Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.     

The actin gene in Paracoccidioides brasiliensis: organization, expression and phylogenetic analyses

PbrACT1, the gene responsible for the synthesis of actin in Paracoccidioides brasiliensis, was found as a single copy, organized into six exons and five introns. Its open reading frame (ORF) codes for a putative protein of 375 amino acids, with a molecular mass of 41.5 kDa and an isoelectric point of 5.6. Analysis of the nucleotide sequence revealed a high homology to other fungal actins, the presence of characteristic fungal actin sequences, and heat shock elements at the 5′ untranslated region (UTR). Phylogenetic analyses with deduced amino acid sequences of fungal actins grouped P. brasiliensis within the phylum Ascomycota, order Onygenales, in concordance with a few previous reports. Patterns of expression through the temperature-induced morphological transitions from mycelial to yeast-like shapes and reverse, suggests that PbrACT1 is regulated in this process. The PbrACT1 gene sequence is available at the GenBank database under accession number AY383732.     

MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.     

Titin; a multidomain protein that behaves as the sum of its parts.

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially "the sum of its parts". From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.     

巴西橡胶树鲨烯合酶基因的克隆与序列分析

鲨烯合酶(SQS )是植物甾醇和三萜化合物生物合成途径中的关键酶。以巴西橡胶树为试验材料,提取胶乳总 RNA,利用 RT-PCR 以及 RACE 的方法克隆橡胶树鲨烯合酶 cDNA 编码区片段,并进行序列分析。结果表明：橡胶树鲨烯合酶 cDNA 编码区为1239 bp,编码413个氨基酸,命名为 HbSQS 。荧光定量分析表明鲨烯合酶基因在不同组织里表达水平存在明显差异,且受乙烯调控。     

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24 600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids (aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.     

A temperature-sensitive N-glycosylation mutant of S. cerevisiae that behaves like a cell-cycle mutant

The temperature-sensitive S. cerevisiae mutant alg1-1, defective in the N-glycosylation of proteins, shows a first cycle arrest at the non-permissive temperature of 36 °C. The cell number increases by 50% and the absorbance approximately doubles. The budding index of 0.4 at 26 °C drops to 0.15 and DNA synthesis quickly comes to a halt at 36 °C. When the temperature is lowered again, budding and DNA synthesis start after a lag of 2–3 h; α-factor prevents both these processes in cells of mating type a. In addition, cells arrested at 26 °C in G1 with α-factor also do not start budding at the non-permissive temperature after removal of α-factor. The results support recent findings obtained with tunicamycin and suggest that at least one glycoprotein is required for G1-S phase transition in yeast.     

Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

Background  

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations.     

Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface

CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43‐Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab′)₂ antibody‐mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43^+/+ versus CD43^?/? macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43‐dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.     

Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa

Summary A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5 and 3 flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.     

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein–protein or protein–carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii .     

The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated

Nontypeable Haemophilus influenzae is a gram-negative commensal organism that is commonly associated with localized respiratory tract disease. The pathogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells. Hia is the major adhesin expressed by a subset of nontypeable H. influenzae strains and promotes efficient adherence to a variety of human epithelial cell lines. Based on previous work, Hia is transported to the surface of Escherichia coli transformants and is capable of mediating E. coli adherence without the assistance of other H. influenzae proteins. In the present study, we examined the mechanism of Hia secretion. PhoA fusions, deletional mutagenesis, and N-terminal amino acid sequencing established that the signal for Hia export from the cytoplasm resides in the first 49 amino acids, including a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelectron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure analysis predicted that the C terminus of Hia forms a beta-barrel with a central hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter proteins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hia is the prototype of an important subfamily of autotransporter proteins.     

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.     

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37 °C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS–PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3–4 h at 37 °C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20 °C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37 °C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.     

The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin

Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the blood stream and eventually invasion of the meninges. The latter two aspects have been well characterized at a molecular level in the last decade. Less is known about the early stages of pathogenesis, i.e. adhesion to and invasion of gastrointestinal cells. Here, the characterization of the Hek protein is reported, which is expressed by neonatal meningitic E. coli (NMEC) and is localized to the outer membrane. It is demonstrated that this protein can cause agglutination of red blood cells and can mediate autoaggregation. Escherichia coli expressing this protein can adhere to and invade epithelial cells. So far, this is the first outer membrane protein in NMEC to be directly implicated in epithelial cell invasion.     

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.     

The role of thymidylate synthase as an RNA binding protein

Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, it is now appreciated that thymidylate synthase functons as an RNA binding protein. Specifically, thymidylate synthase binds with high affinity to its own mRNA, resulting in translational repression. An extensive series of experiments has been performed to elucidate the molecular elements underlying the interaction between thymidylate synthase and its own mRNA. In addition to characterization of the underlying cis- and trans-acting elements, recent studies have shown that thymidylate synthase has the capacity to bind specifically to other cellular RNA species. While the biological significance of these other RNA/thymidylate synthase interactions remains to be defined, this work suggests a potential role for TS in coordinately regulating several critical aspects of cellular metabolism.     

The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids

The PEB1a protein of the gastrointestinal pathogen Campylobacter jejuni mediates interactions with epithelial cells and is an important factor in host colonization. Cell fractionation and immunoblotting showed that PEB1a is most abundant in the periplasm of C. jejuni, and is detectable in the culture supernatant but not in the inner or outer membrane. The protein is homologous with periplasmic-binding proteins associated with ABC transporters and we show by fluorescence spectroscopy that purified recombinant PEB1a binds L-aspartate and L-glutamate with sub microM K(d) values. Binding of L-14C-aspartate or L-14C-glutamate was strongly out-competed by excess unlabelled aspartate or glutamate but only poorly by asparagine and glutamine. A mutant in the Cj0921c gene, encoding PEB1a, was completely unable to transport 5 microM L-14C-glutamate and showed a large reduction (approximately 20-fold) in the rate of L-14C-aspartate transport compared with the wild type. Although microaerobic growth of this mutant was little affected in complex media, growth on aspartate or glutamate in defined media was completely prevented, whereas growth with serine was similar to wild type. 1H-NMR analysis of the culture supernatants of the Cj0921c mutant showed some utilization of aspartate but not glutamate, consistent with the transport data. It is concluded that in addition to the established role of PEB1a as an adhesin, the PEB1 transport system plays a key role in the utilization of aspartate and glutamate, which may be important in vivo carbon sources for this pathogen.