Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide in Streptococcus mutans

We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.     

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.     

D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density

Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.     

Cell Wall Polysaccharide Biosynthesis by Membrane Fragments from Streptococcus pyogenes and Stabilized L-Form

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-¹⁴C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of ¹⁴C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.     

The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall Polysaccharide O-Acetylation

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.The plant cell wall is a complex composite of polysaccharides, glycoproteins, and polyphenols, with the fine structure and quantity of each varying by species, tissue, and developmental time point (Knox, 2008; Burton et al., 2010). Cellulose, hemicelluloses, and pectic polysaccharides are the three major classes of polysaccharides observed in the wall. Current models of the wall have cellulose microfibrils as the major structural component, with hemicelluloses binding to the microfibrils and pectins as an amorphous matrix in which the cellulose/hemicellulose network is embedded (Pauly et al., 1999a; Somerville et al., 2004; Cosgrove, 2005). Unlike the linear β-1,4-glucan chains making up cellulose microfibrils, hemicelluloses and pectins consist of a diverse set of glycosyl units and linkages as well as other modifications such as methylation and acetylation (Caffall and Mohnen, 2009; Scheller and Ulvskov, 2010; Pauly et al., 2013).The O-acetyl substitutions on hemicelluloses and pectins occur on a variety of specific glycosyl residues. The hemicellulose xyloglucan (XyG) consists of a β-1,4-glucan backbone with a regular pattern of xylosyl branches, with additional galactosyl, fucosyl, arabinosyl, and/or galacturonosyl substitution depending on the tissue and plant species (Obel et al., 2009; Pauly et al., 2013; Schultink et al., 2014). XyG O-acetylation has been reported on the β-1,4-glucan backbone (Sims et al., 1996; York et al., 1996) as well as on specific galactosyl or arabinosyl side chains (Kiefer et al., 1989; Vierhuis et al., 2001). The hemicellulose xylan is heavily acetylated at positions O2 and O3 of the backbone β-1,4-xylosyl residues, with the degree of acetylation (O-acetyl groups per backbone of xylosyl residue) ranging from approximately 0.4 to 0.6 depending on the species (Teleman et al., 2002; Evtuguin et al., 2003; Prozil et al., 2012; Chong et al., 2014; Lee et al., 2014). The glycosyl substituents of xylan, including glucuronosyl, arabinosyl, and xylosyl groups, have not been reported to be acetylated. The backbone β-1,4-mannosyl residues of the hemicellulosic polysaccharide mannan also can be acetylated (Manna and McAnalley, 1993). The predominant location of O-acetyl groups in pectin has been reported to be on galacturonic acid residues at positions O2 and O3 (Ralet et al., 2005). O-Acetylation of pectin also has been observed on rhamnosyl (Sengkhamparn et al., 2009), fucosyl, and aceric acid residues (Glushka et al., 2003).The functional significance and biosynthetic pathway of wall polysaccharide O-acetylation are not fully understood. O-Acetylation has been shown to influence the solubility, gelation, and enzymatic accessibility of polysaccharides in vitro (Biely et al., 1986; Huang et al., 2002). These properties are likely to be important for appropriate function in planta. Recently identified Arabidopsis (Arabidopsis thaliana) mutants with polysaccharide O-acetylation deficiencies (reduced wall acetylation [rwa] and trichome birefringence-like [tbl]; Gille and Pauly, 2012) have allowed for testing of the in vivo role of this substituent. The ALTERED XYLOGLUCAN4 (AXY4 [TBL27]) gene from the TBL family was identified in a forward genetic screen of Arabidopsis and is believed to code for a XyG acetyltransferase (Gille et al., 2011). The growth morphology of this mutant, which lacks XyG O-acetylation in leaves, etiolated seedlings, and roots, was not affected under laboratory growth conditions. Arabidopsis mutants deficient for a putative xylan acetyltransferase (TBL29/ESKIMO1 [ESK1]) were reported to have reduced growth and irregular xylem and to be freezing tolerant (Xin et al., 2007; Xiong et al., 2013; Yuan et al., 2013). Arabidopsis mutants deficient for other TBL genes have been reported to exhibit phenotypes such as aberrant trichomes (Bischoff et al., 2010a) and resistance to powdery mildew (Vogel et al., 2004), but polysaccharide acetylation defects have not been demonstrated in these cases. The variation in the morphological phenotypes of different tbl mutants suggests that the function of polysaccharide acetylation is specific to the particular polysaccharide and tissue.While the TBL gene products seem to affect single wall polysaccharides, Arabidopsis mutants defective for one or more of the four RWA genes have decreased acetylation of multiple polysaccharides and growth phenotypes ranging from mild to severe (Lee et al., 2011; Manabe et al., 2011, 2013). For this reason, and because the RWA proteins are integral membrane proteins with 10 predicted transmembrane domains, it has been hypothesized that they may act as transporters for an activated form of acetate into the Golgi apparatus (Manabe et al., 2011). It has been demonstrated that acetyl-CoA is involved in the pathway of pectin acetylation (Pauly and Scheller, 2000); however, it is not clear if acetyl-CoA is transported into the Golgi or there is an alternative donor substrate that acts as a carrier.In this study, we report the identification and characterization of AXY9, an additional component of the plant cell wall polysaccharide acetylation pathway.     

Regulation of Cell Component Production by Growth Rate in the Group B Streptococcus

Group B Streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-h td) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at a td of 1. 4 h than at a td of 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at a td of 1. 4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.     

Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Role of Polysaccharide Synthesis in Elongation Growth and Cell Wall Loosening in Intact Rice Coleoptiles

2,6-Dichlorobenzonitrile (DCB) inhibited only increases in levelsof the cellulosic polysac-charides while monensin and galactoseinhibited increases in levels of both the cellulosic and thematrix polysaccharides in intact rice coleoptiles that weresubmerged in water. Elongation growth of rice coleoptiles wassuppressed by DCB at 10^–6 M, by monensin at 10^–7M, and by galactose at 3 ? 10^–3 M and above. Thus, thesynthesis of both the cellulosic and the matrix polysaccharidesis essential for the elongation of intact rice coleoptiles.These inhibitors increased the minimum stress-relaxation timeand the relaxation rate and they decreased the mechanical extensibilityof the cell wall, indicating that they inhibited cell wall loosening.The concentrations of the inhibitors required for inhibitionof cell wall loosening were higher than those for suppressionof elongation. The data suggest that polysaccharides synthesisplays two roles in elongation. It keeps the cell wall in a "loosened"condition by producing new extensible cell walls, while itsother role is probably related to the fixation or extensionof polymers already present in the cell wall. (Received November 15, 1990; Accepted May 23, 1991)     

A Novel Protein,RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ΔrafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ΔrafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ΔrafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ΔrafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.     

Structure of the Response Regulator NsrR from Streptococcus agalactiae,Which Is Involved in Lantibiotic Resistance

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding.     

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.     

Conjugative R plasmids in Streptococcus agalactiae (group B).

Twenty-one drug-resistant clinical isolates of group B streptococci were investigated for drug-resistance transfer by conjugation. Six strains were resistant to tetracycline, two to chloramphenicol, one to both drugs, and twelve to macrolide antibiotics (erythromycin, oleandomycin and spiramycin), lincomycin, pristinamycin I, and/or chloramphenicol and tetracycline. Ten strains carried R plasmids which were transferable to group B and/or group D recipients by a conjugation-like phenomenon. Six plasmids were transferred at a high frequency (9 × 10^?2 to 4 × 10^?4) and four, at low frequency (5 × 10^?6 to 7 × 10^?8). The molecular weight of one plasmid (pIP501) was investigated after transfer into the new hosts and was found to be similar to that carried by the wild strain (19.8 × 10⁶).     

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.Streptococcus mutans is a facultative colonizer of the human dental plaque, the microbial pellicle that covers the surface of the teeth. It is the major etiological agent of dental caries (17). Sugar metabolism is central to the behavior of S. mutans (4, 7). It can use a variety of sugars. The sugars are fermented by glycolysis with production of organic acids, particularly lactic acid (4, 7). In addition to providing energy, sucrose is used to produce extracellular polysaccharides to form the biofilm matrix that aids in the association of S. mutans with the dental plaque. Once the S. mutans biofilm becomes part of the dental plaque, the acidic by-products of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries (17). The presence of sugars in the dental plaque is periodic and reflects the intake of dietary sugars. If there is excess sugar available, in addition to producing organic acids and matrix, intracellular (iodophilic) polysaccharide (IPS; glycogen) is formed.The IPS of S. mutans is a polymer of the glycogen-amylopectin type, with α-(1, 4)- and α-(1, 6)-linked glucose, and is stored as intracellular granules (10). Intracellular glycogen storage reserves in various bacterial species are synthesized from glucose-1-P via ADP-glucose (1). The synthesis involves at least three enzymes: glycogen synthase, glucose-1-phosphate pyrophosphorylase, and branching enzyme. The genes encoding these enzymes are commonly found in a glg operon, although the order of genes differs between species. In two gram-positive species, Bacillus subtilis and Bacillus stearothermophilus, the gene order is glgB-glgC-glgD-glgA-glgP (15, 29): glgA encodes glycogen synthase, glgB encodes glucan branching enzyme, and glgC and glgD encode subunits of glucose-1-phosphate pyrophosphorylase. The glgP gene encodes glycogen phosphorylase, which is unlikely to be involved in glycogen synthesis (29). Genes putatively encoding similar enzymes are present in the same order in the genome of S. mutans (29); they are thought also to form an operon.The IPS can be used as a source of carbohydrate for fermentation upon nutrient depletion (11, 13). In planktonic cultures, IPS reserves are largely consumed within 12 h of the imposition of sugar starvation (11, 13, 32). In S. mutans, IPS utilization may prolong acid production and hence the period of lowered pH of the resting (between meals) plaque, a factor that contributes to the incidence of caries. Indeed, IPS is implicated in dental caries: a mutant that synthesized elevated levels of IPS was hypercariogenic in germfree rats (27). Strains isolated from human carious lesions were nearly all stable IPS producers, whereas most strains from caries-inactive persons were variable IPS producers (13, 33).Since S. mutans deep in the dental plaque may not have access to nutrients because of competition with the bacteria at the surface of the plaque, the bacteria may need to survive longer periods of nutrient starvation. Previous studies in our laboratory showed that S. mutans can survive under sugar starvation conditions, provided that the pH remains above ∼5.5 (22). The presence of spent medium and mucin significantly prolonged survival of sugar-starved biofilms and batch cultures (22; also unpublished observations). Here we examine the role of IPS.The role of IPS (glycogen) in bacterial survival has been tested for several other bacterial species. It was found to extend survival of Aerobacter aerogenes (8) and Escherichia coli (28). Intracellular glycogen was also shown to support the survival of Streptococcus mitis during stationary-phase starvation (32). In contrast, glycogen-rich Sarcina lutea died at a higher rate during starvation than did bacteria without glycogen (2).In order to test the role of IPS in S. mutans survival, we constructed an IPS-deficient mutant by inactivating glgA (GenBank SMU.1536) (http://www.oralgen.lanl.gov/), putatively encoding the glycogen synthase. We also constructed a mutant potentially altered in IPS metabolism by inactivating the putative pullulanase structural gene, pul (SMU.1541). Pullulanases are responsible for hydrolyzing α-(1,6) linkages (and in some cases 1,4 linkages) in pullulan and in other polysaccharides (35) and may be important in determining the branching in IPS and/or affecting the catabolism of IPS. We studied the persistence of bacteria under conditions of sugar limitation and of sugar excess in both batch cultures and biofilms. We found that IPS can play a role in supporting S. mutans persistence in batch cultures but found no role for IPS in survival in biofilms.     

Immunocytochemical Localization of Enzymes Involved in Lignification of the Cell Wall

O -methyltransferase, and cinnnamyl alcohol dehydrogenase were localized to differentiating xylem. These enzymes are particularly abundant during secondary wall formation. Immunolabeling was observed on polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of anionic peroxidase was also localized to the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough endoplasmic reticulum (r-ER), the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized on the plasma membrane by fusion of the Golgi vesicles to the membrane. Received 3 September 2001/ Accepted in revised form 16 October 2001     

Rapid Antigen Group A Streptococcus Test to Diagnose Pharyngitis: A Systematic Review and Meta-Analysis

Background

Pharyngitis management guidelines include estimates of the test characteristics of rapid antigen streptococcus tests (RAST) using a non-systematic approach.

Objective

To examine the sensitivity and specificity, and sources of variability, of RAST for diagnosing group A streptococcal (GAS) pharyngitis.

Data Sources

MEDLINE, Cochrane Reviews, Centre for Reviews and Dissemination, Scopus, SciELO, CINAHL, guidelines, 2000–2012.

Study Selection

Culture as reference standard, all languages.

Data Extraction and Synthesis

Study characteristics, quality.

Main Outcome(s) and Measure(s)

Sensitivity, specificity.

Results

We included 59 studies encompassing 55,766 patients. Forty three studies (18,464 patients) fulfilled the higher quality definition (at least 50 patients, prospective data collection, and no significant biases) and 16 (35,634 patients) did not. For the higher quality immunochromatographic methods in children (10,325 patients), heterogeneity was high for sensitivity (inconsistency [I²] 88%) and specificity (I² 86%). For enzyme immunoassay in children (342 patients), the pooled sensitivity was 86% (95% CI, 79–92%) and the pooled specificity was 92% (95% CI, 88–95%). For the higher quality immunochromatographic methods in the adult population (1,216 patients), the pooled sensitivity was 91% (95% CI, 87 to 94%) and the pooled specificity was 93% (95% CI, 92 to 95%); however, heterogeneity was modest for sensitivity (I² 61%) and specificity (I² 72%). For enzyme immunoassay in the adult population (333 patients), the pooled sensitivity was 86% (95% CI, 81–91%) and the pooled specificity was 97% (95% CI, 96 to 99%); however, heterogeneity was high for sensitivity and specificity (both, I² 88%).

Conclusions

RAST immunochromatographic methods appear to be very sensitive and highly specific to diagnose group A streptococcal pharyngitis among adults but not in children. We could not identify sources of variability among higher quality studies. The present systematic review provides the best evidence for the wide range of sensitivity included in current guidelines.     

Host Responses to Group A Streptococcus: Cell Death and Inflammation

Infections caused by group A Streptococcus (GAS) are characterized by robust inflammatory responses and can rapidly lead to life-threatening disease manifestations. However, host mechanisms that respond to GAS, which may influence disease pathology, are understudied. Recent works indicate that GAS infection is recognized by multiple extracellular and intracellular receptors and activates cell signalling via discrete pathways. Host leukocyte receptor binding to GAS-derived products mediates release of inflammatory mediators associated with severe GAS disease. GAS induces divergent phagocyte programmed cell death responses and has inflammatory implications. Epithelial cell apoptotic and autophagic components are mobilized by GAS infection, but can be subverted to ensure bacterial survival. Examination of host interactions with GAS and consequences of GAS infection in the context of cellular receptors responsible for GAS recognition, inflammatory mediator responses, and cell death mechanisms, highlights potential avenues for diagnostic and therapeutic intervention. Understanding the molecular and cellular basis of host symptoms during severe GAS disease will assist the development of improved treatment regimens for this formidable pathogen.     

A note on bile tolerance in Streptococcus agalactiae

Streptococcus agalactiae strains isolated from humans were found to be able to grow in the presence of high levels of bile but not in media containing levels of bile salts or surfactants commonly considered equivalent to bile in selective media.     

Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.