Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae β-galactosidase, BgaA.

Bacterial cell-surface proteins play integral roles in host-pathogen interactions. These proteins are often architecturally and functionally sophisticated and yet few studies of such proteins involved in host-pathogen interactions have defined the domains or modules required for specific functions. Streptococcus pneumoniae (pneumococcus), an opportunistic pathogen that is a leading cause of community acquired pneumonia, otitis media and bacteremia, is decorated with many complex surface proteins. These include β-galactosidase BgaA, which is specific for terminal galactose residues β-1-4 linked to glucose or N-acetylglucosamine and known to play a role in pneumococcal growth, resistance to opsonophagocytic killing, and adherence. This study defines the domains and modules of BgaA that are required for these distinct contributions to pneumococcal pathogenesis. Inhibitors of β-galactosidase activity reduced pneumococcal growth and increased opsonophagocytic killing in a BgaA dependent manner, indicating these functions require BgaA enzymatic activity. In contrast, inhibitors increased pneumococcal adherence suggesting that BgaA bound a substrate of the enzyme through a distinct module or domain. Extensive biochemical, structural and cell based studies revealed two newly identified non-enzymatic carbohydrate-binding modules (CBMs) mediate adherence to the host cell surface displayed lactose or N-acetyllactosamine. This finding is important to pneumococcal biology as it is the first adhesin-carbohydrate receptor pair identified, supporting the widely held belief that initial pneumococcal attachment is to a glycoconjugate. Perhaps more importantly, this is the first demonstration that a CBM within a carbohydrate-active enzyme can mediate adherence to host cells and thus this study identifies a new class of carbohydrate-binding adhesins and extends the paradigm of CBM function. As other bacterial species express surface-associated carbohydrate-active enzymes containing CBMs these findings have broad implications for bacterial adherence. Together, these data illustrate that comprehending the architectural sophistication of surface-attached proteins can increase our understanding of the different mechanisms by which these proteins can contribute to bacterial pathogenesis

Molecular Characterization of N-glycan Degradation and Transport in <i>Streptococcus pneumoniae</i> and Its Contribution to Virulence

<div><p>The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen <i>Streptococcus pneumoniae</i> depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man₃GlcNAc₂ core prior to the release of the glycan from the protein by endo-β-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of <i>S</i>. <i>pneumoniae</i> to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that <i>S</i>. <i>pneumoniae</i> also possesses an α-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal α-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man₅GlcNAc₂. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with α-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABC_NG), is able to bind Man₅GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABC_NG to growth of <i>S</i>. <i>pneumoniae</i> on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABC_NG contribute to growth of <i>S</i>. <i>pneumoniae</i>, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in <i>S</i>. <i>pneumoniae</i>. To conclude, we synthesize our findings into a model of N-glycan processing by <i>S</i>. <i>pneumoniae</i> in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABC_NG.</p></div

EndoD and SpGH92 contribute to virulence in a mouse model of pneumonia and sepsis.

<p>Cohorts of mice were infected intranasally with either parental TIGR4 Sm^r, Δ<i>endoD</i>, Δ<i>ngtS-P1-P2</i>, Δ<i>endoD</i> Δ<i>ngtS-P1-P2</i>, Δ<i>gh92</i> or a genetically reconstituted strain, and monitored for survival time and bacterial counts in blood. (A) Median survival times of mice infected with parental TIGR4 Sm^r, deletion mutants of <i>endoD</i> and/or <i>ngtS-P1-P2</i>, and genetically reconstituted strains. Symbols indicate the time that individual mice became severely lethargic and were euthanized; horizontal bars indicate the median survival time (experiment length, and therefore maximum survival time, was 168 hours). (B) Mean bacterial counts in blood 24 and 48 h post-infection with parental TIGR4 Sm^r, deletion mutants of <i>endoD</i> and/or <i>ngtS-P1-P2</i>, and genetically reconstituted strains. Error bars represent the SEM. (C) Median survival times of mice infected with parental TIGR4 Sm^r, Δ<i>gh92</i> and its genetically reconstituted strain. Symbols and bars are the same as in (A). (D) Mean bacterial counts in blood 24 and 36 h post-infection with parental TIGR4 Sm^r, Δ<i>gh92</i> and its genetically reconstituted strain. Error bars represent the SEM. In all panels, asterisks indicate the level of statistical significance between medians/means when compared with the parental TIGR4 Sm^r-infected cohort (* indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 and **** indicates p<0.0001).</p

α-(1,2)-mannosidase activity of SpGH92.

<p>(A) Activity of SpGH92 against α-(1,2), α-(1,3) and α-(1,6)-mannobiose and α-(1,3)(1,6)-mannotriose observed by HPAEC-PAD. Dashed box indicates elution of mannose. (B-D) Activity of SpGH92 on a high-mannose N-glycan (Man₉GlcNAc₂) observed by LC-MS. (B) Extracted ion chromatogram (EIC) of Man₉GlcNAc₂ standard, indicating resolution of both anomers with an <i>m/z</i> value of 942.3304 [M+2H]²⁺ (expected 942.3290, Δ<i>m/z</i> = 1.5 ppm). (C) EIC of Man₅GlcNAc₂ standard, indicating resolution of both anomers with an <i>m/z</i> value of 1235.4322 [M+H]⁺ (expected 1235.4400, Δ<i>m/z</i> = 6.3 ppm). (D) EIC of Man₉GlcNAc₂ treated with SpGH92 showing that the enzyme trims the α-(1,2)-linked mannose residues of Man₉GlcNAc₂, resulting in the formation of Man₅GlcNAc₂.</p

Proposed model of N-glycan metabolism in <i>Streptococcus pneumoniae</i>.

<p>The two arms of N-glycan processing—complex N-glycan (top) and high-mannose N-glycan (bottom)—are shown as graphical representations and converge at ABC_NG. Glycosidases are colour-coded according to their known or predicted activities: sialidase (purple), β-galactosidase (yellow), β-hexosaminidase (blue) and α-mannosidase (green). StrH is shown as a multimodular complex as it contains two catalytic domains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006090#ppat.1006090.ref053" target="_blank">53</a>]. NanA, BgaA, StrH and EndoD are extracellular and all bear LPXTG cell wall anchoring motifs. Complex N-glycan is sequentially depolymerized by NanA, BgaA and StrH [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006090#ppat.1006090.ref018" target="_blank">18</a>], resulting in Man₃GlcNAc₂, which is then released from the glycoconjugate by EndoD [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006090#ppat.1006090.ref020" target="_blank">20</a>]. High-mannose N-glycan is acted on by SpGH92, to produce Man₅GlcNAc₂, and is then released from the glycoconjugate by EndoD. Both Man₃GlcNAc and Man₅GlcNAc are transported by ABC_NG into the cytoplasm, where further depolymerization is carried out by SpGH125 and SpGH38. Dedicated ABC and PTS transporters import the monosaccharides released by NanA, BgaA, StrH and SpGH92 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006090#ppat.1006090.ref051" target="_blank">51</a>]. The proposed architecture of ABC_NG is shown inset. Abbreviations: cytoplasmic membrane (M); peptidoglycan (PG).</p

EndoD and NgtS contribute to growth of <i>S</i>. <i>pneumoniae</i> on a model glycoconjugate.

<p>Growth of deletion mutants of <i>S</i>. <i>pneumoniae</i> TIGR4 Sm^r on the model glycoconjugate fetuin. Deletion mutants of (A) <i>endoD</i>, (B) <i>ngtS-P1-P2</i> and (C) <i>endoD</i> plus <i>ngtS-P1-P2</i> were grown in chemically-defined medium supplemented with 20 mg ml^-1 fetuin as the sole carbon source and compared against their genetically reconstituted strains. All OD_600nm readings shown are the mean from three independent experiments each performed in triplicate. Gray shading indicates the 95% confidence intervals for each strain and statistically significant differences in growth.</p

Deglycosylation of RNase B by SpGH92 and EndoD.

<p>(A) Reconstructed mass spectrum of native RNaseB. The following glycoforms are observed: RNaseB-GlcNAc₂Man₅ (exp: 14899.4 Da, obs: 14900.7 Da), RNaseB-GlcNAc₂Man₆ (exp: 15061.6 Da, obs: 15062.3 Da), RNaseB-GlcNAc₂Man₇ (exp: 15223. Da, obs: 15223.7 Da), RNaseB-GlcNAc₂Man₈ (exp: 15386.0 Da, obs: 15386.7 Da) and RNaseB-GlcNAc₂Man₉ (exp: 15548.2 Da, obs: 15549.2 Da). (B) EndoD cleaves the chitobiose core of Man₅GlcNAc₂, yielding RNaseB-GlcNAc (exp: 13885.2 Da, obs: 13886.2 Da), but cannot act on the Man₆-Man₉ glycoforms. (C) SpGH92 trims the Man₆-Man₉ glycoforms down to the RNaseB-GlcNAc₂Man₅ glycoform (exp: 14899.4 Da, obs: 14900.1 Da). (D) Together, SpGH92 and EndoD act on all glycoforms of RNase B to produce exclusively RNaseB-GlcNAc (exp: 13885.2 Da, obs: 13885.9 Da).</p

<i>S</i>. <i>pneumoniae</i> proteins encoded by the CPL or co-occurring with the CPL in <i>S</i>. <i>pneumoniae</i> and other streptococci.

<p><i>S</i>. <i>pneumoniae</i> proteins encoded by the CPL or co-occurring with the CPL in <i>S</i>. <i>pneumoniae</i> and other streptococci.</p