Calorimetric analysis of AdcR and its interactions with zinc(II) and DNA.

Zinc(II) ions play critical roles in all known life as structurally important stabilizing ions in proteins, catalytically active metals in enzymes, and signaling agents impacting physiological changes. To maintain homeostasis, the intracellular concentration of zinc(II) is strictly controlled by a family of metal-regulatory proteins in both prokaryotic and eukaryotic organisms. In S. pneumoniae, there are two proteins that share responsibility for Zn2+ homeostasis, one of them is the Adhesin Competence Repressor (AdcR) and it binds to a specific double-stranded DNA binding domain (dsDNA). AdcR has been structurally characterized containing two zinc(II) metal centers per monomeric unit. Here we report data collected from differential scanning calorimetry (DSC) experiments aimed to measure the structural stability of AdcR, the fully complimented Zn2AdcR complex, and the protein/DNA complex Zn2AdcR/dsDNA. Thermograms collected from DSC experiments yielded endothermic unfolding events for AdcR, Zn2AdcR, and Zn2AdcR/dsDNA complex at 55.6, 70.2, and 56.6 °C, respectively. A non-two state unfolding model best fits the data, giving ΔH terms associated with these thermal unfolding events of 5.1, 7.1, and 4.9 kcal/mol. These data allow for the development of a thermodynamic cycle connecting both zinc(II) and DNA binding to AdcR. Furthermore, pairing this newly reported data with known association constants for zinc(II) and DNA binding allowed for the generation of thermodynamic profiles for both zinc(II) binding to AdcR and Zn2AdcR binding to DNA, which show both are decisively entropy-driven processes.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with ß-Lactones.

ß-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to ß-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of ß-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four ß-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the ß-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration.

Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His3Asp sites with high affinity. A similar His3Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.

Partial depolymerization of capsular polysaccharides isolated from Streptococcus pneumoniae serotype 2 by various methods.

Partial depolymerization of bacterial capsular polysaccharides (CPS) is an essential process carried out before its use as an antigenic preparation in a vaccine industry. Choice of CPS depolymerization methods depends on the process robustness, reproducibility, yield, retention of CPS bioactivity, etc. Partial depolymerization methods based on chemicals, enzymes, mechanical, thermal, etc. have been subject of many investigations before. Partial depolymerization of Streptococcus pneumoniae serotype 2 purified CPS was conducted by methods such as acid hydrolysis, microfluidization, ultrasonication, thermal and microwave. Partial depolymerization of the CPS was evaluated by size exclusion high performance liquid chromatography, whereas structural identity and conformity of CPS was ensured by 1H NMR spectroscopy. The antigenicity of CPS was assessed by bead based competitive inhibition assay. Microwave and thermal methods effectively depolymerized CPS and reduced the concentration of cell wall polysaccharide (CWPS) impurity, but both methods have a negative impact on the antigenicity of CPS. Whereas the trifluoroacetic acid treatment not only depolymerized the CPS but completely removed the CWPS while retaining the antigenicity of 92 ± 4% and this method is advantageous over other methods.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae.

MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors. Sequence similarity cannot predict whether these transporters are interchangeable. We showed that subtle changes in the central aqueous cavity of the transporter are sufficient to accommodate a different cargo. These changes can occur naturally, suggesting a potential mechanism of expanding substrate selectivity. A directed evolution experiment was performed to identify gain-of-function variants that translocate a noncognate cargo. Coupled with a high-throughput mutagenesis and sequencing (Mut-seq) experiment, residues that are functionally important for the capsule transporter were revealed. Lastly, we showed that the expression of a flippase that can transport unfinished precursors resulted in an increased susceptibility to bacitracin and mild cell shape defects, which may be a driving force to maintain transporter specificity. IMPORTANCE All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens. Most of the CPSs are produced by the Wzx/Wzy mechanism. In this pathway, CPS repeating units are synthesized in the cytoplasm, which must be flipped across the cytoplasmic membrane before polymerization. This step is mediated by the widely conserved MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters. Here, we systematically evaluated the interchangeability of these transporters and identified the residues important for substrate specificity and function. Understanding how CPS is synthesized will inform glycoengineering, vaccine development, and antimicrobial discovery.

A Structural Model for the Ligand Binding of Pneumococcal Serotype 3 Capsular Polysaccharide-Specific Protective Antibodies.

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.

Prediction of human-Streptococcus pneumoniae protein-protein interactions using logistic regression.

Streptococcus pneumoniae is a major cause of mortality in children under five years old. In recent years, the emergence of antibiotic-resistant strains of S. pneumoniae increases the threat level of this pathogen. For that reason, the exploration of S. pneumoniae protein virulence factors should be considered in developing new drugs or vaccines, for instance by the analysis of host-pathogen protein-protein interactions (HP-PPIs). In this research, prediction of protein-protein interactions was performed with a logistic regression model with the number of protein domain occurrences as features. By utilizing HP-PPIs of three different pathogens as training data, the model achieved 57-77 % precision, 64-75 % recall, and 96-98 % specificity. Prediction of human-S. pneumoniae protein-protein interactions using the model yielded 5823 interactions involving thirty S. pneumoniae proteins and 324 human proteins. Pathway enrichment analysis showed that most of the pathways involved in the predicted interactions are immune system pathways. Network topology analysis revealed ß-galactosidase (BgaA) as the most central among the S. pneumoniae proteins in the predicted HP-PPI networks, with a degree centrality of 1.0 and a betweenness centrality of 0.451853. Further experimental studies are required to validate the predicted interactions and examine their roles in S. pneumoniae infection.

Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum.

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F.

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

Harnessing Multiple, Nonproteogenic Substitutions to Optimize CSP:ComD Hydrophobic Interactions in Group†1 Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) is a human pathobiont that causes drastic antibiotic-resistant infections and is responsible for millions of deaths universally. Pneumococcus pathogenicity relies on the competence-stimulating peptide (CSP)-mediated quorum-sensing (QS) pathway that controls competence development for genetic transformation and, consequently, the spread of antibiotic resistance and virulence genes. Modulation of QS in S. pneumoniae can therefore be used to enervate pneumococcal infectivity as well as minimize the susceptibility to resistance development. In this work, we sought to optimize the interaction of CSP1 with its cognate transmembrane histidine kinase receptor (ComD1) through substitution of proteogenic and nonproteogenic amino acids on the hydrophobic binding face of CSP1. The findings from this study not only provided additional structure-activity data that are significant in optimizing CSP1 potency, but also led to the development of potent QS modulators. These CSP-based QS modulators could be used as privileged scaffolds for the development of antimicrobial agents against pneumococcal infections.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Bacterial surface capsular polysaccharides from Streptococcus pneumoniae: A systematic review on structures, syntheses, and glycoconjugate vaccines.

The polysaccharide capsule of Streptococcus pneumoniae constitutes the outermost surface structure of the organism and plays a critical role in virulence. The capsule is the target of current pneumococcal vaccines and glycoconjugates and has important medical and industrial applications. Widespread use of these vaccines is driving changes in serotype prevalence in disease. A massive array of sugars and glycosidic linkages experienced with complete diversity of potential polysaccharide structures. However, it is impossible to collect a sufficient quantity of glycan antigens for the preparation of CPS-based glycoconjugate vaccines from natural sources with high purity and for thorough biological evaluation. So nowadays, the development of a chemical synthetic strategy and their conjugation with a carrier protein to form synthetic glycoconjugate vaccines has been used to gain access on a large scale. This review provides a comprehensive summary of structures, synthesis as well as recent development of synthetic glycoconjugate vaccines, which will support research and may benefit the glycochemical and medical sciences.

Streptococcus pneumoniae, S. mitis, and S. oralis Produce a Phosphatidylglycerol-Dependent, ltaS-Independent Glycerophosphate-Linked Glycolipid.

Lipoteichoic acid (LTA) is a Gram-positive bacterial cell surface polymer that participates in host-microbe interactions. It was previously reported that the major human pathogen Streptococcus pneumoniae and the closely related oral commensals S. mitis and S. oralis produce type IV LTAs. Herein, using liquid chromatography/mass spectrometry-based lipidomic analysis, we found that in addition to type IV LTA biosynthetic precursors, S. mitis, S. oralis, and S. pneumoniae also produce glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a biosynthetic precursor of type I LTA. cdsA and pgsA mutants produce DHDAG but lack (Gro-P)-DHDAG, indicating that the Gro-P moiety is derived from phosphatidylglycerol (PG), whose biosynthesis requires these genes. S. mitis, but not S. pneumoniae or S. oralis, encodes an ortholog of the PG-dependent type I LTA synthase, ltaS By heterologous expression analyses, we confirmed that S. mitisltaS confers poly(Gro-P) synthesis in both Escherichia coli and Staphylococcus aureus and that S. mitisltaS can rescue the growth defect of an S. aureusltaS mutant. However, we do not detect a poly(Gro-P) polymer in S. mitis using an anti-type I LTA antibody. Moreover, Gro-P-linked DHDAG is still synthesized by an S. mitisltaS mutant, demonstrating that S. mitis LtaS does not catalyze Gro-P transfer to DHDAG. Finally, an S. mitisltaS mutant has increased sensitivity to human serum, demonstrating that ltaS confers a beneficial but currently undefined function in S. mitis Overall, our results demonstrate that S. mitis, S. pneumoniae, and S. oralis produce a Gro-P-linked glycolipid via a PG-dependent, ltaS-independent mechanism.IMPORTANCE The cell wall is a critical structural component of bacterial cells that confers important physiological functions. For pathogens, it is a site of host-pathogen interactions. In this work, we analyze the glycolipids synthesized by the mitis group streptococcal species, S. pneumoniae, S. oralis, and S. mitis We find that all produce the glycolipid, glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a precursor for the cell wall polymer type I lipoteichoic acid in other bacteria. We investigate whether the known enzyme for type I LTA synthesis, LtaS, plays a role in synthesizing this molecule in S. mitis Our results indicate that a novel mechanism is responsible. Our results are significant because they identify a novel feature of S. pneumoniae, S. oralis, and S. mitis glycolipid biology.

Development of novel isatin-nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing-bacteria.

Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal ß-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Metabolic biorthogonal labeling and dSTORM imaging of peptidoglycan synthesis in Streptococcus pneumoniae.

Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe that will not perturb the physiological process, and the spatial resolution must reach the nanometer scale to reveal fine details of the synthesis process. This protocol meets both challenges by combining biorthogonal metabolic labeling of peptidoglycan in Streptococcus pneumoniae with super-resolution fluorescence microscopy (dSTORM), also providing cues to adapt it to other bacteria. For complete details on the use and execution of this protocol, please refer to Trouve et al. (2021).

Structural, biosynthetic and serological cross-reactive elucidation of capsular polysaccharides from Streptococcus pneumoniae serogroup 28.

Capsular polysaccharides (CPS) are the key virulent factors in the pathogenesis of Streptococcus pneumoniae. The previously unknown CPS structures of the pneumococcal serotype 28F and 28A were thoroughly characterized by NMR spectroscopy, chemical analysis and AF4-MALS-dRI. The following repeat unit structures were determined: -4)[α-l-Rhap-[4-P-2-Gro]]-(1-3)-α-d-Sug-[6-P-Cho]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 28F: Sug = Glcp, Mw: 540.5 kDa; 28A: Sug = GlcpNAc, Mw: 421.9 kDa; The correlation of CPS structures with biosynthesis showed that glycosyltransferase WciU in serotypes 28F and 28A had different sugar donor specificity toward α-d-Glcp and α-d-GlcNAcp, respectively. Furthermore, latex agglutination tests of de-OAc and de-PO4 CPS were conducted to understand cross-reactions between serogroup 28 with factor antiserum 23d. Interestingly, the de-OAc 28F and 28A CPS can still weakly react with factor antiserum 23d, while de-PO4 CPS did not react with factor antiserum 23d. This indicated that OAc group could affect the affinity and P-2-Gro was crucial for cross-reacting with factor antiserum 23d.

Characterization of the ß-glucuronidase Pn3Pase as the founding member of glycoside hydrolase family GH169.

Paenibacillus sp. 32352 is a soil-dwelling bacterium capable of producing an enzyme, Pn3Pase that degrades the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (Pn3P). Recent reports on Pn3Pase have demonstrated its initial characterization and potential for protection against highly virulent S. pneumoniae serotype 3 infections. Initial experiments revealed this enzyme functions as an exo-ß1,4-glucuronidase cleaving the ß(1,4) linkage between glucuronic acid and glucose. However, the catalytic mechanism of this enzyme is still unknown. Here, we report the detailed biochemical analysis of Pn3Pase. Pn3Pase shows no significant sequence similarity to known glycoside hydrolase (GH) families, thus this novel enzyme establishes a new carbohydrate-active enzyme (CAZy) GH family. Site-directed mutagenesis studies revealed two catalytic residues along with truncation mutants defining essential domains for function. Pn3Pase and its mutants were screened for activity, substrate binding and kinetics. Additionally, nuclear magnetic resonance spectroscopy analysis revealed that Pn3Pase acts through a retaining mechanism. This study exhibits Pn3Pase activity at the structural and mechanistic level to establish the new CAZy GH family GH169 belonging to the large GH-A clan. This study will also serve toward generating Pn3Pase derivatives with optimal activity and pharmacokinetics aiding in the use of Pn3Pase as a novel therapeutic approach against type 3 S. pneumoniae infections.

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.