scholarly journals Positive Correlation between Tyrosine Phosphorylation of CpsD and Capsular Polysaccharide Production in Streptococcus pneumoniae

2003 ◽  
Vol 185 (20) ◽  
pp. 6057-6066 ◽  
Author(s):  
Matthew H. Bender ◽  
Robert T. Cartee ◽  
Janet Yother
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Tyrosine Phosphorylation ◽  
Capsular Polysaccharide ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Regulatory System ◽  
Capsule Production ◽  
Capsule Locus ◽  
Ladder Pattern

ABSTRACT 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∼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∼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.

scholarly journals Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2745-2754 ◽  
Author(s):  
Alistair J. Standish ◽  
Jonathan J. Whittall ◽  
Renato Morona
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Capsular Polysaccharide ◽  
Regulatory Mechanism ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Regulatory Proteins ◽  
Amidase Activity ◽  
The Past

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

scholarly journals Early signalling mechanism in colonic epithelial cell response to gastrin

1995 ◽  
Vol 311 (3) ◽  
pp. 945-950 ◽  
Author(s):  
R R Yassin ◽  
K M Little
Keyword(s):  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Promoting Effect ◽  
Kinase C ◽  
Growth Promoting ◽  
Colonic Cells ◽  
Signalling Cascade ◽  
Isolation Buffer

The hormone gastrin exerts a growth-promoting effect on gastrointestinal cells. The molecular mechanisms by which colonic epithelial cells respond to gastrin are still poorly understood. In this study, we demonstrate a novel feature of the action of gastrin on normal colonic cells, namely the rapid phosphorylation on tyrosine of phospholipase C gamma 1 (PLC gamma 1). Tyrosine phosphorylation of PLC gamma 1, elicited by gastrin, was transient, concentration-dependent, and was abrogated by pretreating the colonic cells with the gastrin-receptor antagonist proglumide, the tyrosine kinase inhibitor genistein, and by removal of the tyrosine phosphatase inhibitor orthovanadate from the isolation buffer. Tyrosine phosphorylation of PLC gamma 1 correlated with the time- and concentration-dependent decrease in the mass of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and the increase in the epithelial concentration of inositol 1,4,5-trisphosphate (IP3). Likewise, the stimulated increase in IP3 was also prevented by proglumide and genistein. Gastrin induced a definite but transient increase in the intracellular concentration of free Ca2+ [Ca2+]i, and increased membrane-translocation of immunoreactive alpha- and beta-protein kinase C. The data thus indicate that gastrin elicits at least one signalling cascade, through rapid tyrosine phosphorylation of PLC gamma 1, leading to the activation of a PIP2-specific PLC pathway.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

2010 ◽  
Vol 299 (4) ◽  
pp. C844-C853 ◽  
Author(s):  
Signe Skyum Kirkegaard ◽  
Ian Henry Lambert ◽  
Steen Gammeltoft ◽  
Else Kay Hoffmann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Regulatory Volume Decrease ◽  
Janus Kinase ◽  
Cell Swelling ◽  
Hek 293 ◽  
Set Point ◽  
Phosphatase Inhibitor

The swelling-activated K+ currents ( IK,vol) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K2p), TWIK-related acid-sensitive K+ channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by IK,vol indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K+ efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K+ efflux 1.3 times. To exclude K+ efflux via a KCl cotransporter, cellular Cl− was substituted with NO3−. Also under these conditions K+ efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K+ channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.

scholarly journals Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi

2005 ◽  
Vol 289 (3) ◽  
pp. C748-C756 ◽  
Author(s):  
Rachel J. Webb ◽  
Jacob D. Judah ◽  
Lee-Chiang Lo ◽  
Geraint M. H. Thomas
Keyword(s):  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Phosphorylation ◽  
Albumin Secretion ◽  
Protein Tyrosine ◽  
Constitutive Secretion

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process.

scholarly journals Changes in Availability of Oxygen Accentuate Differences in Capsular Polysaccharide Expression by Phenotypic Variants and Clinical Isolates of Streptococcus pneumoniae

2001 ◽  
Vol 69 (9) ◽  
pp. 5430-5439 ◽  
Author(s):  
Jeffrey N. Weiser ◽  
Deborah Bae ◽  
Henry Epino ◽  
Stephen B. Gordon ◽  
Miki Kapoor ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tyrosine Phosphorylation ◽  
Capsular Polysaccharide ◽  
Fold Increase ◽  
Growth Conditions ◽  
Inhibitory Effect ◽  
Invasive Infection ◽  
Environmental Concentration ◽  
Key Factor ◽  
Effect Of Oxygen

ABSTRACT Most isolates of Streptococcus pneumoniae are mixed populations of transparent (T) and opaque (O) colony phenotypes. Differences in the production of capsular polysaccharide (CPS) between O and T variants were accentuated by changes in the environmental concentration of oxygen. O variants demonstrated a 5.2- to 10.6-fold increase in amounts of CPS under anaerobic compared to atmospheric growth conditions, while CPS production remained low under all conditions for T variants. Increased amounts of CPS in O compared to T pneumococci were associated with increased expression ofcps-encoded proteins. The inhibitory effect of oxygen on expression of CPS in O variants correlated with decreased tyrosine phosphorylation of CpsD, a tyrosine kinase and regulator of CPS synthesis. Modulation of CpsD expression and its activity by tyrosine phosphorylation may allow the pneumococcus to adapt to the requirements of both colonization, where decreased CPS allows for adherence, and bacteremia, where increased CPS may be required to escape from opsonic clearance. In patients with invasive infection, paired isolates from the same patient were shown to have predominately a T colony phenotype without phosphotyrosine on CpsD when cultured from the nasopharynx, and an O phenotype that phosphorylates CpsD in response to oxygen when cultured from the blood. Differences in the availability of oxygen, therefore, may be a key factor in allowing for the selection of distinct phenotypes in these two host environments.

scholarly journals Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene

2018 ◽  
Author(s):  
Sam Manna ◽  
Eileen M. Dunne ◽  
Belinda D. Ortika ◽  
Casey L. Pell ◽  
Mike Kama ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Latex Agglutination ◽  
Reference Sequence ◽  
Middle Income ◽  
Capsule Locus ◽  
Vaccine Impact ◽  
Quellung Reaction

AbstractObjectivesAs part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated ‘33F-1’). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results.MethodsWhole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence.ResultsAll ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence.ConclusionsWe have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.

scholarly journals Transformation of nonencapsulated Streptococcus pneumoniae during systemic infection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jessica L. Bradshaw ◽  
Iftekhar M. Rafiqullah ◽  
D. Ashley Robinson ◽  
Larry S. McDaniel
Keyword(s):  
Streptococcus Pneumoniae ◽  
Systemic Infection ◽  
Dna Uptake ◽  
Pathogenic Potential ◽  
Bacterial Surface ◽  
Capsule Production ◽  
Virulent Strains ◽  
Capsule Locus ◽  
Ear Infections ◽  
Complement Deposition

Abstract Streptococcus pneumoniae (pneumococcus) is a principal cause of bacterial middle ear infections, pneumonia, and meningitis. Capsule-targeted pneumococcal vaccines have likely contributed to increased carriage of nonencapsulated S. pneumoniae (NESp). Some NESp lineages are associated with highly efficient DNA uptake and transformation frequencies. However, NESp strains lack capsule that may increase disease severity. We tested the hypothesis that NESp could acquire capsule during systemic infection and transform into more virulent pneumococci. We reveal that NESp strains MNZ67 and MNZ41 are highly transformable and resistant to multiple antibiotics. Natural transformation of NESp when co-administered with heat-killed encapsulated strain WU2 in a murine model of systemic infection resulted in encapsulation of NESp and increased virulence during bacteremia. Functional capsule production increased the pathogenic potential of MNZ67 by significantly decreasing complement deposition on the bacterial surface. However, capsule acquisition did not further decrease complement deposition on the relatively highly pathogenic strain MNZ41. Whole genome sequencing of select transformants demonstrated that recombination of up to 56.7 kbp length occurred at the capsule locus, along with additional recombination occurring at distal sites harboring virulence-associated genes. These findings indicate NESp can compensate for lack of capsule production and rapidly evolve into more virulent strains.

scholarly journals Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

1984 ◽  
Vol 160 (2) ◽  
pp. 386-397 ◽  
Author(s):  
L S McDaniel ◽  
G Scott ◽  
J F Kearney ◽  
D E Briles
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Minimum Amount ◽  
Binding Studies ◽  
Fatal Infection ◽  
Rough Strain ◽  
Type 3

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

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