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Metabolism

The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements.

Sci Rep. 2017 Aug 8;7(1):7609. doi: 10.1038/s41598-017-07716-5.

The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements.

Sebastián M1,2, Lira-Navarrete E2,3, Serrano A1,2,4, Marcuello C5,6, Velázquez-Campoy A1,2,7,8, Lostao A5,7, Hurtado-Guerrero R2,7, Medina M9,10, Martínez-Júlvez M11,12.

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Abstract

Prokaryotic bifunctional FAD synthetases (FADSs) catalyze the biosynthesis of FMN and FAD, whereas in eukaryotes two enzymes are required for the same purpose. FMN and FAD are key cofactors to maintain the flavoproteome homeostasis in all type of organisms. Here we shed light to the properties of the hitherto unstudied bacterial FADS from the human pathogen Streptococcus pneumoniae (SpnFADS). As other members of the family, SpnFADS catalyzes the three typical activities of prokaryotic FADSs: riboflavin kinase (RFK), ATP:FMN:adenylyltransferase (FMNAT), and FAD pyrophosphorylase (FADpp). However, several SpnFADS biophysical properties differ from those of other family members. In particular; i) the RFK activity is not inhibited by the riboflavin (RF) substrate, ii) the FMNAT and FADSpp activities require flavin substrates in the reduced state, iii) binding of adenine nucleotide ligands is required for the binding of flavinic substrates/products and iv) the monomer is the preferred state. Collectively, our results add interesting mechanistic differences among the few prokaryotic bifunctional FADSs already characterized, which might reflect the adaptation of the enzyme to relatively different environments. In a health point of view, differences among FADS family members provide us with a framework to design selective compounds targeting these enzymes for the treatment of diverse infectious diseases.

PMID: 28790457 PMCID: PMC5548840 DOI: 10.1038/s41598-017-07716-5

The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions.

mSystems. 2017 Jun 6;2(3). pii: e00019-17. doi: 10.1128/mSystems.00019-17. eCollection 2017 May-Jun.

The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions.

Wuchty S1,2,3,4, Rajagopala SV5, Blazie SM5, Parrish JR6, Khuri S1,2, Finley RL Jr6, Uetz P7.

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Abstract

The functions of roughly a third of all proteins in Streptococcus pneumoniae, a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein's function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins.

KEYWORDS:

functional prediction; protein-protein interactions

PMID: 28744484 PMCID: PMC5513735 DOI: 10.1128/mSystems.00019-17

Effect of decreased BCAA synthesis through disruption of ilvC gene on the virulence of Streptococcus pneumoniae.

Arch Pharm Res. 2017 Jul 22. doi: 10.1007/s12272-017-0931-0. [Epub ahead of print]

Effect of decreased BCAA synthesis through disruption of ilvC gene on the virulence of Streptococcus pneumoniae.

Kim GL1, Lee S1, Luong TT1, Nguyen CT1, Park SS1, Pyo S1, Rhee DK2.

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Abstract

Streptococcus pneumoniae (pneumococcus) is responsible for significant morbidity and mortality worldwide. It causes a variety of life-threatening infections such as pneumonia, bacteremia, and meningitis. In bacterial physiology, the metabolic pathway of branched-chain amino acids (BCAAs) plays an important role in virulence. Nonetheless, the function of IlvC, one of the enzymes involved in the biosynthesis of BCAAs, in S. pneumoniae remains unclear. Here, we demonstrated that downregulation of BCAA biosynthesis by ilvC ablation can diminish BCAA concentration and expression of pneumolysin (Ply) and LytA, and subsequently attenuate virulence. Infection with an ilvC mutant showed significantly reduced mortality and colonization in comparison with strain D39 (serotype 2, wild type), suggesting that ilvC can potentiate S. pneumoniae virulence due to adequate BCAA synthesis. Taken together, these results suggest that the function of ilvC in BCAA synthesis is essential for virulence factor and could play an important role in the pathogenesis of respiratory infections.

KEYWORDS:

BCAA; Colonization; Streptococcus pneumoniae; Virulence; ilvC

PMID: 28735462 DOI: 10.1007/s12272-017-0931-0

 

 

Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.

Mol Microbiol. 2017 Jul 15. doi: 10.1111/mmi.13748. [Epub ahead of print]

Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.

Stamsås GA1, Straume D1, Ruud Winther A1, Kjos M1, Frantzen CA1, Håvarstein LS1.

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Abstract

In a screen for mutations suppressing the lethal loss of PBP2b in Streptococcus pneumoniae we identified Spr1851 (named EloR), a cytoplasmic protein of unknown function whose inactivation removed the requirement for PBP2b as well as RodA. It follows from this that EloR and the two elongasome proteins must be part of the same functional network. This network also includes StkP, as this serine/threonine kinase phosphorylates EloR on threonine 89 (T89). We found that ΔeloR cells, and cells expressing the phosphoablative form of EloR (EloRT89A ), are significantly shorter than wild-type cells. Furthermore, the phosphomimetic form of EloR (EloRT89E ) is not tolerated unless the cell in addition acquires a truncated MreC or non-functional RodZ protein. By itself, truncation of MreC as well as inactivation of RodZ gives rise to less elongated cells, demonstrating that the stress exerted by the phosphomimetic form of EloR is relieved by suppressor mutations that reduce or abolish the activity of the elongasome. Of note, it was also found that loss of elongasome activity caused by truncation of MreC elicits increased StkP-mediated phosphorylation of EloR. Together, the results support a model in which phosphorylation of EloR stimulates cell elongation, while dephosphorylation has an inhibitory effect.

© 2017 John Wiley & Sons Ltd.

PMID: 28710862 DOI: 10.1111/mmi.13748

J Org Chem. 2017 Aug 4;82(15):8123-8140. doi: 10.1021/acs.joc.7b01264. Epub 2017 Jul 19.

Structure-Reactivity Relationships of Conformationally Armed Disaccharide Donors and Their Use in the Synthesis of a Hexasaccharide Related to the Capsular Polysaccharide from Streptococcus pneumoniae Type 37.

Angles d'Ortoli T1, Hamark C1, Widmalm G1.

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Abstract

To advance the field of glycobiology, efficient synthesis methods of oligosaccharides and glycoconjugates are a requisite. In glycosylation reactions using superarmed donors, both selectivity and reactivity issues must be considered, and we herein investigate these aspects for differently protected β-linked 2-O-glycosylated glucosyl donors carrying bulky tert-butyldimethylsilyl groups to different extents. The acceptors in reactions being secondary alcohols presents a challenging situation with respect to steric crowding. Conformational pyranose ring equilibria of the superarmed disaccharide donors with axial-rich substituents contained skew and boat conformations, and three-state models were generally assumed. With NIS/TfOH as the promotor, 2,6-di-tert-butyl-4-methylpyridine as the base, and a dichloromethane/toluene solvent mixture, ethyl 1-thio-β-d-glucosyl disaccharide donors having 6-O-benzyl group(s) besides tert-butyldimethylsilyl groups were efficiently coupled at -40 °C to the hydroxyl group at position 3 of glucopyranosyl acceptors to form β-(1 → 2),β-(1 → 3)-linked trisaccharides, isolated in excellent 95% yield. The more axial-rich donors in skew and boat conformations are thus preorganized closer to the assumed transition state in these glycosylation reactions. The developed methodology was subsequently applied in the synthesis of a multibranched hexasaccharide related to the capsular polysaccharide from Streptococcus pneumoniae type 37, which consists of a β-(1 → 3)-linked backbone and a β-(1 → 2)-linked side chain of d-glucosyl residues in disaccharide repeating units.

PMID: 28682619 DOI: 10.1021/acs.joc.7b01264

Chromosome segregation drives division site selection in Streptococcus pneumoniae.

Proc Natl Acad Sci U S A. 2017 Jul 3. pii: 201620608. doi: 10.1073/pnas.1620608114. [Epub ahead of print]

Chromosome segregation drives division site selection in Streptococcus pneumoniae.

van Raaphorst R1,2, Kjos M1,3, Veening JW4,2.

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Abstract

Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae(pneumococcus) is an oval-shaped, symmetrically dividing opportunistic human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus, the question remains as to what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, before FtsZ. Interestingly, Z-ring formation occurs coincidently with initiation of DNA replication. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting, or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation, and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells.

KEYWORDS:

FtsZ; SMC; Streptococcus pneumoniae; cell division; chromosome organization

PMID: 28674002 DOI: 10.1073/pnas.1620608114

Proteomic comparisons of opaque and transparent variants of Streptococcus pneumoniae by two dimensional-differential gel electrophoresis.

Sci Rep. 2017 May 26;7(1):2453. doi: 10.1038/s41598-017-02465-x.

Proteomic comparisons of opaque and transparent variants of Streptococcus pneumoniae by two dimensional-differential gel electrophoresis.

Chai MH1, Weiland F2,3,4, Harvey RM1, Hoffmann P2,3, Ogunniyi AD5,6, Paton JC1.

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Abstract

Streptococcus pneumoniae (the pneumococcus) is a human pathogen, accounting for massive global morbidity and mortality. Although asymptomatic colonization of the nasopharynx almost invariably precedes disease, the critical determinants enabling pneumococcal progression from this niche to cause invasive disease are poorly understood. One mechanism proposed to be central to this transition involves opacity phase variation, whereby pneumococci harvested from the nasopharynx are typically transparent, while those simultaneously harvested from the blood are opaque. Here, we used two dimensional-differential gel electrophoresis (2D-DIGE) to compare protein expression profiles of transparent and opaque variants of 3 pneumococcal strains, D39 (serotype 2), WCH43 (serotype 4) and WCH16 (serotype 6A) in vitro. One spot comprising a mixture of capsular polysaccharide biosynthesis protein and other proteins was significantly up-regulated in the opaque phenotype in all 3 strains; other proteins were differentially regulated in a strain-specific manner. We conclude that pneumococcal phase variation is a complex and multifactorial process leading to strain-specific pathogenicity.

PMID: 28550292 PMCID: PMC5446427 DOI: 10.1038/s41598-017-02465-x

Proteomic variation and diversity in clinical Streptococcus pneumoniaeisolates from invasive and non-invasive sites.

PLoS One. 2017 Jun 2;12(6):e0179075. doi: 10.1371/journal.pone.0179075. eCollection 2017.

Proteomic variation and diversity in clinical Streptococcus pneumoniaeisolates from invasive and non-invasive sites.

Bittaye M1, Cash P1, Forbes K1.

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Abstract

Streptococcus pneumoniae is responsible for a variety of invasive and non-invasive human infections. There are over 90 serotypes of S. pneumoniae differing in their ability to adapt to the different niches within the host. Two-dimensional gel electrophoresis was used to discriminate clinical S. pneumoniae isolates recovered from either blood cultures (invasive site isolates) or other sites, including sputum, tracheal aspirate, ear, eye and skin swabs (non-invasive site isolates). Global protein expression profiles for five invasive site and six non-invasive site isolates representing five different serotypes (serotypes 4, 6, 9, 14 and 23) were obtained for each isolate and combined into a single data set using Progenesis SameSpots™ software. One-hundred and eighty six protein spots (39% of the protein spots in the dataset) differed significantly (ANOVA, p<0.05) in abundance between the invasive site (101 upregulated protein spots) and non-invasive site (85 upregulated protein spots) isolates. Correlations between the bacterial proteomes and their sites of isolation were determined by Principal Component Analysis (PCA) using the significantly different protein spots. Out of the 186 variable protein spots, 105 exhibited a serotype-associated pattern of variability. The expression of the remaining 81 protein spots was concluded to be uniquely linked to the site of bacterial isolation. Mass spectrometry was used to identify selected protein spots that showed either constant or differential abundance levels. The identified proteins had a diverse range of functions including, capsule biogenesis, DNA repair, protein deglycation, translation, stress response and virulence as well as amino acid, carbohydrate, lipid and nucleotide metabolism. These findings provide insight on the proteins that contribute towards the adaptation of the bacteria to different sites within the host.

PMID: 28575057 PMCID: PMC5456405 DOI: 10.1371/journal.pone.0179075

Integrated proteomic and metabolomic analysis reveals that rhodomyrtone reduces the capsule in Streptococcus pneumoniae.

Sci Rep. 2017 Jun 2;7(1):2715. doi: 10.1038/s41598-017-02996-3.

Integrated proteomic and metabolomic analysis reveals that rhodomyrtone reduces the capsule in Streptococcus pneumoniae.

Mitsuwan W1, Olaya-Abril A2, Calderón-Santiago M3, Jiménez-Munguía I2, González-Reyes JA4, Priego-Capote F3, Voravuthikunchai SP1, Rodríguez-Ortega MJ5.

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Abstract

The emergence of antibiotic-resistant pathogenic bacteria is a healthcare problem worldwide. We evaluated the antimicrobial activity of rhodomyrtone, an acylphloroglucinol present in Rhodomyrtus tomentosa leaves, against the human Gram-positive pathogen Streptococcus pneumoniae. The compound exhibited pronounced anti-pneumococcal activity against a broad collection of clinical isolates. We studied the effects at the molecular level by integrated proteomic and metabolomic analysis. The results revealed alterations in enzymes and metabolites involved in several metabolic pathways including amino acid biosynthesis, nucleic acid biosynthesis, glucid, and lipid metabolism. Notably, the levels of two enzymes (glycosyltransferase and UTP-glucose-1-phosphate uridylyltransferase) and three metabolites (UDP-glucose, UDP-glucuronic acid and UDP-N-acetyl-D-galactosamine) participating in the synthesis of the pneumococcal capsule clearly diminished in the bacterial cells exposed to rhodomyrtone. Rhodomyrtone-treated pneumococci significantly possessed less amount of capsule, as measured by a colorimetric assay and visualized by electron microscopy. These findings reveal the utility of combining proteomic and metabolomic analyses to provide insight into phenotypic features of S. pneumoniae treated with this potential novel antibiotic. This can lead to an alternative antibiotic for the treatment of S. pneumoniae infections, because of the growing concern regarding antimicrobial resistance.

PMID: 28578394 PMCID: PMC5457420 DOI: 10.1038/s41598-017-02996-3

Transition Metal Homeostasis in Streptococcus pyogenes and Streptococcus pneumoniae.

Adv Microb Physiol. 2017;70:123-191. doi: 10.1016/bs.ampbs.2017.01.002. Epub 2017 Feb 20.

Transition Metal Homeostasis in Streptococcus pyogenes and Streptococcus pneumoniae.

Turner AG1, Ong CY1, Walker MJ1, Djoko KY1, McEwan AG2.

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Abstract

Trace metals such as Fe, Mn, Zn and Cu are essential for various biological functions including proper innate immune function. The host immune system has complicated and coordinated mechanisms in place to either starve and/or overload invading pathogens with various metals to combat the infection. Here, we discuss the roles of Fe, Mn and Zn in terms of nutritional immunity, and also the roles of Cu and Zn in metal overload in relation to the physiology and pathogenesis of two human streptococcal species, Streptococcus pneumoniae and Streptococcus pyogenes. S. pneumoniae is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the population; however, transition to internal sites can cause a range of diseases such as pneumonia, otitis media, meningitis and bacteraemia. S. pyogenes is a human pathogen responsible for diseases ranging from pharyngitis and impetigo, to severe invasive infections. Both species have overlapping capacity with respect to metal acquisition, export and regulation and how metal homeostasis relates to their virulence and ability to invade and survive within the host. It is becoming more apparent that metals have an important role to play in the control of infection, and with further investigations, it could lead to the potential use of metals in novel antimicrobial therapies.

© 2017 Elsevier Ltd. All rights reserved.

KEYWORDS:

Copper; Group A Streptococcus; Iron; Manganese; Metal homeostasis; Streptococcus pneumoniae; Streptococcus pyogenes; Zinc

PMID: 28528647 DOI: 10.1016/bs.ampbs.2017.01.002

Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D-N-acetylglucosamine.

Proc Natl Acad Sci U S A. 2017 May 30;114(22):5695-5700. doi: 10.1073/pnas.1620431114. Epub 2017 May 11.

Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D-N-acetylglucosamine.

Larson TR1, Yother J2.

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Abstract

For many bacteria, including those important in pathogenesis, expression of a surface-localized capsular polysaccharide (CPS) can be critical for survival in host environments. In Gram-positive bacteria, CPS linkage is to either the cytoplasmic membrane or the cell wall. Despite the frequent occurrence and essentiality of these polymers, the exact nature of the cell wall linkage has not been described in any bacterial species. Using the Streptococcus pneumoniae serotype 2 CPS, which is synthesized by the widespread Wzy mechanism, we found that linkage occurs via the reducing end glucose of CPS and the β-D-N-acetylglucosamine (GlcNAc) residues of peptidoglycan (PG). Hydrofluoric acid resistance, 31P-NMR, and 32P labeling demonstrated the lack of phosphodiester bonds, which typically occur in PG-polysaccharide linkages. Component sugar analysis of purified CPS-PG identified only CPS and PG sugars in the appropriate ratios, suggesting the absence of an oligosaccharide linker. Time of flight mass spectrometry confirmed a direct glycosidic linkage between CPS and PG and showed that a single CPS repeat unit can be transferred to PG. The linkage was acetolysis susceptible, indicative of a 1,6 glycosidic bond between CPS and the GlcNAc C-6. The acetylation state of GlcNAc did not affect linkage. A direct glycosidic linkage to PG was also demonstrated for serotypes 8 and 31, whose reducing end sugars are glucose and galactose, respectively. These results provide the most detailed descriptions of CPS-PG linkages for any microorganism. Identification of the linkage is a first step toward identifying the linking enzyme and potential inhibitors of its activity.

KEYWORDS:

Gram positive; Wzy; capsule; glycobiology; pneumococcus

PMID: 28495967 PMCID: PMC5465879 [Available on 2017-11-30] DOI: 10.1073/pnas.1620431114

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae.

Front Cell Infect Microbiol. 2017 Mar 9;7:70. doi: 10.3389/fcimb.2017.00070. eCollection 2017.

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae.

Afzal M1, Kuipers OP2, Shafeeq S3.

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Abstract

NAD (Nicotinamide Adenine Dinucleotide) biosynthesis is vital for bacterial physiology and plays an important role in cellular metabolism. A naturally occurring vitamin B complex, niacin (nicotinic acid), is a precursor of coenzymes NAD and NADP. Here, we study the impact of niacin on global gene expression of Streptococcus pneumoniae D39 and elucidate the role of NiaR as a transcriptional regulator of niaX, nadC, and pnuC. Transcriptome comparison of the D39 wild-type grown in chemically defined medium (CDM) with 0 to 10 mM niacin revealed elevated expression of various genes, including niaX, nadC, pnuC, fba, rex, gapN, pncB, gap, adhE, and adhB2 that are putatively involved in the transport and utilization of niacin. Niacin-dependent expression of these genes is confirmed by promoter lacZ-fusion studies. Moreover, the role of transcriptional regulator NiaR in the regulation of these genes is explored by DNA microarray analysis. Our transcriptomic comparison of D39 ΔniaR to D39 wild-type revealed that the transcriptional regulator NiaR acts as a transcriptional repressor of niaX, pnuC, and nadC. NiaR-dependent regulation of niaX, nadC, and pnuC is further confirmed by promoter lacZ-fusion studies. The putative operator site of NiaR (5'-TACWRGTGTMTWKACASYTRWAW-3') in the promoter regions of niaX, nadC, and pnuC is predicted and further confirmed by promoter mutational experiments.

KEYWORDS:

NiaR; Pneumococcus; nadC; niaX; niacin; pnuC

PMID: 28337428 PMCID: PMC5343564 DOI: 10.3389/fcimb.2017.00070