A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.     

Mouse antibody response to group A streptococcal carbohydrate

In an attempt to more fully understand the generation of antibody diversity to carbohydrate (CHO) Ag, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal CHO from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein Ag). We have analyzed the use of H and L chain V region genes in the early (day 7) and late response (hyperimmune) and have sequenced the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results indicate that the level of somatic mutation in the hyperimmune response is generally low in comparison with the response to haptens and protein Ag. These data also suggest that there is a positive selection for mutation in CDR 1 during the hyperimmune response to group A streptococcal CHO.     

Immune response to superoxide dismutase in group A streptococcal infection

Extracellular localisation of manganese-dependent superoxide dismutase (SodA) by group A streptococcus (GAS) may have a role in protection of this pathogenic bacterium from exogenously produced reactive oxygen species. In this study we show that SodA is found both in surface protein extracts and in culture supernatants of GAS. To investigate whether SodA is a possible vaccine candidate outbred Quackenbush mice were subcutaneously vaccinated with recombinant SodA. Strong antibody responses which were moderately opsonic were elicited. These antibodies were unable to protect mice from intraperitoneal challenge with M1 GAS. We also show that SodA and p145 (a conserved peptide from the M-protein) antibodies are present at significantly higher levels amongst patients with rheumatic heart disease than in control subjects from the same endemic region. The higher SodA antibody levels in patients may be indicative of a role for this protein in pathogenesis of rheumatic heart disease but are more likely to be a marker of recent or recurrent streptococcal infection.     

Two isoforms of the Drosophila RNA binding protein, how, act in opposing directions to regulate tendon cell differentiation

Differential RNA metabolism regulates a wide array of developmental processes. Here, we describe a mechanism that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation.     

Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood

Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci.     

Two subunits of the Drosophila mediator complex act together to control cell affinity

The organizing centers for Drosophila imaginal disc development are created at straight boundaries between compartments; these are maintained by differences in cell affinity controlled by selector genes and intercellular signals. skuld and kohtalo encode homologs of TRAP240 and TRAP230, the two largest subunits of the Drosophila mediator complex; mutations in either gene cause identical phenotypes. We show here that both genes are required to establish normal cell affinity differences at the anterior-posterior and dorsal-ventral compartment boundaries of the wing disc. Mutant cells cross from the anterior to the posterior compartment, and can distort the dorsal-ventral boundary in either the dorsal or ventral direction. The Skuld and Kohtalo proteins physically interact in vivo and have synergistic effects when overexpressed, consistent with a skuld kohtalo double-mutant phenotype that is indistinguishable from either single mutant. We suggest that these two subunits do not participate in all of the activities of the mediator complex, but form a submodule that is required to regulate specific target genes, including those that control cell affinity.     

Production of hybridomas synthesizing monoclonal antibodies to streptococcal group A polysaccharide

Nine stable hybridomas synthetizing monoclonal antibodies to the antigenic determinants of polysaccharide A of group A streptococcus were obtained. Three monoclonal antibodies possessed precipitating properties. The formation of hybridomas was found to be influenced by the presence of immune splenocytes and the standard conditions of cell fusion. The highest yield of hybridomas was observed under the conditions ensuring the growth of cell in 80-100% of the wells. Rapid and specific screening was found to be an important stage in obtaining hybridomas.     

Resistance of the group A streptococcal L form to sonic oscillation

The mechanical fragility of the L form of two group A streptococcal strains was assessed by subjecting L-form suspensions to sonic oscillation (60 W, 20 kc/sec). To evaluate the effect of the sonic energy applied, the original streptococcal strains and aSerratia marcescens strain were subjected to the same treatment. The killing of the organisms by sonic oscillation followed the expression log N_o/N_t=–K·t^1/2. The mortality rate constants K of the streptococcus,S. marcescens and the L form of the AED and GL-8 streptococcal strains were –0.19,–0.98,–1.14 and–1.52 min^–1/2. respectively. The mortality rates of the L form of the two streptococcal strains differed significantly (P=0.01). From a comparison of these data, and taking into account the difference in cell envelope between the bacterial and the L form, it is concluded that the limiting envelopes of the group A streptococcal L-form elements probably possess a relatively marked stability. The factors which might be responsible for the difference in mortality rates between the L form of the streptococcal strains are discussed.The author wishes to thank Dr. W. Hijmans (Institute for Rheumatism Research, Leiden) for his contribution to and constant interest in the work and Mr. J. C. Houwelingen (Department of Mathematical Statistics, Utrecht) for the statistical analysis. This study could not have been accomplished without the able and conscientious technical assistance of Miss H. L. Ensering (Institute for Rheumatism Research, Leiden), which is gratefully acknowledged.     

Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development

Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host’s immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation.     

Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier

Group A Streptococcus pyogenes (GAS) is a human pathogen that causes local suppurative infections and severe invasive diseases. Systemic dissemination of GAS is initiated by bacterial penetration of the epithelial barrier of the pharynx or damaged skin. To gain insight into the mechanism by which GAS penetrates the epithelial barrier, we sought to identify both bacterial and host factors involved in the process. Screening of a transposon mutant library of a clinical GAS isolate recovered from an invasive episode allowed identification of streptolysin S (SLS) as a novel factor that facilitates the translocation of GAS. Of note, the wild type strain efficiently translocated across the epithelial monolayer, accompanied by a decrease in transepithelial electrical resistance and cleavage of transmembrane junctional proteins, including occludin and E-cadherin. Loss of integrity of intercellular junctions was inhibited after infection with a deletion mutant of the sagA gene encoding SLS, as compared with those infected with the wild type strain. Interestingly, following GAS infection, calpain was recruited to the plasma membrane along with E-cadherin. Moreover, bacterial translocation and destabilization of the junctions were partially inhibited by a pharmacological calpain inhibitor or genetic interference with calpain. Our data indicate a potential function of SLS that facilitates GAS invasion into deeper tissues via degradation of epithelial intercellular junctions in concert with the host cysteine protease calpain.     

SPA1 and DET1 act together to control photomorphogenesis throughout plant development

The COP1/SPA complex and DET1 function to suppress photomorphogenesis in dark-grown Arabidopsis seedlings. Additionally, they inhibit flowering under non-inductive short-day conditions. The COP1/SPA complex and DET1, as part of the CDD complex, represent distinct high-molecular-weight complexes in Arabidopsis. Here, we provide genetic evidence that these complexes co-act in regulating plant development. We report the isolation of a spa1 enhancer mutation that represents a novel, very weak allele of det1. This det1 ^esp1 mutation caused no detectable mutant phenotype in the presence of wild-type SPA1, but showed strongly synergistic genetic interaction with the spa1 mutation in the control of seedling photomorphogenesis, anthocyanin accumulation, plant size as well as flowering time. On the biochemical level, the det1 ^esp1 spa1 double mutant showed higher HY5 protein levels than either single mutant or the wild type. The genetic interaction of spa1 and det1 mutations was further confirmed in the spa1 det1-1 double mutant which carries a strong allele of det1. Taken together, these results show that SPA1 and DET1 act together to control photomorphogenesis throughout plant development. Hence, this suggests that COP1/SPA complexes and the CDD complex co-act in controlling the protein stability of COP1/SPA target proteins.     

X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound to a peptide from the group A streptococcal surface protein PAM

The crystal structure of the human Pg-derived angiogenesis inhibitor, angiostatin, complexed to VEK-30, a peptide from the group A streptococcal surface protein, PAM, was determined and refined to 2.3 A resolution. This is the first structure of angiostatin bound to a ligand and provides a model of the interaction between Pg and streptococcal-derived pathogenic proteins. VEK-30 contains a "through-space isostere" for C-terminal lysine, wherein Arg and Glu side chains, separated by one helical turn, bind within the bipolar angiostatin kringle 2 (K2) domain lysine-binding site. VEK-30 also makes several contacts with K2 residues that exist outside of the canonical LBS and are not conserved among the other Pg kringles, thus providing a molecular basis for the selectivity of VEK-30 for K2. The structure also shows that Pg kringle domains undergo significant structural rearrangement relative to one another and reveals dimerization between two molecules of angiostatin and VEK-30 related by crystallographic symmetry. This dimerization, which exists only in the crystal structure, is consistent with the parallel coiled-coil full-length PAM dimer expected from sequence similarities and homology modeling.     

A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division

Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.