Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D approximately P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D approximately P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis.     

Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase

One of the main virulence factors of the pathogenic bacterium Streptococcus pneumoniae is the capsule, present at the bacterial surface, surrounding the entire cell. Virtually all the 90 different capsular serotypes of S. pneumoniae, which vary in their chemical composition, express two conserved proteins, Wzd and Wze, which regulate the rate of the synthesis of capsule. In this work, we show that Wzd, a membrane protein, and Wze, a cytoplasmic tyrosine kinase, localize at the bacterial division septum, when expressed together in pneumococcal cells, without requiring the presence of additional proteins encoded in the capsule operon. The interaction between the two proteins and their consequent septal localization was dependent on a functional ATP binding domain of Wze. In the absence of either Wzd or Wze, capsule was still produced, linked to the cell surface, but it was absent from the division septum. We propose that Wzd and Wze are spatial regulators of capsular polysaccharide synthesis and, in the presence of ATP, localize at the division site, ensuring that capsule is produced in co‐ordination with cell wall synthesis, resulting in full encapsulation of the pneumococcal cells.     

肺炎链球菌糖代谢蛋白CcpA对荚膜多糖的调控作用

摘要:【目的】研究肺炎链球菌糖代谢蛋白CcpA对肺炎链球菌荚膜多糖(CPS)的调控作用。【方法】利用大肠杆菌(Escherichia coli)BL21(DE3)工程菌原核表达CcpA蛋白,使用Ni2+亲和层析的方法纯化蛋白。利用纯化后的CcpA蛋白免疫昆明小鼠并制备多克隆抗体;采用ELISA法测定抗CcpA抗体效价。随后,利用Westertn blot方法分析CcpA蛋白在肺炎链球菌中的保守性。另外,利用EMSA方法分析CcpA与cps基因座启动子区域片段的结合。最后,构建ccpA基因缺失株和ccpA基因回复株;利用ELISA法测定野生D39菌 株、ccpA基因缺失株和ccpA基因回复株的荚膜多糖含量。【结果】Western blot结果显示CcpA蛋白在多种血清型的肺炎链球菌均有表达,CcpA蛋白可与cps基因座启动子区域结合,且呈剂量依赖性;ccpA基因缺失时,细菌CPS含量升高,回复表达CcpA蛋白后,CPS含量显著降低。【结论】CcpA是肺炎链球菌中一种保守表达的蛋白,可通过调节cps基因座启动子负性调控肺炎链球菌荚膜多糖的表达。     

Key role of amino acid residues in the dimerization and catalytic activation of the autolysin LytA, an important virulence factor in Streptococcus pneumoniae

LytA, the main autolysin of Streptococcus pneumoniae, was the first member of the bacterial N-acetylmuramoyl-l-alanine amidase (NAM-amidase) family of proteins to be well characterized. This autolysin degrades the peptidoglycan bonds of pneumococcal cell walls after anchoring to the choline residues of the cell wall teichoic acids via its choline-binding module (ChBM). The latter is composed of seven repeats (ChBRs) of approximately 20 amino acid residues. The translation product of the lytA gene is the low-activity E-form of LytA (a monomer), which can be "converted" (activated) in vitro by choline into the fully active C-form at low temperature. The C-form is a homodimer with a boomerang-like shape. To study the structural requirements for the monomer-to-dimer modification and to clarify whether "conversion" is synonymous with dimerization, the biochemical consequences of replacing four key amino acid residues of ChBR6 and ChBR7 (the repeats involved in dimer formation) were determined. The results obtained with a collection of 21 mutated NAM-amidases indicate that Ile-315 is a key amino acid residue in both LytA activity and folding. Amino acids with a marginal position in the solenoid structure of the ChBM were of minor influence in dimer stability; neither the size, polarity, nor aromatic nature of the replacement amino acids affected LytA activity. In contrast, truncated proteins were drastically impaired in their activity and conversion capacity. The results indicate that dimerization and conversion are different processes, but they do not answer the questions of whether conversion can only be achieved after a dimer formation step.     

Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3

A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 by EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD⁺-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or scrotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.     

The influence of the O-antigen and the capsular polysaccharide on the establishment of PlCmts lysogeny in Klebsiella pneumoniae

The O-antigen of the lipopolysaccharide in Klebsiella pneumoniae caused a significant reduction in the frequency of establishment of PlCmts lysogeny, while the capsular polysaccharide showed no effect on this frequency. The bacterial receptor for PlCmts are the lipopolysaccharide-core oligosaccharides, the results suggest that K. pneumoniae strains with an O-antigen in their lipopolysaccharide have a poorly accessible lipopolysaccharide-core (the PlCmts bacterial receptor), while K. pneumoniae strains lacking the O-antigen have a highly accessible lipopolysaccharide-core. The accessibility of the receptor is independent of the K antigen (capsular polysaccharide).     

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.     

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae; Serologic distinction between the receptors by blocking antibodies

Abstract Group A and B streptococci ( Streptococcus pyogenes and Streptococcus agalactiae ) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding epitopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.     

Interrelationship between the thiamine content, thiamine diphosphate-dependent enzyme activity and reduced glutathione level in the rat liver

Changes in the amount of thiamine, reduced glutathione, thiamine diphosphate-dependent dehydrogenase activity has been traced after thiamine injection to thiamine-deficient rats and oxythiamine to normal rats. The obtained data show that a drop in reduced glutathione level was a primary reason of the alpha-keto-acid dehydrogenase activity reduction under conditions of the thiamine deficiency. The existence of immediate connection between thiamine and glutathione metabolism is supposed.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

银杏园土壤酶活性与土壤肥力的关系研究

研究了银杏土壤酶活性与土壤肥力的关系,试图探索利用土壤酶活性作为评价银杏果园土壤肥力的指标。结果表明,有３种酶的活性与土壤肥力存在着极为密切的关系,酶活性随土壤肥力的高低而有规律地变化。根据相关分析,该土壤的脲酶、蛋白酶、转化酶的活性均与土壤有机质、全氮、全磷、碱解氮、速效磷、速效钾及银杏果实产量呈极显著相关。不同土壤酶活性之间也存在极显著的相关性。因此,土壤酶活性作为评价银杏土壤肥力水平的指标是可行的。     

Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major)

In teleosts, estradiol-17beta (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450(arom)) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450(arom) during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450(arom) is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450(arom) mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450(arom) mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450(arom) enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHbeta, but not FSHbeta, correlated with increased P450(arom) mRNA levels during the course of ovarian development. In addition, the levels of P450(arom) mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450(arom) mRNA during oocyte development of red seabream.     

The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. II. Influence of time after BCG inoculation on production of interferon and cytotoxin by capsular polysaccharide of Klebsiella pneumoniae or by bacterial lipopolysaccharide and on hyperreactivity to their lethal effects.

The time course of the occurrence of hyperreactivity in interferon and cytotoxin responses to the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) and bacterial lipopolysaccharide (LPS) and of the hyperreactivity to their lethal effects was followed after infection with BCG in SMA and ICR strains of mice. The duration of these hyperreactivities of BCG-infected mice depended on the inoculum doses of BCG. The time patterns of the hyperreactivity to the lethal effects of neutral CPS-K and LPS were similar in both strains of mice, although the maximum toxicity of LPS by the intraperitoneal route in BCG-infected mice on a weight basis was stronger than that of neutral CPS-K. Irrespective of inducer and mouse strain, the time pattern of the hyperreactivity to produce cytotoxin was similar to that of the hyperreactivity to produce interferon. The patterns for these phenomena when neutral CPS-K was used as an inducer were also similar to those when LPS was used. In ICR mice the hyperreactivity in interferon and cytotoxin responses to either neutral CPS-K or LPS decayed significantly earlier than the hyperreactivity to their lethal effects, whereas in SMA mice the occurrence of both types of hyperreactivities seemed to be associated. Therefore, it is suggested that the mechanism for the hyperreactivity in interferon and cytotoxin responses to neutral CPS-K or LPS in BCG-infected mice is not necessarily the same as that for the hyperreactivity to their lethal effects.     

Effect of glutamine or glucose deprivation on the expression of cyclin and cyclin-dependent kinase genes in glioma cell line U87 and its subline with suppressed activity of signaling enzyme of endoplasmic reticulum-nuclei-1

Ischemia has been shown to induce a set of complex intracellular signaling events known as the unfolded protein response, which is mediated by endoplasmic reticulum-nuclei-1 sensing enzyme. We have studied the expression of several cyclin and cyclin-dependent kinase genes which participate in the control of cell cycle and proliferation under ischemic conditions (glucose or glutamine deprivation) in endoplasmic reticulum-nuclei-1-deficient glioma cells. It was shown that blockade of endoplasmic reticulum-nuclei signaling enzyme-1, the key endoplasmic reticulum stress sensor, leads to an increase of the expression levels of cyclin-dependent kinase-2 and cyclin A2, D3, E2 and G2 genes but suppresses cyclin D1. Moreover, the expression level of cyclin-dependent kinase-2 as well as cyclin A2, D3 and E2 mRNAs is significantly decreased under glucose or glutamine deprivation conditions both in control and endoplasmic reticulum-nuclei-1-deficient glioma cells. However, cyclin-dependent kinase-4 and -5 mRNA expressions is increased, but in glucose deprivation conditions only. Results of this study have shown that the expression of most tested genes of encoded cyclins and cyclin-dependent kinases is dependent on endoplasmic reticulum-nuclei-1 signaling enzyme function both in normal and glutamine and glucose deprivation conditions and possibly participates in cell adaptive response to endoplasmic reticulum stress associated with ischemia.     

Effects of post-injury hypothermia and nerve growth factor infusion on antioxidant enzyme activity in the rat: implications for clinical therapies

The pathological sequelae of traumatic brain injury (TBI) include increased oxidative stress due to the production of reactive oxygen species (ROS). Regulation of ROS levels following TBI is determined primarily by antioxidant enzyme activity that in turn can be influenced by nerve growth factor (NGF). Hypothermia is one of the current therapies designed to combat the deleterious effects of TBI. However, it has been shown to suppress post-trauma increases in NGF levels in rat brain. The present study sought to determine whether post-injury hypothermia also impairs the antioxidant response to injury, and if such an effect could be reversed by infusion of exogenous NGF. We employed a lateral controlled cortical impact injury model in rat, followed by moderate hypothermia treatment with supplemental intracerebroventricular infusion of NGF or vehicle. The time course of changes in post-injury/intervention levels of NGF and activity of three major enzymes responsible for ROS scavenging, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), was determined in the hippocampus. Relative to levels in injured, normothermic animals, hypothermia treatment not only suppressed NGF levels, but also attenuated CAT and GPx activity, and increased SOD activity. Infusion of NGF in injured, hypothermia-treated animals was ineffective in restoring hippocampal antioxidant enzymes activity to levels produced after injury under normothermic conditions, although it was able to increase septal cholinergic (choline acetyltransferase) enzyme activity. These results have implications for clinical treatment of TBI, demonstrating that moderate hypothermia suppresses NGF and the antioxidant response after TBI; the latter cannot be countered by exogenous NGF administration.     

A regenerative approach for bone repair in congenital pseudarthrosis of the tibia associated or not associated with type 1 neurofibromatosis: correlation between laboratory findings and clinical outcome

Background and aimsCongenital pseudarthrosis of the tibia (CPT) is a rare orthopedic disease presenting spontaneous fractures that do not heal. The treatment of CPT is characterized by repeated surgical procedures that often fail, with the inevitable outcome of severe disability and amputation. We tested the hypothesis that CPT may benefit from regenerative strategies based on mesenchymal stromal cells (MSC) combined with platelet-rich fibrin (PRF) as a source of growth factors. The aim of the study was to verify whether laboratory testing to assess the osteogenic properties of MSC and the osteo-inductive activity of PRF correlated with the clinical outcome.MethodsTen patients affected by refractory CPT were treated by using MSC derived from the iliac crest (IC-MSC), PRF and lyophilized bone. In six patients, CPT was associated with type 1 neurofibromatosis (NF1). Biochemical, functional and molecular assays were performed to assess the intrinsic osteogenic potential of IC-MSC (cells cultured with fetal calf serum) and the osteo-inductive properties of PRF (cells cultured with autologous serum).ResultsBone consolidation was obtained in three patients who had CPT and NF1. In these patients, the IC-MSC exposed to autologous serum were able to form mineral nodules in vitro, while the mineralizing ability was totally abrogated in patients with a poor clinical outcome.ConclusionsCell therapy may be a useful tool for the treatment of refractory CPT because it increases the opportunity to achieve effective bone tissue regeneration. Our data suggest that the presence of pro-osteogenic growth factors is an essential requirement for bone healing.