scholarly journals Comparative Virulence of Streptococcus pneumoniaeStrains with Insertion-Duplication, Point, and Deletion Mutations in the Pneumolysin Gene

1999 ◽  
Vol 67 (2) ◽  
pp. 981-985 ◽  
Author(s):  
Anne M. Berry ◽  
A. David Ogunniyi ◽  
David C. Miller ◽  
James C. Paton
Keyword(s):  
Amino Acids ◽  
Streptococcus Pneumoniae ◽  
Complement Activation ◽  
Point Mutations ◽  
Cytolytic Activity ◽  
Toxin Gene ◽  
Wild Type ◽  
Gene Encoding ◽  
Deletion Mutations

ABSTRACT Pneumolysin is a 471-amino-acid toxin produced byStreptococcus pneumoniae which has both cytolytic and complement activation properties. We have constructed a derivative of the type 2 S. pneumoniae strain D39 in which the portion of the pneumolysin gene encoding amino acids 55 to 437 has been deleted in-frame. The virulence of this strain (ΔPly) was compared with those of wild-type D39, a pneumolysin insertion-duplication mutant (PLN-A), and a derivative (PdT) carrying a toxin gene with three point mutations known to abolish both cytolytic activity and complement activation. PdT was intermediate in virulence between D39 and either PLN-A or ΔPly in a mouse intraperitoneal challenge model. This provides unequivocal evidence that pneumolysin has an additional property that is not abolished by point mutations which reduce cytotoxicity and complement activation to virtually undetectable levels.

scholarly journals Role of Manganese-Containing Superoxide Dismutase in Oxidative Stress and Virulence of Streptococcus pneumoniae

2000 ◽  
Vol 68 (5) ◽  
pp. 2819-2826 ◽  
Author(s):  
Hasan Yesilkaya ◽  
Aras Kadioglu ◽  
Neill Gingles ◽  
Janet E. Alexander ◽  
Tim J. Mitchell ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Streptococcus Pneumoniae ◽  
Anaerobic Growth ◽  
Wild Type ◽  
Gene Encoding ◽  
Lower Growth ◽  
Redox Active ◽  
Lower Growth Rate

ABSTRACT Streptococcus pneumoniae was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The sodA gene, encoding MnSOD, of virulent S. pneumoniae type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the sodA mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.

scholarly journals Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin

2011 ◽  
Vol 79 (6) ◽  
pp. 2440-2450 ◽  
Author(s):  
Shaper Mirza ◽  
Landon Wilson ◽  
William H. Benjamin ◽  
Jan Novak ◽  
Stephen Barnes ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptococcus Pneumoniae ◽  
Serine Protease ◽  
Human Lactoferrin ◽  
Free Form ◽  
Capsular Type ◽  
Gene Encoding ◽  
Content Type ◽  
Killing Activity

ABSTRACTIt is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, includingStreptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease,prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P< 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin killsStreptococcus pneumoniaerequires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

scholarly journals Pneumococcal Surface Protein C Contributes to Sepsis Caused by Streptococcus pneumoniae in Mice

2004 ◽  
Vol 72 (5) ◽  
pp. 3077-3080 ◽  
Author(s):  
Francesco Iannelli ◽  
Damiana Chiavolini ◽  
Susanna Ricci ◽  
Marco Rinaldo Oggioni ◽  
Gianni Pozzi
Keyword(s):  
Streptococcus Pneumoniae ◽  
Protein C ◽  
Lethal Dose ◽  
Surface Protein ◽  
Infection Model ◽  
Wild Type ◽  
Mutant Strains ◽  
Type 3

ABSTRACT The role of pneumococcal surface protein C (PspC; also called SpsA, CbpA, and Hic) in sepsis by Streptococcus pneumoniae was investigated in a murine infection model. The pspC gene was deleted in strains D39 (type 2) and A66 (type 3), and the mutants were tested by being injected intravenously into mice. The animals infected with the mutant strains showed a significant increase in survival, with the 50% lethal dose up to 250-fold higher than that for the wild type. Our findings indicate that PspC affords a decisive contribution to sepsis development.

scholarly journals Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity

1995 ◽  
Vol 311 (2) ◽  
pp. 699-704 ◽  
Author(s):  
A Muraoka ◽  
M Hashiramoto ◽  
A E Clark ◽  
L C Edwards ◽  
H Sakura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Structural Features ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Wild Type Clone

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

scholarly journals Role of Pneumolysin’s Complement-Activating Activity during Pneumococcal Bacteremia in Cirrhotic Rats

1999 ◽  
Vol 67 (6) ◽  
pp. 2862-2866 ◽  
Author(s):  
Rosemarie B. Alcantara ◽  
Laurel C. Preheim ◽  
Martha J. Gentry
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mortality Rate ◽  
Cytolytic Activity ◽  
Wild Type ◽  
Isogenic Mutant ◽  
Pneumococcal Bacteremia ◽  
Unit Increase

ABSTRACT We investigated the role of pneumolysin’s complement-activating activity during Streptococcus pneumoniae bacteremia in a hypocomplementemic, cirrhotic host. Isogenic mutant pneumococcal strains, in which pneumolysin was expressed from a plasmid, were used. These strains included H+C+, expressing wild-type pneumolysin with both cytolytic and complement-activating activity; PLY−, carrying the plasmid without the pneumolysin gene; and, H+C−, expressing pneumolysin with cytolytic activity only. In control rats, intravenous infection with 2.0 × 107 CFU of H+C+ per ml of blood resulted in a decrease in bacteremia of 3.5 log units by 18 h postinfection and 55% mortality. By contrast, cirrhotic rats infected similarly with the H+C+ strain demonstrated a 0.2-log-unit increase in bacteremia by 18 h postinfection and 100% mortality. Both control and cirrhotic rats cleared the PLY− strain more effectively from their bloodstreams by 18 h postinfection (6.2 and 5.6 log unit decreases, respectively). Infection with the PLY− strain also resulted in low mortality (0 and 14%, respectively) for control and cirrhotic rats. When infected with the H+C− strain (without complement-activating activity), both groups cleared the organism from their bloodstreams nearly as well as they did the PLY− strain. Furthermore, the mortality rate for control and cirrhotic rats was identical after infection with the H+C− strain. These studies suggest that pneumolysin production contributes to decreased pneumococcal clearance from the bloodstream and higher mortality in both control and cirrhotic rats. However, pneumolysin’s complement-activating activity may uniquely enhance pneumococcal virulence in the hypocomplementemic, cirrhotic host.

scholarly journals Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth

2007 ◽  
Vol 81 (21) ◽  
pp. 11798-11808 ◽  
Author(s):  
Aisling S. Dugan ◽  
Megan L. Gasparovic ◽  
Natia Tsomaia ◽  
Dale F. Mierke ◽  
Bethany A. O'Hara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sialic Acid ◽  
Burst Size ◽  
Point Mutations ◽  
Bk Virus ◽  
Host Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Virus Propagation

ABSTRACT BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing α(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

scholarly journals A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

2012 ◽  
Vol 78 (24) ◽  
pp. 8595-8600 ◽  
Author(s):  
Xiuzhen Gao ◽  
Xi Chen ◽  
Weidong Liu ◽  
Jinhui Feng ◽  
Qiaqing Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enantiomeric Excess ◽  
Acid Synthesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Amino Acid Synthesis ◽  
Content Type

ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.

scholarly journals Mutation of luxS of Streptococcus pneumoniae Affects Virulence in a Mouse Model

2003 ◽  
Vol 71 (6) ◽  
pp. 3206-3212 ◽  
Author(s):  
Uwe H. Stroeher ◽  
Adrienne W. Paton ◽  
A. David Ogunniyi ◽  
James C. Paton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mouse Model ◽  
Bacterial Species ◽  
Systemic Disease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Pneumococcal Infections ◽  
Wide Range

ABSTRACT The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a defined luxS mutation on the virulence of Streptococcus pneumoniae. Although the luxS mutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, the luxS mutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that the luxS mutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation of luxS had pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product of luxS having a regulatory role in S. pneumoniae. This is the first report of a direct role for luxS (and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis of luxS does not completely attenuate S. pneumoniae has implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.

Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1911-1917 ◽  
Author(s):  
Daniel Llull ◽  
Patrick Veiga ◽  
Josselyne Tremblay ◽  
Saulius Kulakauskas
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Agar Medium ◽  
Upper Respiratory Tract ◽  
Gene Encoding ◽  
Capsule Production ◽  
Bacterial Immobilization ◽  
Reliable Procedure

The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0·04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS− mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS− phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS− mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.

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