Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0348321 (Haemophilus)
 15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To facilitate species level identification of bacteria without the requirement of presumptive identification, the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S-23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus. Three pairs of primer targeting conserved sequences flanking the 3' end of 16S and the 5' end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains. The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon). The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kitjj Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer. Then those sequences were compared with known sequences on the GenBank. The alignment of nucleotide sequence, evolutionary distances and phylogenetic trees were analyzed by software DANMAN version 4.0. The PCR products were showed polymorphism patterns with agarose gel. One band was contained in streptococcus genus. The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446 bp. All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera. It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable, three regions showed significant spatial variation. Most of the differences between the sequences came from several bases insertions/deletions and substitutions. There are two major bands in the Haemophilus biotypes (515 and 884 bp), the small ISR amplicon contained one tDNA coding for tRNA(Glu). In contrast to the large one contained two tRNA genes coding for tRAN(Ala) and tRNA(Ile). Two regions of repeating motifs with only A or T were present in higher copy numbers between tRAN(Ala) and tRNA(Ile). The phylogenetic trees varied from 97.5 to 98.8%. The PCR and direct sequencing of 16S-23S rRAN ISR were successful in the pathogen species identification.
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PMID:[Identification of Streptococcus species and Haemophilus influenzae by direct sequencing of PCR products from 16S-23S rRNA itergenic sacer regions]. 1563 51

Bacterial prolyl-tRNA synthetases and some smaller paralogs, YbaK and ProX, can hydrolyze misacylated Cys-tRNA Pro or Ala-tRNA Pro. To assess the significance of this quality control editing reaction in vivo, we tested Escherichia coli ybaK for its ability to suppress the E. coli thymidylate synthase thyA:146CCA missense mutant strain, which requires Cys-tRNA(Pro) for growth in the absence of thymine. Missense suppression was observed in a ybaK deletion background, suggesting that YbaK functions as a Cys-tRNA Pro deacylase in vivo. In vitro studies with the full set of 20 E. coli aminoacyl-tRNAs revealed that the Haemophilus influenzae and E. coli YbaK proteins are moderately general aminoacyl-tRNA deacylases that preferentially hydrolyze Cys-tRNA Pro and Cys-tRNA Cys and are also weak deacylases that cleave Gly-tRNA, Ala-tRNA, Ser-tRNA, Pro-tRNA, and Met-tRNA. The ProX protein acted as an aminoacyl-tRNA deacylase that cleaves preferentially Ala-tRNA and Gly-tRNA. The potential of H. influenzae YbaK to hydrolyze in vivo correctly charged Cys-tRNA Cys was tested in E. coli strain X2913 (ybaK+). Overexpression of H. influenzae ybaK decreased the in vivo ratio of Cys-tRNA Cys to tRNA Cys from 65 to 35% and reduced the growth rate of strain X2913 by 30% in LB medium. These data suggest that YbaK-mediated hydrolysis of aminoacyl-tRNA can influence cell growth.
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PMID:The bacterial YbaK protein is a Cys-tRNAPro and Cys-tRNA Cys deacylase. 1588 96

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.
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PMID:The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases. 1594 83

Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (K (m)) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine (0%). Examination of the pH dependence of the kinetic constants k (cat) and K (m) for E134D-DapE revealed ionizations at pH 6.4, 7.4, and approximately 9.7. Isothermal titration calorimetry experiments revealed a significant weakening in metal K (d) values of E134D-DapE. D134 and A134 perturb the second divalent metal binding site significantly more than the first, but both altered enzymes can still bind two divalent metal ions. Structural perturbations of the dinuclear active site of DapE were also examined for two E134-substituted forms, namely E134D-DapE and E134A-DapE, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E134D-DapE and E134A-DapE did not reveal any significant changes in the electronic absorption spectra, suggesting that both Co(II) ions in E134D-DapE and E134A-DapE reside in distorted trigonal bipyramidal coordination geometries. EPR spectra of [Co_(E134D-DapE)] and [Co_(E1341A-DapE] are similar to those observed for [CoCo(DapE)] and somewhat similar to the spectrum of [Co(H(2)O)(6)](2+) which typically exhibit E/D values of approximately 0.1. Computer simulation returned an axial g-tensor with g ((x,y))=2.24 and E/D=0.07; g ( z ) was only poorly determined, but was estimated as 2.5-2.6. Upon the addition of a second Co(II) ion to [Co_(E134D-DapE)] and [Co_(E134A-DapE)], a broad axial signal was observed; however, no signals were observed with B (0)||B (1) ("parallel mode"). On the basis of these data, E134 is intrinsically involved in the hydrolysis reaction catalyzed by DapE and likely plays the role of a general acid and base.
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PMID:Kinetic and spectroscopic characterization of the E134A- and E134D-altered dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae. 1642 26

Peptidoglycan-associated lipoprotein (Pal) is a potential vaccine candidate from Haemophilus influenzae that is highly conserved in Gram-negative bacteria and anchored to the outer membrane through an N-terminal lipid attachment. Pal stabilizes the outer membrane by providing a noncovalent link to the peptidoglycan (PG) layer through a periplasmic domain. Using NMR spectroscopy, we determined the three-dimensional structure of a complex between the periplasmic domain of Pal and a biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-alpha-d-Glu-m-Dap-D-Ala-d-Ala (m-Dap is meso-diaminopimelate). Pal has a binding pocket lined with conserved surface residues that interacts exclusively with the peptide portion of the ligand. The m-Dap residue, which is mainly found in the cell walls of Gram-negative bacteria, is sequestered in this pocket and plays an important role by forming hydrogen bond and hydrophobic contacts to Pal. The structure provides insight into the mode of cell wall recognition for a broad class of Gram-negative membrane proteins, including OmpA and MotB, which have peptidoglycan-binding domains homologous to that of Pal.
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PMID:Peptidoglycan recognition by Pal, an outer membrane lipoprotein. 1647 1

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children, characterized by inflammation mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. Synthetic peptides corresponding to loops L5, L6, and L7 activate JNK and p38 mitogen-activated protein kinase (MAPK) pathways, L7 being the most active peptide. Therefore, sequence-activity relationships and key residues were identified by elongating sequence to different extents, designing cyclic peptides, and performing an alanine scan of L7. The ability of mutant peptides to induce activation of signal transduction pathways and release of TNF-alpha and IL-6 has been determined, and, in conjunction with CD spectra, bioinformatics analysis, and molecular dynamics data, showed that 6 out of 8 amino acids contribute significantly to the overall activity. Molecular dynamics showed that L7 modifications increased loop rigidity and helicity after Gly6 mutation, thus, providing a possible structural explanation for observed loss of bioactivity. This work provides insights into essential molecular details of P2 that may impact on the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.
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PMID:Structural requirements for proinflammatory activity of porin P2 Loop 7 from Haemophilus influenzae. 1658 85

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure-function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.
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PMID:The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC. 1673 27

The bacterial cell wall provides essential protection from the external environment and confers strength and rigidity to counteract internal osmotic pressure. Without this layer the cell would be easily ruptured and it is for this reason that biosynthetic pathways leading to the formation of peptidoglycan have for many years been a prime target for effective antibiotics. Central to this pathway are four similar ligase enzymes which add peptide groups to glycan moieties. As part of a program to better understand the structure-function relationships in these four enzymes, the crystal structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC) has been determined to 2.6 A resolution. The structure was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and refined to a crystallographic R factor of 0.212 (R(free) = 0.259). The enzyme has a modular multi-domain structure very similar to those of other members of the mur family of ATP-dependent amide-bond ligases. Detailed comparison of these four enzymes shows that considerable conformational changes are possible. These changes, together with the recruitment of two different N-terminal domains, allow this family of enzymes to bind a substrate which is identical at one end and at the other has the growing peptide tail which will ultimately become part of the rigid bacterial cell wall. Comparison of the E. coli and Haemophilus influenzae structures and analysis of the sequences of known MurC enzymes indicate the presence of a ;dimerization' motif in almost 50% of the MurC enzymes and points to a highly conserved loop in domain 3 that may play a key role in amino-acid ligand specificity.
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PMID:Structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC). 1713 82

Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.
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PMID:Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid. 1769 92

The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated "hypothetical protein" in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase "hot dog" fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.
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PMID:Structure of YciA from Haemophilus influenzae (HI0827), a hexameric broad specificity acyl-coenzyme A thioesterase. 1826 Jun 43


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