Structure of the pneumococcal l,d-carboxypeptidase DacB and pathophysiological effects of disabled cell wall hydrolases DacA and DacB.

Bacterial cell wall hydrolases are essential for peptidoglycan turnover and crucial to preserve cell shape. The d,d-carboxypeptidase DacA and l,d-carboxypeptidase DacB of Streptococcus pneumoniae function in a sequential manner. Here, we determined the structure of the surface-exposed lipoprotein DacB. The crystal structure of DacB, radically different to that of DacA, contains a mononuclear Zn(2+) catalytic centre located in the middle of a large and fully exposed groove. Two different conformations were found presenting a different arrangement of the active site topology. The critical residues for catalysis and substrate specificity were identified. Loss-of-function of DacA and DacB altered the cell shape and this was consistent with a modified peptidoglycan peptide composition in dac mutants. Contrary, an lgt mutant lacking lipoprotein diacylglyceryl transferase activity required for proper lipoprotein maturation retained l,d-carboxypeptidase activity and showed an intact murein sacculus. In addition we demonstrated pathophysiological effects of disabled DacA or DacB activities. Real-time bioimaging of intranasal infected mice indicated a substantial attenuation of ΔdacB and ΔdacAΔdacB pneumococci, while ΔdacA had no significant effect. In addition, uptake of these mutants by professional phagocytes was enhanced, while the adherence to lung epithelial cells was decreased. Thus, structural and functional studies suggest DacA and DacB as optimal drug targets.

Characterization of central carbon metabolism of Streptococcus pneumoniae by isotopologue profiling.

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-(13)C(6)]- or [1,2-(13)C(2)]glucose. GC/MS analysis of protein-derived amino acids showed lack of (13)C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high (13)C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-(13)C(2)]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci.

Impact of glutamine transporters on pneumococcal fitness under infection-related conditions.

The genomic analysis of Streptococcus pneumoniae predicted six putative glutamine uptake systems, which are expressed under in vitro conditions, as shown here by reverse transcription-PCR. Four of these operons consist of glnHPQ, while two lack glnH, which encodes a soluble glutamine-binding protein. Here, we studied the impact of two of these glutamine ATP-binding cassette transporters on S. pneumoniae D39 virulence and phagocytosis, which consist of GlnQ and a translationally fused protein of GlnH and GlnP. Mice infected intranasally with D39Δgln0411/0412 showed significantly increased survival times and a significant delay in the development of pneumococcal pneumonia compared to those infected with D39, as observed in real time using bioluminescent pneumococci. In a mouse sepsis model, the mutant D39Δgln0411/0412 showed only moderate but significant attenuation. In contrast, the D39Δgln1098/1099 knockout strain was massively attenuated in the pneumonia and septicemia mouse infection model. To cause pneumonia or sepsis with D39Δgln1098/1099, infection doses 100- to 10,000-fold higher than those used for wild-type strain D39 were required. In an experimental mouse meningitis model, D39Δgln1098/1099 produced decreased levels of white blood cells in cerebrospinal fluid and showed decreased numbers of bacteria in the bloodstream compared to D39 and D39Δgln0411/0412. Phagocytosis experiments revealed significantly decreased intracellular survival rates of mutants D39Δgln1098/1099 and D39Δgln0411/0412 compared to wild-type D39, suggesting that the deficiency of Gln uptake systems impairs resistance to oxidative stress. Taken together, our results demonstrate that both glutamine uptake systems are required for full virulence of pneumococci but exhibit different impacts on the pathogenesis of pneumococci under in vivo conditions.

PavB is a surface-exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections.

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose-dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with DeltapavB pneumococci showed significantly increased survival times compared with wild-type bacteria. Importantly, the pavB-mutant showed a delay in transmigration to the lungs as observed in real-time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co-infection experiments the wild-type out-competed the pavB-mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface-exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.

Pneumococcal Metabolic Adaptation and Colonization Are Regulated by the Two-Component Regulatory System 08.

Streptococcus pneumoniae two-component regulatory systems (TCS) enable adaptation and ensure its maintenance in host environments. This study deciphers the impact of TCS08 on pneumococcal gene expression and its role in metabolic and pathophysiological processes. Transcriptome analysis and real-time PCR demonstrated a regulatory effect of TCS08 on genes involved mainly in environmental information processing, intermediary metabolism, and colonization by S. pneumoniae D39 and TIGR4. Striking examples are genes for fatty acid biosynthesis, genes of the arginine deiminase system, and the psa operon encoding the manganese ABC transport system. In silico analysis confirmed that TCS08 is homologous to Staphylococcus aureus SaeRS, and a SaeR-like binding motif is displayed in the promoter region of pavB, the upstream gene of the tcs08 operon encoding a surface-exposed adhesin. Indeed, PavB is regulated by TCS08 as confirmed by immunoblotting and surface abundance assays. Similarly, pilus-1 of TIGR4 is regulated by TCS08. Finally, in vivo infections using the acute pneumonia and sepsis models showed a strain-dependent effect. Loss of function of HK08 or TCS08 attenuated D39 virulence in lung infections. The RR08 deficiency attenuated TIGR4 in pneumonia, while there was no effect on sepsis. In contrast, lack of HK08 procured a highly virulent TIGR4 phenotype in both pneumonia and sepsis infections. Taken together, these data indicate the importance of TCS08 in pneumococcal fitness to adapt to the milieu of the respiratory tract during colonization.IMPORTANCEStreptococcus pneumoniae interplays with its environment by using 13 two-component regulatory systems and one orphan response regulator. These systems are involved in the sensing of environmental signals, thereby modulating pneumococcal pathophysiology. This study aimed to understand the functional role of genes subject to control by the TCS08. The identified genes play a role in transport of compounds such as sugars or amino acids. In addition, the intermediary metabolism and colonization factors are modulated by TCS08. Thus, TCS08 regulates genes involved in maintaining pneumococcal physiology, transport capacity, and adhesive factors to enable optimal colonization, which represents a prerequisite for invasive pneumococcal disease.

Challenges and Opportunities in the Development of Aptamers for TNFα.

RNA aptamers for tumor necrosis factor-alpha (TNFα), for which functionality was demonstrated in L929 cells, show only little affinity for the protein in vitro. Detailed investigation of the aptamer-protein interaction by surface plasmon resonance and quartz crystal microbalance analysis revealed that affinity is not the only crucial parameter for efficacy and functionality of those aptamers. Instead, the sensitive equilibrium of the monomeric and homotrimeric form of soluble TNFα decides on aptamer binding. Our results show that the field of application and the source of TNFα have to be carefully defined before selection of aptamer sequences.

Analysis of the zwf-pgl-eda-operon in Pseudomonas putida strains H and KT2440.

A 3.9-kb fragment of the genome of Pseudomonas putida H, containing the complete zwf-pgl-eda-operon, encoding glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 2-keto-3-deoxy-6-phosphogluconate-aldolase, respectively, and part of the divergently transcribed regulatory gene, hexR, was cloned and analyzed. The nucleotide sequences of these genes showed high similarities to the corresponding DNA sequences of P. putida KT2440 and also to sequences of Pseudomonas aeruginosa PAO1. Derivatives of strains H and KT2440, containing transcriptional lacZ fusions to P(zwf) were generated and used to study the expression of these operons. In both strains, this operon was induced by carbohydrates such as glucose, gluconate, fructose and glycerol. The transcription rate of the zwf-pgl-eda-operon was found to be about three times higher in the KT2440 background than in strain H. In both strains the induction of the zwf-pgl-eda-operon by carbohydrates during growth on carboxylic acids was not affected by carbon catabolite repression.

Regulation of the arginine deiminase system by ArgR2 interferes with arginine metabolism and fitness of Streptococcus pneumoniae.

UNLABELLED: Streptococcus pneumoniae is auxotrophic for arginine, and molecular analysis of the pneumococcal genome showed that the gene encoding an arginine-ornithine antiporter (ArcD) is organized in a cluster together with the arcABC genes encoding the arginine deiminase system (ADS) of pneumococci. The ADS consists of the arginine deiminase (AD), the catabolic ornithine carbamoyltransferase (cOCT), and the carbamate kinase (CK). Pneumococcal genomes contain three ArgR-type regulators (ArgR1, ArgR2, and AhrC) that are supposed to be involved in the regulation of arginine metabolism. Here, we identified ArgR2 of TIGR4 as the regulator of the ADS and ArcD. ArgR2 binds to promoter sequences of the arc operon, and the deficiency of ArgR2 in TIGR4 abrogates expression of the ADS, including the arginine-ornithine antiporter ArcD. Intranasal infection of mice and real-time bioimaging revealed that deletion of the arcABCDT genes attenuates TIGR4. However, the acute-pneumonia model and coinfection experiments indicated that the arginine-ornithine antiporter ArcD is essential to maintain fitness, while the deficiency of ADS enzymes has a minor impact on pneumococcal fitness under in vivo conditions. Strikingly, argR2 mutant TIGR4 outcompeted the wild type in the respiratory tract, suggesting an increase in fitness and further regulatory functions of ArgR2. In contrast to TIGR4, other pneumococci, such as D39, lacking expression of ArgR2, constitutively express the ADS with a truncated nonfunctional AD. On the basis of these results, we propose that the arginine-ornithine antiporter is essential to maintain pneumococcal fitness and that the genes of the ADS cluster are positively regulated in a strain-specific manner by ArgR2. IMPORTANCE: Pneumococci are the major etiologic agents of community-acquired pneumonia, causing more than 1.5 million deaths annually worldwide. These versatile pathogens are highly adapted to the nutrients provided by the host niches encountered. Physiological fitness is of major importance for colonization of the nasopharyngeal cavity and dissemination during invasive infections. This work identifies the regulator ArgR2 as the activator of the S. pneumoniae TIGR4 ADS and the arginine-ornithine transporter ArcD, which is needed for uptake of the essential amino acid arginine. Although ArgR2 activates ArcD expression and uptake of arginine is required to maintain pneumococcal fitness, the deficiency of ArgR2 increases TIGR4 virulence under in vivo conditions, suggesting that other factors regulated by ArgR2 counterbalance the reduced uptake of arginine by ArcD. Thus, this work illustrates that the physiological homeostasis of pneumococci is complex and that ArgR2 plays a key role in maintaining bacterial fitness.

Molecular architecture of Streptococcus pneumoniae surface thioredoxin-fold lipoproteins crucial for extracellular oxidative stress resistance and maintenance of virulence.

The respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we characterize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their redox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2 O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the way for the design of novel antimicrobials.

Recognition of drug degradation products by target proteins: isotetracycline binding to Tet repressor.

Tetracycline antibiotics and their degradation products appear in medically treated tissues, food, soil, and manure sludge in the environment. In the context of protein interactions with various tetracyclines we performed crystal structure analyses of the tetracycline repressor in complex with weak or noninducing tetracycline derivatives. Isotetracyclines are degradation products of tetracyclines, which occur under physiological conditions. The typical framework of the antibiotic is irreversibly broken at the BC-ring connection, leading to a modified orientation of the AB to the new C*D ring fragments. The shape of the zwitterionic AB-ring fragment is unchanged and still binds to the TetR recognition site in a manner comparable to the intact antibiotic but without typical Mg(2+) chelation. This work is an example that drug degradation products can still bind to specific targets and should be discussed in light of potential and critical side effects.

Analysis of the proteome of Pseudomonas putida KT2440 grown on different sources of carbon and energy.

Using 2D electrophoresis the protein expression pattern during growth on carbon sources with different impact on carbon catabolite repression of phenol degradation was analysed in a derivative of Pseudomonas putida KT2440. The cytosolic protein pattern of cells growing on phenol or the non-repressive substrate pyruvate was almost identical, but showed significant differences to that of cells growing with the repressive substrates succinate or glucose. Proteins, which were mainly expressed in the presence of phenol or pyruvate, could be assigned to the functional groups of transport, detoxification, stress response, amino acid, energy, carbohydrate and nucleotide metabolism. The addition of succinate to cells growing with phenol ('shift-up') resulted in the inhibition of the synthesis of these proteins. Proteins with enhanced expression at growth with succinate or glucose were proteins for de novo synthesis of nucleotides, amino acids and enzymes of the TCA cycle. The synthesis of proteins, necessary for phenol catabolism was regulated in different manners following the addition of succinate. Whereas the synthesis of Phl-proteins (subunits of the phenolhydroxylase) only decreased slowly, was the translation of the Cat-proteins (catechol 1,2-dioxygenase, cis,cis-muconate cycloisomerase and muconolactone isomerase) repressed immediately and the synthesis of the Pca-proteins (beta-ketoadipate enolactone hydrolase, beta-ketoadipate succinyl-CoA transferase and beta-ketoadipyl CoA thiolase) remained unaffected.