scholarly journals Aeromonas caviae motility and glycosylation

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Laila Allihaybi
Keyword(s):  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Bacterial Motility ◽  
Aquatic Environments ◽  
Wound Infections ◽  
Bacterial Colonisation ◽  
Filament Assembly ◽  
Aeromonas Caviae ◽  
Pseudaminic Acid ◽  
Polar Type

Aeromonas are Gram-negative facultative anaerobic rods, which inhabit various aquatic environments and are pathogens of both warm and cold-blooded animals. In humans they cause gastro-enteritis and wound infections. They are motile in liquid environments by a single polar type of flagellum. The flagellum plays an important role for the bacterial colonisation and the adhesion to the host cells. The Aeromonaspolar flagella filament is a polymer composed of two flagellins, FlaA and FlaB. The flagellins are O-linked glycosylated through the addition of the unusual bacterial sugar pseudaminic acid to serine and threonine residues within the flagellins D2/D3 domain. The addition of this sugar is essential for flagella filament assembly and bacterial motility. The flagellin’s are modified by between 6 – 8 sugar residues that occupy the potential 14 sites of attachment. Motility accessory factors (Maf proteins) are candidate enzymes for transferring glycan molecules to the flagellin (glycosyltransferases transferring sugar to flagellin) due to their genetic location and motility phenotypes associated with disruption mutants. This study utilised site-directed mutagenesis to change the potential sites of flagellin glycosylation to assess the effect of these mutations on motility by swimming assays and flagella filament formation by electron microscopy. The analysis of different numbers of site-directed mutants suggest that some sites are more important than others and that the removal of 4 sites results in greatly reduced motility.

scholarly journals Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

2005 ◽  
Vol 187 (8) ◽  
pp. 2582-2591 ◽  
Author(s):  
Yasuo Mitani ◽  
XianYing Meng ◽  
Yoichi Kamagata ◽  
Tomohiro Tamura
Keyword(s):  
Cell Wall ◽  
Culture Media ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Wall Structure ◽  
Synthetase Activity ◽  
Glutamine Amidotransferase ◽  
Glutaminase Activity

ABSTRACT The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH4Cl, indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.

scholarly journals Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tümay Capraz ◽  
Nikolaus Ferdinand Kienzl ◽  
Elisabeth Laurent ◽  
Jan W Perthold ◽  
Esther Föderl-Höbenreich ◽  
...  
Keyword(s):  
Viral Entry ◽  
Structural Models ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Directed Mutagenesis ◽  
Binding Affinities ◽  
Clinical Tests ◽  
Angiotensin Converting Enzyme 2 ◽  
Decoy Receptor ◽  
Dynamics Simulations

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

How Bacteria Bind More Strongly Under Mechanical Force: The Catch-Bond FimH

2003 ◽  
Author(s):  
Wendy E. Thomas ◽  
Evgeni V. Sokurenko ◽  
Viola Vogel
Keyword(s):  
Shear Stress ◽  
Structural Model ◽  
Intestinal Bacteria ◽  
Mechanical Force ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Structural Variations ◽  
Catch Bond ◽  
Static Conditions

We study a protein that responds to mechanical force in most striking manner. We demonstrate that Escherichia coli bacteria need shear stress to bind to certain tissues and model surfaces; they bind strongest precisely when the body tries to wash them off. We have determined that the protein responsible for this behavior is FimH, a ubiquitous adhesion protein in intestinal bacteria that mediates adhesion to host cells via the carbohydrate mannose. Although mechanical force noramlly decreases bond lifetimes, we have shown that the bond betweeen FimH and simple mono-mannose receptors is s “catch-bond” that lasts longer under shear stress. In contrast, structural variations in either FimH or the receptor cause a stronger mode of adhesion in static conditions with little or no activation under force. We derive a structural for how mechanical force switches FimH to a strong binding mode by using steered molecular dynamics simulations, and validate the predictions with subsequent site-directed mutagenesis. The physiological consequences as well as the engineering principles suggested by the structural model will be discussed.

scholarly journals EnP1, a Microsporidian Spore Wall Protein That Enables Spores To Adhere to and Infect Host Cells In Vitro

2007 ◽  
Vol 6 (8) ◽  
pp. 1354-1362 ◽  
Author(s):  
Timothy R. Southern ◽  
Carrie E. Jolly ◽  
Melissa E. Lester ◽  
J. Russell Hayman
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Spore Wall ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Binding Motif ◽  
Heparin Binding ◽  
Cell Infection ◽  
Host Cell Surface

ABSTRACT Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

scholarly journals Membrane trafficking of the bacterial adhesin GspB and the accessory Sec transport machinery

2018 ◽  
Vol 294 (5) ◽  
pp. 1502-1515 ◽  
Author(s):  
Cierra Spencer ◽  
Barbara A. Bensing ◽  
Nagendra N. Mishra ◽  
Paul M. Sullam
Keyword(s):  
Signal Peptide ◽  
Membrane Trafficking ◽  
Lipid Membranes ◽  
Large Cell ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Hydrophobic Core ◽  
Gram Positive Bacteria ◽  
Anionic Lipids

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are large, cell wall–anchored adhesins that mediate binding to many host cells and proteins and are associated with bacterial virulence. SRR glycoproteins are exported to the cell surface by the accessory Sec (aSec) system comprising SecA2, SecY2, and 3–5 additional proteins (Asp1 to Asp5) that are required for substrate export. These adhesins typically have a 90-amino acid-long signal peptide containing an elongated N-region and a hydrophobic core. Previous studies of GspB (the SRR adhesin ofStreptococcus gordonii) have shown that a glycine-rich motif in its hydrophobic core is essential for selective, aSec-mediated transport. However, the role of this extended N-region in transport is poorly understood. Here, using protein–lipid co-flotation assays and site-directed mutagenesis, we report that the N-region of the GspB signal peptide interacts with anionic lipids through electrostatic forces and that this interaction is necessary for GspB preprotein trafficking to lipid membranes. Moreover, we observed that protein–lipid binding is required for engagement of GspB with SecA2 and for aSec-mediated transport. We further found that SecA2 and Asp1 to Asp3 also localize selectively to liposomes that contain anionic lipids. These findings suggest that the GspB signal peptide electrostatically binds anionic lipids at the cell membrane, where it encounters SecA2. After SecA2 engagement with the signal peptide, Asp1 to Asp3 promote SecA2 engagement with the mature domain, which activates GspB translocation.

scholarly journals Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

2003 ◽  
Vol 69 (8) ◽  
pp. 4830-4836 ◽  
Author(s):  
Takeharu Tsuge ◽  
Tamao Hisano ◽  
Seiichi Taguchi ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Chain Length ◽  
Acyl Chain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Acyl Chain Length ◽  
Aeromonas Caviae

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1 Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

Site-Directed Mutagenesis of Asp313, Glu315, and Asp391 Residues in Chitinase of Aeromonas Caviae

IUBMB Life ◽  
1999 ◽  
Vol 48 (2) ◽  
pp. 199-204 ◽  
Author(s):  
Fu-Pang Lin ◽  
Hsing-Chen Chen ◽  
Chung-Saint Lin
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Aeromonas Caviae

scholarly journals Insight into motility-dependent pathogenicity of the zoonotic spirochete Leptospira

2020 ◽  
Author(s):  
Jun Xu ◽  
Nobuo Koizumi ◽  
Shuichi Nakamura
Keyword(s):  
Host Tissue ◽  
Solid Surfaces ◽  
Host Cells ◽  
Bacterial Motility ◽  
Kidney Cells ◽  
Pathogenic Species ◽  
Tissue Surfaces ◽  
Crawling Motility ◽  
Insight Into ◽  
Host Tissues

AbstractBacterial motility is crucial for many pathogenic species in the process of invasion and/or dissemination. The spirochete bacteria Leptospira spp. cause symptoms, such as hemorrhage, jaundice, and nephritis, in diverse mammals including humans. Although loss-of-motility attenuate the spirochete, the mechanism of the motility-dependent pathogenicity is unknown. Here, focusing on that Leptospira spp. swim in liquid and crawl on solid surfaces, we investigated the spirochetal dynamics on the host tissues by infecting cultured kidney cells from various species with pathogenic and nonpathogenic leptospires. We found that, in the case of the pathogenic leptospires, a larger fraction of bacteria attached to the host cells and persistently traveled long distances using the crawling mechanism. Our results associate the kinetics and kinematic features of the spirochetal pathogens with their virulence.One Sentence SummaryAdhesivity and crawling motility over host tissue surfaces are closely related to the pathogenicity of a zoonotic spirochete.

scholarly journals Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells

1998 ◽  
Vol 72 (3) ◽  
pp. 2213-2223 ◽  
Author(s):  
Moulay Hicham Alaoui-Ismaili ◽  
Christopher D. Richardson
Keyword(s):  
Insect Cells ◽  
Interaction Analysis ◽  
Recombinant Baculovirus ◽  
Host Cells ◽  
Filament Assembly ◽  
Late Protein ◽  
Structural And Functional Properties ◽  
Occlusion Bodies ◽  
Infected Insect ◽  
Infected Cells

ABSTRACT Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta mooreientomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.

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