Stable-isotope analysis of a deep-sea benthic-fish assemblage: evidence of an enriched benthic food web.

In this study, fishes and invertebrates collected from the continental slope (1000 m) of the eastern North Pacific Ocean were analysed using stable-isotope analysis (SIA). Resulting trophic positions (T(P) ) were compared to known diets and habitats from the literature. Dual isotope plots indicated that most species groups (invertebrates and fishes) sorted as expected along the carbon and nitrogen axes, with less intraspecific variability than interspecific variability. Results also indicated an isotopically distinct benthic and pelagic food web, as the benthic food web was more enriched in both nitrogen and carbon isotopes. Trophic positions from SIA supported this finding, resulting in the assignment of fishes to different trophic positions from those expected based on published dietary information. These differences can be explained largely by the habitat of the prey and the percentage of the diet that was scavenged. A mixing model estimated dietary contributions of prey similar to those of the known diet of Bathyraja trachura from stomach-content analysis (SCA). Linear regressions indicated that trophic positions calculated from SIA and SCA, when plotted against B. trachura total length for 32 individuals, exhibited similar variation and patterns. Only the T(P) from SCA yielded significant results (stomach content: P < 0·05, stable isotope: P > 0·05).

Results of the premature birth national need-gap study.

OBJECTIVE: Family-centered care is a standard of practice in neonatal intensive care units (NICUs). The purpose of the study was to assess successes and opportunities for improvement with parents' experiences and involvement in their premature infants' care in NICUs. STUDY DESIGN: Researchers' surveyed 502 parents whose children were currently < or =30 months old, had been born at a gestational age < or =36 weeks and had gone through or were currently in NICUs. RESULT: Most parents of premature infants were reasonably satisfied with the access, attention and information received from physicians and nurses in the NICU. However, approximately one-fourth were only moderately satisfied and nearly 10% were dissatisfied. CONCLUSION: While progress has been made in meeting the needs of parents in the NICU, more work needs to be carried out to improve family-centered care efforts. Specific attention should be given to providing more information and interaction opportunities for families, which may ultimately improve NICU outcomes.

Similarities between complement-mediated and streptolysin S-mediated hemolysis.

The oxygen-stable hemolysin streptolysin S (SLS) of Streptococcus pyogenes is encoded in part by the pel/sagA gene product. Antibodies to a synthetic peptide from the C terminus of the Pel/SagA open reading frame inhibited hemolysis mediated by both culture supernatants from multiple M serotypes of S. pyogenes isolates or a commercially available SLS preparation. Analysis of the SLS-mediated hemolytic reaction demonstrated that it was temperature- and concentration-dependent. Like complement-mediated hemolysis it conforms to the prediction of a one-hit mechanism of hemolysis. A number of intermediates in the SLS-mediated hemolysis of sheep erythrocytes could be distinguished. SLS could bind to erythrocytes below 17 degrees C; however, lysis could only occur at temperatures >23 degrees C. Following binding of SLS and washing, a papain-sensitive intermediate could be distinguished prior to insertion of the SLS complex into the erythrocyte membrane, which resulted in formation of a transmembrane pore and led to irreversible osmotic lysis of the cell. These intermediates were similar to those described previously during complement-mediated hemolysis.

Streptococcal pyrogenic exotoxin B enhances tissue damage initiated by other Streptococcus pyogenes products.

This study compared the pathology and infection pattern of streptococcal pyrogenic exotoxin B-positive (SpeB(+)) and SpeB-negative (SpeB(-)) isogenic variants of an M1 isolate of Streptococcus pyogenes in a mouse skin air sac model. SpeB(+) strains resulted in severe local tissue damage that extended from the epidermis through the subcutaneous layers, whereas isogenic SpeB(-) variants had reduced gross pathology. At the histologic level, differences in necrosis and host responses to each variant were apparent. Injection of purified SpeB alone into a skin air sac failed to induce any significant tissue damage; however, coinjection of the enzyme with either the wild-type or the speB mutant resulted in increased and accelerated tissue necrosis. Surprisingly, coinjection of the enzyme with the spleen-recovered SpeB(-) variant failed to induce a lesion.

Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes.

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr approximately 41,000 and the fully active enzyme Mr approximately 28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.

Isolation of human plasma-inducible, growth phase- and temperature-regulated gene fusions in Streptococcus pyogenes using a Tn917-lacZ transposon.

Streptococcus pyogenes is capable of causing a variety of human diseases ranging from superficial or deep tissue infections to non-infectious post-streptococcal infection sequelae. In this paper, we report the use of a Tn917-lacZ transposon to isolate random lacZ transcription fusions in the S. pyogenes chromosome. Libraries of random Tn917-lacZ mutants were generated in a representative opacity factor positive strain CS101 (M49) and an opacity factor negative strain 1881 (M1). Several different mutant phenotypes were isolated. These included: temperature-regulated promoters, growth phase/cell density-regulated promoters and a human plasma-inducible promoter. Expression of the temperature-regulated fusions was 5-10-fold higher when grown at 30 degrees C compared to growth at 37 degrees C. The growth phase-regulated fusions were induced 30-fold at late exponential phase and were repressed by a diffusible S. pyogenes factor(s). Expression of the human plasma-inducible fusion was induced 10-15-fold by human plasma or sera, 4-fold by rabbit sera and was repressed by horse and mouse sera. In addition, hemolysin negative and capsule over expression mutants were isolated. These results demonstrate the utility of Tn917-lacZ mutagenesis for the identification of S. pyogenes promoters.

Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation.

Mouse skin passage of a Streptococcus pyogenes Tn917 mutant of sagA/pel restores virulence, beta-hemolysis and sagA/pel expression without altering the position or sequence of the transposon.

BACKGROUND: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene. Mutants lacking expression of this gene were less virulent in a dermonecrotic mouse infection model. Inactivation of the sagA/pel gene affect the expression of a variety of virulence factors in addition to the hemolysin. Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein. RESULTS: In this study a mouse skin air sac model was utilized to analyze the effect of biological pressures on expression of SLS and other sagA/pel regulated gene products. The insertion delayed the lethal effect of S. pyogenes in a mouse skin infection model. Despite this, bacteria could be cultured from the kidneys 72 hours post infection. These kidney-recovered isolates were beta-hemolytic despite the transposon being present in its original location and had equivalent virulence to the wild type isolate when re-injected into naive mice. Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels. CONCLUSIONS: These results show that biological pressure present in the mouse can select for variants with altered expression of key virulence factor genes in S. pyogenes.

Analysis of expression of a cytosolic enzyme on the surface of Streptococcus pyogenes.

The normally cytosolic glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, (GAPDH) has been reported to be expressed on the surface of Streptococcus pyogenes, group A, where it can act as a plasmin binding protein (Plr), and potentially a signaling molecule. In studies of wild-type and isogenic mutants, an association between surface expression of antigenic GAPDH/Plr and M and M-related fibrinogen-binding proteins was identified. Inactivation of the mga gene, whose product controls expression of M and M-related proteins also influenced expression of surface GAPDH/Plr. Revertants or pseudorevertants of mga mutants led to concomitant re-expression of surface GAPDH/Plr and M and M-related proteins. Using surface enhanced laser desorption ionization (SELDI) mass spectroscopy, a physical association between GAPDH/Plr and streptococcal fibrinogen-binding proteins was demonstrated. These studies support the hypothesis that surface M and M-related proteins are involved in anchoring GAPDH/Plr on the surface of group A streptococci.

Interaction of group A streptococci with human plasmin(ogen) under physiological conditions.

A series of methods for analyzing the interaction of group A streptococci with the human plasminogen system are described. Examples of group A streptococcal isolates capable of assembling surface plasminogen activator activity when grown in human plasma are presented and the key requirements for this process are evaluated. The stabilities of cell-associated plasmin and plasminogen activator complexes are compared and a model for the interaction of group A streptococci with the plasminogen system in an infected host is presented.

The potential role for nephritis-associated plasmin receptor in acute poststreptococcal glomerulonephritis.

Immunoglobulin G from a patient convalescing from acute poststreptococcal glomerulonephritis (APSGN) bound specific antigenic sites in early APSGN glomeruli. A streptococcal cytoplasmic antigen (preabsorbing antigen, PA-Ag), could selectively preabsorb fluorescein isothiocyanate (FITC)-labeled IgG and prevented glomerular staining. The antigen was purified and identified as an M(r) approximately 43,000 protein with a pI of 4.7 that strongly activated complement C3 (N. Yoshizawa, S. Oshima, I. Sagel, J. Shimizu, and G. Treser, 1992, J. Immunol. 148, 3110-3116). In the present study, a nephritogenic antigen was purified by affinity chromatography using APSGN IgG-immobilized Sepharose followed by chromatography on an anion-exchange resin. Purification was monitored by ELISA and Western blotting using the binding characteristics of the specific antibodies present in APSGN serum. The molecular weight of the purified antigen, named nephritis-associated plasmin receptor (NAPlr), was an M(r) approximately 43,000 protein and the internal amino acid sequence was found to be homologous to those of the plasmin receptor (Plr) of group A streptococci strain 64/14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The purified NAPlr exhibited GAPDH activity and plasmin(ogen) binding activity. Using FITC-labeled rabbit anti-NAPlr, the antigen was found to be present in the glomeruli of 22 of 22 patients in the early stage of APSGN. Bacterial Plr was also demonstrated in human APSGN glomeruli for the first time using monoclonal antibody to the recombinant Plr protein. Antibody to NAPlr was found in the sera of 46 of 50 (92%) patients within 3 months of onset. These results led us to speculate that NAPlr bound to the glomeruli may contribute to the pathogenesis of APSGN via plasmin and complement activation.

A pivotal role for interferon-gamma in protection against group A streptococcal skin infection.

Administration of exogenous recombinant interleukin-12 (rIL-12) either prophylactically or therapeutically provides significant protection against lethal group A streptococcal skin infection in a mouse model. Treatment of mice with rIL-12 before infection with group A streptococci induced expression of interferon-gamma (IFN-gamma) at the infection site. In vivo neutralization of IFN-gamma increased susceptibility to lethal infection and completely abrogated the protective effects of rIL-12. IFN-gamma knockout mice were also more susceptible to lethal infection. Although IL-12 treatment provided protection, higher doses induced significantly elevated levels of IFN-gamma transcription that were associated with increased susceptibility to lethal infection. These results support the hypothesis that IFN-gamma at the infection site is critical for protection but suggest that increased systemic levels are detrimental to survival after infection with group A streptococci.

Absence of SpeB production in virulent large capsular forms of group A streptococcal strain 64.

Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties.

Species specificity of plasminogen activation and acquisition of surface-associated proteolytic activity by group C streptococci grown in plasma.

Our laboratory previously demonstrated that group C streptococcal isolates from humans and horses secrete streptokinases that preferentially activate plasminogens reflecting the origin of the isolates. To analyze the significance of these findings, series of streptokinase-producing Streptococcus equisimilis isolates recovered from humans and horses were examined. Southern blot analysis revealed that chromosomal DNA of the streptococcal isolates from humans reacted exclusively with a skc(hu) probe and that chromosomal DNA of streptococcal isolates from horses reacted preferentially with an skc(eq) probe in a distinct pattern. The streptococcal isolates were examined for the ability to acquire surface-bound plasmin-like activity when grown in the presence of human or equine plasma. Each of eight isolates from humans acquired significant enzymatic activity only when grown in the presence of human plasma, while each of eight isolates from horses acquired activity only when grown in the presence of equine plasma. Analysis of bacterial and host protein requirements indicated critical roles for streptokinase, activatable plasminogen, and fibrinogen. These requirements may explain why certain streptococcal isolates cause disease only in a limited number of mammalian hosts.

Identification of pel, a Streptococcus pyogenes locus that affects both surface and secreted proteins.

A Tn917 insertion mutant of an M49 serotype, opacity factor-positive Streptococcus pyogenes, was isolated. It had the following phenotypes: decreased beta-hemolysis mediated by streptolysin S, reduction in the activity of a secreted cysteine protease and streptokinase, and an altered immunoglobulin and fibrinogen-binding phenotype. The site of insertion of Tn917 into the chromosome and the surrounding sequence, the pel region (pleiotropic effect locus), was determined. Phage A25 transduction confirmed that the pleiotropic changes in phenotype could be cotransduced with Tn917. The pel region was cloned and sequenced, and the transposon was found to be inserted upstream of a single open reading frame which led to a failure to transcribe a 500-base mRNA. The loss of this transcript decreased the transcription of emm and speB genes and reduced the secretion of streptokinase. Enhanced Pel expression from a nisin-inducible plasmid resulted in increased message levels for emm in a wild-type organism. Characterization of the pel mutant provides evidence for the coordinated regulation of secreted and surface proteins and suggests the existence of a new global regulatory factor in S. pyogenes.

Interaction between group A streptococci and the plasmin(ogen) system promotes virulence in a mouse skin infection model.

Group A streptococci are capable of acquiring a surface-associated, unregulatable plasmin-like enzymatic activity when incubated in human plasma. The effect of this enzymatic activity on virulence of group A isolate CS101 was examined in a mouse skin infection model. Initial studies demonstrated enhanced virulence for bacteria preincubated in human plasma but not in plasminogen-depleted plasma. A direct correlation between surface-associated enzymatic activity and virulence was not observed; however, an association between virulence and the assembly of a surface-associated plasminogen activator that could activate mouse plasminogen was noted. This activity enhanced virulence in wild type but not in plg-/- plasminogen-deficient mice. These results support the hypothesis that acquisition of a surface-associated plasmin(ogen)-dependent enzymatic activity can contribute to the virulence of group A streptococcal invasive infections.

A secreted streptococcal cysteine protease can cleave a surface-expressed M1 protein and alter the immunoglobulin binding properties.

Previous studies of recent clinical isolates of serotype M1 group A streptococci indicated that they display two patterns of non-immune human IgG subclass binding reactivity associated with their M1 protein. One group reacted with all four IgG subclasses (type IIo), while the second group expressed an M1 protein reacting preferentially with human IgG3 (type IIb). In this study, we have demonstrated that a cysteine protease, SpeB, present in culture supernatants of M1 serotype group A streptococcal isolates expressing type IIb IgG binding protein, can convert a recombinant Emm1 protein from a type IIo functional profile to a type IIb profile by removal of 24 amino acids from the N-terminus of the mature M1 protein. Furthermore, SpeB can convert bacteria expressing IgG binding proteins of the type IIo phenotype into those expressing type IIb proteins. The role of the cysteine protease as the central bacterial enzyme in this posttranslational modification event was confirmed by generation of an isogenic SpeB-negative mutant.

Interaction of a group A Streptococcus within human plasma results in assembly of a surface plasminogen activator that contributes to occupancy of surface plasmin-binding structures.

Group A streptococcal isolate 187061 incubated in human plasma or serum reconstituted with fibrinogen but not plasminogen-depleted plasma or serum alone acquired a surface plasminogen activator activity. Assembly of the surface plasminogen activator was inhibited by the presence of neutralizing antibodies to streptokinase. Once assembled, the bacterial-associated plasminogen activator could generate plasmin when incubated in human plasminogen, plasmin or serum which could bind to bacterial surface plasmin-binding structures despite the presence of host physiological inhibitors. These studies provide evidence that the pathways by which group A isolates interact with human plasmin(ogen) are potentially linked and may provide a mechanism for bacteria to acquire host enzymatic activity efficiently in the infected host.

Clot formation by group A streptococci.

Group A streptococci of several different M serotypes can cause human plasma to clot in nutrient-poor media. Addition of glucose to the medium prevents clot formation. Once formed, clots are stable for several days and can be lysed on addition of exogenous streptokinase or urokinase. Clot lysis can also be achieved by addition of glucose to a clot containing wild-type group A streptococci but not clots containing an isogenic mutant in which the ska gene was inactivated.