Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases

In the human host, Streptococcus pneumoniae encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface. S. pneumoniae is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), β-galactosidase (BgaA), and N-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides S. pneumoniae with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that S. pneumoniae is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to S. pneumoniae colonization and pathogenesis.     

Recognition of Streptococcus pneumoniae by the innate immune system

Streptococcus pneumoniae is both a frequent colonizer of the upper respiratory tract and a leading cause of life-threatening infections such as pneumonia, meningitis and sepsis. The innate immune system is critical for the control of colonization and for defence during invasive disease. Initially, pneumococci are recognized by different sensors of the innate immune system called pattern recognition receptors (PRRs), which control most subsequent host defence pathways. These PRRs include the transmembrane Toll-like receptors (TLRs) as well as the cytosolic NOD-like receptors (NLRs) and DNA sensors. Recognition of S. pneumoniae by members of these PRR families regulates the production of inflammatory mediators that orchestrate the following immune response of infected as well as neighbouring non-infected cells, stimulates the recruitment of immune cells such as neutrophils and macrophages, and shapes the adaptive immunity. This review summarizes the current knowledge of the function of different PRRs in S. pneumoniae infection.     

Interference between Streptococcus pneumoniae and Staphylococcus aureus: In vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae

The bactericidal activity of Streptococcus pneumoniae toward Staphylococcus aureus is mediated by hydrogen peroxide. Catalase eliminated this activity. Pneumococci grown anaerobically or genetically lacking pyruvate oxidase (SpxB) were not bactericidal, nor were nonpneumococcal streptococci. These results provide a possible mechanistic explanation for the interspecies interference observed in epidemiologic studies.     

Species-specific interaction of Streptococcus pneumoniae with human complement factor H

Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.     

Electrotransformation of Streptococcus pneumoniae

Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm^-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm^-1 field strength, 200 Ω resistance and 25 μF capacitance.     

Transglycosylation and transfer reaction activities of endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus (Streptococcus) pneumoniae

Endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae was shown to have transglycosylation and transfer reaction (reversed hydrolysis) activities. Treatment of asialoglycoproteins having Gal beta 1----3GalNAc alpha 1----Ser/Thr linkages with enzyme preparations containing glycerol resulted in formation of nonreducing trisaccharides. The structure of the main trisaccharide (approximately 80%) was deduced to be Gal beta 1----3GalNAc alpha 1----1(3)-glycerol by analysis of sugar composition and the results of exoglycosidase treatment and periodate oxidation. The ability of the endoglycosidase to catalyze transfer of Gal beta 1----3GalNAc to various acceptors was also demonstrated by incubation of the enzyme with the disaccharide and the test compound. The following were found to show acceptor activity: glycerol, Tris, p-nitrophenol, threonine, serine, D-glucose, D-galactose, D-fucose, and 6-O-methylgalactose. Transfer to the primary hydroxyl groups of glycerol and hexoses appears to be favored since the major glycerol product was 1(3)-substituted and transfer to D-fucose and 6-O-methyl-D-galactose was less than that to D-galactose. In order to avoid spurious results, it is necessary to carry out incubations with this enzyme in the absence of glycerol and other hydroxy compounds. The potential use of this endoglycosidase in the synthesis of glycosides is indicated by our studies.     

环介导恒温扩增法检测肺炎链球菌的研究

目的 建立环介导恒温扩增(LAMP)检测肺炎链球菌的方法.方法 用LAMP技术扩增肺炎链球菌菌株,并应用50例临床标本采用传统培养法、PCR法、LAMP法进行检测,比较3种方法的检出率,同时检测方法特异性和灵敏度.结果 所测肺炎链球菌均获扩增产物,对其他非肺炎链球菌无交叉反应.LAMP检测灵敏度可达102 CFU/mL.50例临床标本使用LAMP法检出9例肺炎链球菌阳性(18.0％),使用传统培养法检出阳性4例(8.0％).结论 LAMP法较传统培养检测方法特异性强、灵敏度高、操作方便、快速,适合临床标本的肺炎链球菌检测.     

The uptake of choline by Streptococcus pneumoniae.

Uptake of choline, a structural component of pneumococcal C- and F-teichoic acids, into bacteria growing in a defined medium was very efficient with an uptake constant ([S]10 5) of 3.2 microns. It was inhibited by iodoacetate, dinitrophenol and oligomycin but not by structural analogues of choline. Ethanolamine, however, was transported in the absence of choline but with a reduced affinity ([S]0.5 71.4 microns). The same constitutive system was probably used by both ethanolamine and choline. It is suggested that this system required ATP and probably involved choline kinase.     

Blood clearance of Streptococcus pneumoniae by C-reactive protein

C-reactive protein (CRP) has long been known to be an acute phase protein associated with infection and various forms of tissue damage. Recent studies have shown that human CRP can be used to passively protect mice from lethal infection with Streptococci pneumoniae. In this study we have undertaken a detailed examination of the ability of human CRP and rabbit CRP (CxRP) to mediate the blood clearance of pneumococci in mice. We have shown that the optimal activity of these acute-phase proteins requires a functioning complement system, and it can take place even in the xid mouse, which has virtually no naturally occurring anti-pneumococcal antibody in its serum. These studies provide additional evidence that CRP may play a protective role in pneumococcal infections, and it may help postpone the development of fatal levels of pneumococci in the blood, long enough for an effective anti-pneumococcal antibody response to be generated.     

MKP1 regulates the induction of MCP1 by Streptococcus pneumoniae pneumolysin in human epithelial cells

Epithelial cells act as the first line of host defense against microbes by producing a range of different molecules for clearance. Chemokines facilitate the clearance of invaders through the recruitment of leukocytes. Thus, upregulation of chemokine expression represents an important innate host defense response against invading microbes such as Streptococcus pneumoniae. In this study, we report that the expression of Monocyte Chemotactic Protein 1 (MCP1) was highly induced in response to S. pneumoniae in vitro and in vivo. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for this induction. Furthermore, MCP1 induction was mediated by the p38 mitogen-activated protein kinase (MAPK) whose activation was controlled by MAPK phosphatase 1 (MKP1). Therefore, this study reveals novel roles of pneumolysin in mediating MKP1 expression for the regulation of MCP1 expression in human epithelial cells.     

Alteration of Streptococcus pneumoniae membrane properties by the folate analog methotrexate

The antifolate compound methotrexate (MTX) is toxic to the gram-positive bacterium Streptococcus pneumoniae. Interaction of MTX with this bacterium resulted in an increase in the electric transmembrane potential (delta psi) and enhanced the delta psi-dependent uptake of isoleucine and MTX. In contrast, delta psi-independent uptake of glutamine was not changed. Folate, a nontoxic analog of MTX, did not exhibit these membrane effects, nor did it prevent the effect of MTX, suggesting that the NH2 in position 4 of the pteridine ring of the MTX molecule is involved in the MTX response. A strain bearing the nonsense mutation amiA9, selected for MTX resistance, did not exhibit increased membrane potential after MTX pretreatment. This suggests that MTX interacts with a specific membrane component in S. pneumoniae. A resulting change in ion permeability could lead to changes in the magnitude of the delta psi. The MTX-sensitive component is altered or absent in mutant amiA9.     

Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.     

Inhibition of transformation in Streptococcus pneumoniae by lysogeny.

Streptococcus pneumoniae R6X was lysogenized with bacteriophage 304 isolated after mitomycin induction of an ungrouped alpha-hemolytic streptococcus. Lysogenized pneumococci lost their capacity to undergo genetic transformation: transformability was restored after cells were spontaneously cured of their prophage. Both lysogens and nonlysogens produced activator substance (competence factor), and both bound deoxyribonucleic acid in a deoxyribonuclease-resistant form. However, nonlysogens retained deoxyribonucleic acid after washing, whereas lysogens did not. The latter did not liberate phage nor (unlike nonlysogens) degrade transforming deoxyribonucleic acid and contained normal levels of endonuclease.     

Identification of the optochin-resistant Streptococcus pneumoniae strains

So far the prevalent method of the identification of pneumococci in the routine diagnostic practice has included three tests: sensitivity to optoxin, solubility in biliary salts and reaction with omniserum. At present optoxin-resistant pneumococcal strains, as well as those giving dual reaction to solubility in biliary salts, occur sufficiently often thatconsiderably complicates the identification of this infective agent. We have summarized experience in the isolation of S. pneumoniae strains, resistant to optoxin and not soluble in biliary salts; in addition, the prospects of introducing the complex approach to the identification of a given culture with the use of molecular diagnostic methods into daily practice are evaluated.