plots of G. mellonella upon injection with 4 x 10⁷ CFU/larva of E. faecalis JH2-2, or JHCit^-. L. lactis IL1403 (4 x 10⁷ CFU/larva) was employed as a control. (B, C, D and E) Kaplan-Meier survival plots of insects upon injection with 1 x 10⁷ CFU/larva of E. faecalis JH2-2-OadB^-, JH2-2-OadA^-, JH2-2-OadA^-/pOadA, or JH2-2-AlsS^-, respectively; E. faecalis JH2-2 1 x 10⁷ CFU/larva was used as pathogenic control. (F) Images of innoculated larvae showing different degrees of disease. G. mellonella last instar larvae inoculated with L. lactis or E. faecalis citrate-deficient strains showed a typical healthy creamy color, conversely larvae infected with E. faecalis JH2-2 or oadA^- complemented strain showed different stages of disease.

Fig 4. Induction of cit promoters in blood and urine.

Fluorescence microscopy at different time points of E. faecalis JH2-2 cells harboring pTLGR or pTLGR-Pcit plasmids grown in defibrinated blood (A) or urine (B). Two independent experiments were carried out and representative images acquired of three technical replicates are shown. Scale bar 5 μm.

Fig 5

E. faecalis strains growth in G. mellonela larvae (A), blood (B) and urine (C). (A) G. mellonella was inoculated with 9 x 10⁶ CFU/larvae. Bacterial burden was quantified using pools of hemolymph extracted from different larvae at the time points indicated. Growth was monitored by measuring colony forming units per milliliter (CFU/ml). JH2-2-OadA^- (purple), JH2-2-oadB^- (orange), JH2-2-Cit^- (green) and JH2-2 (red). Data points correspond to the mean ± standard error of six replicates, significative difference is indicated by * or **. (B and C) Growth of E. faecalis strains in blood and urine, respectively; E. faecalis JH2-2 (red circle), JH2-2-OadA^- (purple square), JH2-2-oadB^- (orange up triangle), JH2-2-Cit^- (green down triangle) and JH2-2-OadA^-/pOadA (cyan diamond). Data points correspond to the mean ± standard error of four and three replicates, respectively.