Marie-Claude Trombe

The NADH oxidase of Streptococcus pneumoniae : its involvement in competence and virulence

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase‐deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence‐regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

The ami locus of the gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The Ami A, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R‐factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.

Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic‐type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese‐dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild‐type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP‐N‐acetylglucosamine, an essential common precursor to cell envelope components.

Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in Streptococcus pneumoniae

ABSTRACT In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.

Competence regulation by oxygen in Streptococcus pneumoniae: involvement of ciaRH and comCDE.

Anaerobic aerotolerant Streptococcus pneumoniae modulates its genetic transformability and its virulence in response to the oxygen concentration. The activity of a single protein encoded by nox and showing NADH oxidase activity is involved in these adaptive responses to O2. Northern blot analysis of wild‐type cultures grown under aerobic and microaerobic conditions indicated transcriptional control of comCDE by O2. An O2‐independent mutant strain carrying the gain‐of‐function mutation comE38KE was isolated and its analysis showed that ComE is a key point in competence stimulation by O2. Plasmid insertion mutations in ciaRH revealed that this two component signal‐transducing system negatively regulates comCDE transcription. The level of comCDE transcripts appears as a major control point in competence regulation by O2 and also by growth phase and cell density.

Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB

In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO(4) in vitro. Thus, mutations which either reduced the stability of MicA-PO(4) or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO(4) is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.

Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection

In the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Strepto‐coccus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in α‐galactoside meta‐bolism and galactose transport. DLDH is an enzyme that is classically involved in the three‐step conversion of 2‐oxo acids to their respective acyl‐CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N‐terminal lipoyl domain. DLDH‐negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. These DLDH‐negative bacteria grew normally in vitro but were avirulent in sepsis and lung infection models in mice, indicating that DLDH activity is necessary for the survival of pneumococci within the host. The lack of virulence was not associated with a loss of 2‐oxo acid dehydrogenase activity, as the wild‐type pneumococcal strains did not contain activity of any of the known 2‐oxo acid enzyme complexes. Instead, studies of carbohydrate utilization demonstrated that the DLDH‐negative bacteria were impaired for α‐galactoside and galactose metabolism. The DLDH mutants lost their ability to oxidize or grow with galactose or melibiose as sole carbon source and showed reduced oxidation and growth on raffinose or stachyose. The bacteria had an 85% reduction in α‐galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH‐negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism.

RegR, a global LacI/GalR family regulator, modulates virulence and competence in Streptococcus pneumoniae

ABSTRACT The homolactic and catalase-deficient pathogen Streptococcus pneumoniae is not only tolerant to oxygen but requires the activity of its NADH oxidase, Nox, to develop optimal virulence and competence for genetic transformation. In this work, we show that the global regulator RegR is also involved in these traits. Genetic dissection revealed that RegR regulates competence and the expression of virulence factors, including hyaluronidase. In bacteria grown in vitro, RegR represses hyaluronidase. At neutral pH, it increases adherence to A549 epithelial cells, and at alkaline pH, it acts upstream of the CiaRH two-component signaling system to activate competence. These phenotypes are not associated with changes in antibiotic resistance, central metabolism, and carbohydrate utilization. Although the RegR0 (where 0 indicates the loss of the protein) mutation is sufficient to attenuate experimental virulence of strain 23477 in mice, the introduction of an additional hyl0 (where 0 indicates the loss of function) mutation in the RegR0 strain 23302 dramatically reduces its virulence. This indicates that residual virulence of the RegR0 Hyl+ derivative is due to hyaluronidase and supports the dual role of RegR in virulence. This LacI/GalR regulator, not essential for in vitro growth in rich media, is indeed involved in the adaptive response of the pneumococcus via its control of competence, adherence, and virulence.

The MicAB Two-Component Signaling System Is Involved in Virulence of Streptococcus pneumoniae

ABSTRACT In Streptococcus pneumoniae, the two-component signaling system MicAB was previously shown to contribute to repression of competence when oxygen is limited. In virulent strains expressing the serotype 2 and 6 capsule, mutation of the MicB kinase reduced the lag period of growth when cultures were switched from an aerobic to anaerobic atmosphere. After intranasal challenge of mice, the micB::km mutation decreased virulence, as shown by the absence of symptoms and by a lower level of recovery of CFU from lungs and blood. It is proposed that MicAB is involved in the adaptive response of the bacteria to changes in oxygen level during the course of infection.

Lipid composition of aminopterin-resistant and sensitive strains of Streptococcus pneumoniae. Effect of aminopterin inhibition

The polar lipids of Streptococcus pneumoniae wild type and aminopterin-resistant strains were analysed. The membrane contained only two acid phospholipids, phosphatidylglycerol and cardiolipin, and a large amount of two glycolipids, glucosyldiglyceride and galactosylglucosyldiglyceride. The unsaturated acyl chains ranged from 58 to 87% of total fatty acids, depending on the strain and on growth conditions. No relation could be established between aminopterin resistance and polar lipid or fatty acid compositions. However, in the presence of bacteriostatic concentrations of aminopterin, the wild type and the resistant mutant did not have the same behavior. The resistant strain maintained its fatty acid composition and a normal [32P]phosphate distribution among phospholipids while the wild type shifted to a higher content in unsaturated fatty acids and to a high relative cardiolipin labelling. Such a differencein [32P] distribution was not observed when bacteriostatic concentrations of chloramphenicol were used, or when growth was stopped after amino acid deprivation induced by high concentrations of isoleucine. The biochemical basis of the aminopterin resistant character of the amiA mutants are not yet well understood but the present study establishes that the mutation confers a certain insensitivity of the lipid metabolism to aminopterin.