Melanie A. Higgins

Identification and structural basis of binding to host lung glycogen by streptococcal virulence factors

The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal α-glucan–metabolizing machinery as virulence factors.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

Significance The pneumococcus accounts for 25% of deaths in children under 5 y of age in developing countries. One of the most important virulence factors expressed by this pathogen is the pore-forming toxin, pneumolysin (Ply), an example of a Gram-positive cholesterol-dependent cytolysin (CDC). We show that Ply interacts with the Lewis histo-blood group antigen sialyl LewisX and that blocking this interaction can protect RBCs from lysis. We also identify glycan receptors on RBCs for the CDC streptolysin O from group A streptococcus. Our study supports the emerging paradigm shift that CDCs have cellular receptors other than cholesterol that define target cell tropism. The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae

The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in α‐glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette‐transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous α‐glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX‐ and SpuA‐dependent manner. SpuA is able to degrade glycogen into a ladder of α‐1,4‐glucooligosaccharides while the high‐affinity interaction (Ka ∼ 106 M−1) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X‐ray crystallographic analyses of apo‐ and complexed MalX illuminate the proteins specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal α‐glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaeα‐glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.

Differential recognition and hydrolysis of host carbohydrate-antigens by Streptococcus pneumoniae family 98 glycoside hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Inhibition of the Pneumococcal Virulence Factor Strh and Molecular Insights Into N-Glycan Recognition and Hydrolysis.

The complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-β-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.

The Conformation and Function of a Multimodular Glycogen-Degrading Pneumococcal Virulence Factor

SpuA is a large multimodular cell wall-attached enzyme involved in the degradation of glycogen by the pathogenic bacterium Streptococcus pneumoniae. The deletion of the gene encoding SpuA from the bacterium resulted in a strain with reduced competitiveness in a mouse model of virulence relative to the parent strain, linking the degradation of host-glycogen to the virulence of the bacterium. Through the combined use of X-ray crystallography, small-angle X-ray scattering, and inhibitor binding, the molecular features involved in substrate recognition by this complex protein are revealed. This uniquely illustrates the complexity of the active site, the conformational changes incurred during carbohydrate binding by this protein, and the interaction and cooperation of its composite modules during this process. New insight into the function of this particular pneumococcal virulence factor is provided along with substantial contributions to the nascent framework for understanding the structural and functional interplay between modules in multimodular carbohydrate-active enzymes.

Blood-group antigen recognition by a solute-binding protein from a serotype 3 strain of Streptococcus pneumoniae

Streptococcus pneumoniae is a common bacterial pathogen that is well known for its ability to cause acute respiratory disease (pneumonia), ear infections, and other serious illnesses. This Gram-positive bacterium relies on its carbohydrate-metabolizing capabilities for full virulence in its host; however, the range of glycan targets that it can attack is presently not fully appreciated. S. pneumoniae is known to have a fucose utilization operon that in the TIGR4 strain plays a role in its virulence. Here we identify a second type of fucose utilization operon that is present in a subset of S. pneumoniae strains, including the serotype 3 strain SP3-BS71. This operon contains a transporter with a solute-binding protein, FcsSBP (fucose solute-binding protein), that interacts tightly (Ka approximately 1 x 10(6) M(-1)) and specifically with soluble A- and B-antigen trisaccharides but displays no selectivity between these two sugars. The structure of the FcsSBP in complex with the A-trisaccharide antigen, determined to 2.35 A, reveals its mode of binding to the reducing end of this sugar, thus highlighting this proteins requirement for soluble blood group antigen ligands. Overall, this report exposes a heretofore unknown capability of certain S. pneumoniae strains to transport and potentially metabolize the histo-blood group antigen carbohydrates of its host.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

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.

The Overall Architecture and Receptor Binding of Pneumococcal Carbohydrate-Antigen-Hydrolyzing Enzymes

The TIGR4 and SP3-BS71 strains of Streptococcus pneumoniae each produce family 98 glycoside hydrolases, called Sp4GH98 and Sp3GH98, respectively, which have different modular architectures and substrate specificities. Sp4GH98 degrades the Lewis(Y) antigen and possesses three C-terminal family 47 carbohydrate-binding modules (CBMs) that bind to this substrate. Sp3GH98 degrades the blood group A/B antigens and has two N-terminal family 51 CBMs that are of unknown function. Here, we examine the complex carbohydrate-binding specificity of the family 51 CBMs from Sp3GH98 (referred to as CBM51-1 and CBM51-2), the structural basis of this interaction, and the overall solution conformations of both Sp3GH98 and Sp4GH98, which are shown to be fully secreted proteins. Through glycan microarray binding analysis and isothermal titration calorimetry, CBM51-1 is found to bind specifically to the blood group A/B antigens. However, due to a series of relatively small structural rearrangements that were revealed in structures determined by X-ray crystallography, CBM51-2 appears to be incapable of binding carbohydrates. Analysis of small-angle X-ray scattering data in combination with the available high-resolution X-ray crystal structures of the Sp3GH98 and Sp4GH98 catalytic modules and their CBMs yielded models of the biological solution structures of the full-length enzymes. These studies reveal the complex architectures of the two enzymes and suggest that carbohydrate recognition by the CBMs and the activity of the catalytic modules are not directly coupled.