scholarly journals Comparison of Cell Wall Localization among Pir Family Proteins and Functional Dissection of the Region Required for Cell Wall Binding and Bud Scar Recruitment of Pir1p

2005 ◽  
Vol 4 (11) ◽  
pp. 1872-1881 ◽  
Author(s):  
Toru Sumita ◽  
Takehiko Yoko-o ◽  
Yoh-ichi Shimma ◽  
Yoshifumi Jigami
Keyword(s):  
Cell Wall ◽  
Repetitive Sequence ◽  
Functional Role ◽  
Fluorescent Protein ◽  
Microscopic Analysis ◽  
Red Fluorescent Protein ◽  
Terminal Sequence ◽  
Cell Wall Binding ◽  
Covalently Linked

ABSTRACT We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1Δ cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars. Stepwise deletion of the eight units of the repetitive sequence of Pir1p revealed that one unit is enough for the protein to bind bud scars and that the extent of binding of Pir1p to the cell wall depends on the number of these repetitive units. The localization of a chimeric Pir1p in which the repetitive sequence of Pir1p was replaced with that of Pir4p revealed the functional role of the different protein regions, specifically, that the repetitive sequence is required for binding to the cell wall and that the C-terminal sequence is needed for recruitment to bud scars. This is the first report that bud scars contain proteins like Pir1p as internal components.

scholarly journals The Chitin Connection

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
David L. Goldman ◽  
Alfin G. Vicencio
Keyword(s):  
Cell Wall ◽  
Cell Membrane ◽  
Fungal Infections ◽  
Allergic Inflammation ◽  
Chitinase Activity ◽  
Chitin Deacetylase ◽  
Fungal Cell ◽  
Content Type ◽  
Covalently Linked

ABSTRACTChitin, a polymer ofN-acetylglucosamine, is an essential component of the fungal cell wall. Chitosan, a deacetylated form of chitin, is also important in maintaining cell wall integrity and is essential forCryptococcus neoformansvirulence. In their article, Gilbert et al. [N. M. Gilbert, L. G. Baker, C. A. Specht, and J. K. Lodge, mBio 3(1):e00007-12, 2012] demonstrate that the enzyme responsible for chitosan synthesis, chitin deacetylase (CDA), is differentially attached to the cell membrane and wall. Bioactivity is localized to the cell membrane, where it is covalently linked via a glycosylphosphatidylinositol (GPI) anchor. Findings from this study significantly enhance our understanding of cryptococcal cell wall biology. Besides the role of chitin in supporting structural stability, chitin and host enzymes with chitinase activity have an important role in host defense and modifying the inflammatory response. Thus, chitin appears to provide a link between the fungus and host that involves both innate and adaptive immune responses. Recently, there has been increased attention to the role of chitinases in the pathogenesis of allergic inflammation, especially asthma. We review these findings and explore the possible connection between fungal infections, the induction of chitinases, and asthma.

The role of rpoS on the survival of a p-nitrophenol degrading Pseudomonas putida strain in planktonic and biofilm phases

2009 ◽  
Vol 55 (10) ◽  
pp. 1176-1186 ◽  
Author(s):  
M. L. Maki ◽  
J. R. Lawrence ◽  
G. D.W. Swerhone ◽  
K. T. Leung
Keyword(s):  
Pseudomonas Putida ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Red Fluorescent Protein ◽  
Generation Times ◽  
Impact On Survival ◽  
Green Fluorescent ◽  
Biofilm Development

The survival of and interactions between a Pseudomonas putida strain labelled with a red fluorescent protein gene (WT-rfp) and its green fluorescent protein gene-labelled rpoS– mutant (KO-gfp) were examined. The generation times of the planktonic WT-rfp and KO-gfp in trypticase soy broth were not significantly different (i.e., p > 0.05) from each other at 30 °C. However, the biovolume of the KO-gfp biofilm was about 7 times larger than its WT-rfp counterpart after 48 h of growth. Furthermore, the presence of WT-rfp suppressed the biofilm development of KO-gfp significantly in co-culture biofilms. In planktonic conditions, the pre-carbon-starved WT-rfp achieved a 3-fold greater survival than the pre-carbon-starved KO-gfp in 0.85% saline after a 13-day incubation. In a 1:1 ratio co-culture, the pre-carbon-starved WT-rfp outcompeted the pre-carbon-starved KO-gfp by 20-fold. However, the survival of WT-rfp and KO-gfp were not significantly different from each other in biofilm conditions. Additionally, 11.4% and 61.2% of the WT-rfp and KO-gfp biofilms, respectively, remained intact after washing in 0.2% SDS for 60 min. In conclusion, the rpoS had a significant impact on survival and competitiveness of planktonic P. putida, and on biofilm development, being implicated in competitive suppression of biofilm development in co-culture biofilms and decreased biofilm cohesiveness.

scholarly journals Perturbation of the T4 molecule transmits a negative signal to T cells.

1985 ◽  
Vol 162 (4) ◽  
pp. 1294-1303 ◽  
Author(s):  
I Bank ◽  
L Chess
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Functional Role ◽  
Cell Activation ◽  
Target Cells ◽  
U937 Cells ◽  
Accessory Cells ◽  
Covalently Linked ◽  
Negative Signal

We investigated the functional role of the T4 molecule in the activation of T cells by OKT3. T4+ cells were induced to proliferate by OKT3 and erythrocyte rosette-negative accessory cells in the presence or absence of OKT4C, OKT4, and OKT1. OKT4C (IgG1), and not OKT4 (IgG2) or OKT1 (IgG1) inhibited proliferation when OKT4C was added during the first 24 h of cell culture. The inhibition of OKT3 activation by OKT4C did not require Ia+ accessory cells, since T4+ cells could be activated by OKT3 in the presence of Ia- U937 cells, and this activation was markedly inhibited by OKT4C. Furthermore, T4+ cells could be induced to proliferate by OKT3 covalently linked to Sepharose beads, in the absence of any accessory cells. Under these conditions, OKT4C, but not OKT4 or OKT1 significantly inhibited proliferation. These data demonstrate that at least one mechanism by which anti-T4 antibodies inhibit T cell activation is independent of any putative role of T4 molecules in the recognition of Ia on target cells. The data are compatible with the idea that perturbation of the T4 molecules can transmit a negative signal to T4+ cells.

scholarly journals Functional Role of C-Terminal Sequence Elements in the Transporter Associated with Antigen Processing

2004 ◽  
Vol 174 (1) ◽  
pp. 328-339 ◽  
Author(s):  
Sarah Ehses ◽  
Ralf M. Leonhardt ◽  
Guido Hansen ◽  
Michael R. Knittler
Keyword(s):  
Functional Role ◽  
Antigen Processing ◽  
Terminal Sequence ◽  
Sequence Elements

scholarly journals The Auxiliary Role of the Amidase Domain in Cell Wall Binding and Exolytic Activity of Staphylococcal Phage Endolysins

Viruses ◽  
2018 ◽  
Vol 10 (6) ◽  
pp. 284 ◽  
Author(s):  
Bokyung Son ◽  
Minsuk Kong ◽  
Sangryeol Ryu
Keyword(s):  
Cell Wall ◽  
Cell Wall Binding ◽  
Auxiliary Role

scholarly journals LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Minsuk Kong ◽  
Hongjun Na ◽  
Nam-Chul Ha ◽  
Sangryeol Ryu
Keyword(s):  
Cell Wall ◽  
Catalytic Domain ◽  
Binding Activity ◽  
Binding Domain ◽  
Lytic Activity ◽  
Content Type ◽  
Cell Wall Binding ◽  
A Cell ◽  
Cell Wall Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.

scholarly journals Role of Net Charge on Catalytic Domain and Influence of Cell Wall Binding Domain on Bactericidal Activity, Specificity, and Host Range of Phage Lysins

2011 ◽  
Vol 286 (39) ◽  
pp. 34391-34403 ◽  
Author(s):  
Lieh Yoon Low ◽  
Chen Yang ◽  
Marta Perego ◽  
Andrei Osterman ◽  
Robert Liddington
Keyword(s):  
Cell Wall ◽  
Host Range ◽  
Bactericidal Activity ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Net Charge ◽  
Cell Wall Binding ◽  
Cell Wall Binding Domain ◽  
Phage Lysins

scholarly journals A mammalian fatty acid hydroxylase responsible for the formation of α-hydroxylated galactosylceramide in myelin

2005 ◽  
Vol 388 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Matthias ECKHARDT ◽  
Afshin YAGHOOTFAM ◽  
Simon N. FEWOU ◽  
Inge ZÖLLER ◽  
Volkmar GIESELMANN
Keyword(s):  
Fatty Acid ◽  
Functional Role ◽  
Time Course ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mammalian Brain ◽  
Fatty Acid Hydroxylase

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the α-C atom of the fatty acid moiety. α-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of α-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)–FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid α-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2–3 weeks of age. This is in agreement with the reported time course of fatty acid α-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of α-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of α-hydroxylation modification in myelin, skin and other organs.

scholarly journals Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

2012 ◽  
Vol 194 (23) ◽  
pp. 6498-6506 ◽  
Author(s):  
Marcel R. Eugster ◽  
Martin J. Loessner
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Teichoic Acid ◽  
Single Copy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Binding Domains ◽  
Cell Wall Binding ◽  
Wall Teichoic Acids

ABSTRACTThe C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. TheListeria monocytogenesPly118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure ofListeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding usingL. monocytogenesserovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundanttagOhomologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of eithertagO1(EGDelmo0959) ortagO2(EGDelmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficientListeriacells. Substitution oftagOfrom an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from otherListeriaphage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.

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