Role of Dihydrolipoamide Dehydrogenase in Regulation of Raffinose Transport in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50-to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDHnegative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.Dihydrolipoamide dehydrogenases (DLDH) are enzymes classically involved in the reoxidation of dihydrolipoamide during conversion of 2-oxo acids (such as pyruvate) in several multienzyme complexes in the central metabolism (34, 52). However, previous work from our and another laboratory have shown that DLDH is not involved in metabolizing 2-oxo acids in the pneumococcus, indicating that DLDH must serve other functions (41,42). This is supported by evidence that organisms that lack 2-oxo acid dehydrogenases still express a DLDH protein (9, 10). One such function was suggested by Richarme in the 1980s when he showed that Escherichia coli strains that fail to make lipoic acid or strains treated with an inhibitor that mainly inhibits DLDH function resulted in reduced import of galactose, maltose, and ribose through ATP-binding cassette (ABC) transporters (35). We then showed that inactivation of DLDH in the pneumococcus results in an inability of the bacteria to import and utilize galactose and the alpha-galactoside sugars raffinose and stachyose and that a lack of DLDH is associated with an almost complete attenuation of this strain in animal infection experiments (41).Transport of carbohydrates and other energy sources is important for many aspects of bacterial life and therefore highly regulated. Fitness in the bacterial host ...

Identification of an ATPase, MsmK, Which Energizes Multiple Carbohydrate ABC Transporters in Streptococcus pneumoniae

“…The exact contribution of each of the six transporters is currently unknown; however, previous data suggest that individual transporters vary in their contributions to both colonization and pathogenesis (4,11,16,23,29,37). While we did not assess the contribution of msmK to pathogenesis here, during the revision of the manuscript Tyx et al demonstrated a role for this gene in raffinose transport and pneumococcal pneumonia (40). While further studies are required, current data certainly indicate that msmK and therefore carbohydrate transport are important during pneumococcal pathogenesis.…”

Pneumococcal Interactions with Epithelial Cells Are Crucial for Optimal Biofilm Formation and Colonization In Vitro and In Vivo

“…The absence of symptoms associated with pneumonia and invasive disease was confirmed by lack of visual signs such as ruffled fur, inactivity, and labored breathing, as well as lack of fever measured through the abdominal surface temperature, as described previously (6). At 48 h postinoculation, animals were euthanized by CO 2 asphyxiation and nasopharyngeal tissue was dissected as described previously (70).…”

“…Mice were colonized as described previously (70). In short, 20 l of a bacterial suspension containing 5 ϫ 10 6 CFU of pneumococci in phosphate-buffered saline (PBS) was pipetted into the nares of nonanesthetized mice.…”

“…Pneumococcal strains were grown in synthetic medium (CDM) or in Todd-Hewitt medium containing 0.5% yeast extract (THY) as described previously (70). The study used the serotype 19F strains EF3030 and BG8826 (2), the serogroup 6 strain SP670 (31), the serotype 3 strain WU2 (14), the serotype 2 strain D39 (3), and versions of D39 lacking capsule (44), pneumococcal surface protein C (PspC) (4), pneumococcal surface protein A (PspA) (46), pneumolysin (9), and autolysin (LytA) (8).…”

“…Pellets were resuspended in 0.5 ml of 0.9% NaCl, 1 ml of RNAprotect (Qiagen, Valencia, CA) was added, and the mixture was incubated at room temperature for 5 min. Cells were then pelleted at 9,000 ϫ g for 2 min at room temperature, and RNA was purified using Qiashreeder columns and the RNeasy minikit as described previously (70).…”