scholarly journals Antigen 84, an Effector of Pleiomorphism in Mycobacterium smegmatis

2007 ◽  
Vol 189 (21) ◽  
pp. 7896-7910 ◽  
Author(s):  
Liem Nguyen ◽  
Nicole Scherr ◽  
John Gatfield ◽  
Anne Walburger ◽  
Jean Pieters ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Cell Shape ◽  
Gene Disruption ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Asymmetrically Distributed ◽  
Cell Wall Expansion

ABSTRACT While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.

scholarly journals Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant ofMycobacterium smegmatis

2000 ◽  
Vol 182 (23) ◽  
pp. 6854-6856 ◽  
Author(s):  
Aimee E. Belanger ◽  
Joelle C. Porter ◽  
Graham F. Hatfull
Keyword(s):  
Recombinant Proteins ◽  
Mycobacterium Smegmatis ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Gene Encoding ◽  
Enzymatic Assays ◽  
Peptidoglycan Biosynthesis

ABSTRACT A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation inddlA, the gene encodingd-alanine:d-alanine ligase. Enzymatic assays using recombinant proteins and d-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.

How Flies Find Fungal Foes

2007 ◽  
Vol 2007 (369) ◽  
pp. tw21-tw21
Author(s):  
L. Bryan Ray
Keyword(s):  
Cell Wall ◽  
Virulence Factors ◽  
Defense Mechanism ◽  
Fungal Infections ◽  
Toll Pathway ◽  
Online Journal ◽  
Gene Encoding ◽  
Fungal Virulence

Responses of the immune systems of plants and animals show what appears to be evidence of ancient attacks and counterattacks by pathogens and their hosts in the battle for survival. Drosophila have developed receptors that recognize constituents of bacterial cell walls and mount an immune response that causes proteolytic cleavage of the cytokine Spätzle. The Spätzle fragment then activates Toll receptors and leads to production of antimicrobial peptides. Gottar et al. explored the response of Drosophila to fungal infections and found a similar defense mechanism but also unveiled a second signaling pathway that detects a virulence factor produced by the fungus. The authors infected flies by pricking them with a needle dipped in fungus-containing solution and monitored survival or Toll-dependent expression of the gene encoding an antifungal peptide. They found that the receptor GNBP3 (Gram-negative binding protein 3) was required for detection of cell wall components of the fungi and consequent activation of Toll receptors. However, cells with a mutated GNB3 protein could still respond to fungi and activate Toll, but in this case cell wall-derived components were not the trigger. This response depended on the presence of live fungi and, presumably, the production of virulence factors. One such factor is the protease PR1, and the authors showed that expression of PR1 alone led to activation of the Toll pathway. Knowing that a fly protease PSH (Persephone), which is thought to participate in a cascade of proteases that lead to Spätzle cleavage and activation of the Toll pathway in response to fungi, itself requires proteolytic removal of a prodomain for activity, the authors tested whether PR1 might activate PSH. Indeed, studies in vitro and in vivo indicated that PSH appears to be a direct substrate of PR1. The fungi use the PR1 protease to break down the protective cuticle of the insect and allow infection. The authors propose that PSH acts like a sensor to monitor the status of the cuticle. If the presence of PR1 shows that the defense barrier is being broken, PSH is cleaved and the antimicrobial signaling is initiated. Whether humans have such a sensor system to recognize fungal virulence factors remains unknown.M. Gottar, V. Gobert, A. A. Matskevich, J.-M. Reichhart, C. Wang, T. M. Butt, M. Belvin, J. A. Hoffmann, D. Ferrandon, Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell127, 1425-1437 (2006). [Online Journal]

scholarly journals CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 932-943 ◽  
Author(s):  
Cédric Absalon ◽  
Michal Obuchowski ◽  
Edwige Madec ◽  
Delphine Delattre ◽  
I. Barry Holland ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Regulatory Network ◽  
Sensor Kinase ◽  
Translation Factor ◽  
Shape Determination ◽  
Pasta Domain ◽  
Cell Wall Expansion

The conserved prpC, prkC, cpgA locus in Bacillus subtilis encodes respectively a Ser/Thr phosphatase, the cognate sensor kinase (containing an external PASTA domain suggested to bind peptidoglycan precursors) and CpgA, a small ribosome-associated GTPase that we have shown previously is implicated in shape determination and peptidoglycan deposition. In this study, in a search for targets of PrkC and PrpC, we showed that, in vitro, CpgA itself is phosphorylated on serine and threonine, and another GTPase, the translation factor EF-Tu, is also phosphorylated by the kinase on the conserved T384 residue. Both substrates are dephosphorylated by PrpC in vitro. In addition, we identified YezB, a 10.3 kDa polypeptide, and a component of the stressosome, as a substrate for both enzymes in vitro and apparently in vivo. We propose that the PrpC/PrkC/CpgA system constitutes an important element of a regulatory network involved in the coordination of cell wall expansion and growth in B. subtilis.

scholarly journals FERONIA’s sensing of cell wall pectin activates ROP GTPase signaling in Arabidopsis

2018 ◽  
Author(s):  
Wenwei Lin ◽  
Wenxin Tang ◽  
Charles T. Anderson ◽  
Zhenbiao Yang
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Cell Surface Receptor ◽  
Pavement Cell ◽  
Signaling Mechanism ◽  
Rop Gtpase ◽  
Surface Receptor ◽  
A Cell

ABSTRACTPlant cells need to monitor the cell wall dynamic to control the wall homeostasis required for a myriad of processes in plants, but the mechanisms underpinning cell wall sensing and signaling in regulating these processes remain largely elusive. Here, we demonstrate that receptor-like kinase FERONIA senses the cell wall pectin polymer to directly activate the ROP6 GTPase signaling pathway that regulates the formation of the cell shape in the Arabidopsis leaf epidermis. The extracellular malectin domain of FER directly interacts with de-methylesterified pectin in vivo and in vitro. Both loss-of-FER mutations and defects in the pectin biosynthesis and de-methylesterification caused changes in pavement cell shape and ROP6 signaling. FER is required for the activation of ROP6 by de-methylesterified pectin, and physically and genetically interacts with the ROP6 activator, RopGEF14. Thus, our findings elucidate a cell wall sensing and signaling mechanism that connects the cell wall to cellular morphogenesis via the cell surface receptor FER.

scholarly journals Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum ofSaccharomyces cerevisiae:In Vivo and In Vitro Evidence

1999 ◽  
Vol 10 (4) ◽  
pp. 1019-1030 ◽  
Author(s):  
Olga Castro ◽  
Ling Yun Chen ◽  
Armando J. Parodi ◽  
Claudia Abeijón
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Glucose Transport ◽  
Subcellular Location ◽  
Functional Expression ◽  
Uridine Diphosphate ◽  
Gene Encoding ◽  
Sugar Nucleotide

It has been proposed that synthesis of β-1,6-glucan, one ofSaccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparentKmof 46 μM and a Vmaxof 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) inS. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected inalg6 or alg5 mutant cells, which transfer Man9GlcNAc2to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

scholarly journals Protein phosphatase SppA regulates apical growth and dephosphorylates cell polarity determinant DivIVA in Streptomyces coelicolor

2021 ◽  
Author(s):  
Fanny Passot ◽  
Stuart Cantlay ◽  
Klas Flardh
Keyword(s):  
Cell Wall ◽  
Cell Polarity ◽  
Protein Phosphatase ◽  
Cell Shape ◽  
Streptomyces Coelicolor ◽  
Polar Growth ◽  
Hyphal Branching ◽  
Tip Extension

Bacteria that exhibit polar growth, i.e. build their peptidoglycan cell walls in restricted zones at cell poles, often show large morphological diversity and plasticity. However, their mechanisms for regulation of cell shape and cell wall assembly are poorly understood. The Gram-positive Streptomyces bacteria, like other Actinobacteria, depend on the essential coiled coil protein DivIVA for establishment of cell polarity and direction of polar growth. Streptomycetes grow as filamentous hyphae that exhibit tip extension. New hyphal tips are generated by lateral branching. Cell shape is largely determined by the control of cell wall growth at these hyphal tips. The Ser/Thr protein kinase AfsK is involved in controlling polar growth and directly phosphorylates DivIVA. Here, we identify a protein phosphatase in Streptomyces coelicolor , SppA, that dephosphorylates DivIVA in vivo and in vitro and affects growth and cell shape. An sppA mutant shows reduced rate of hyphal tip extension, altered hyphal branching patterns, and exhibits frequent spontaneous hyphal growth arrests, all contributing to the unusually dense mycelial structure and slow growth rate that characterize sppA mutants. These phenotypes are largely suppressed in an afsK sppA double mutant, showing that AfsK and SppA partially affect the same regulatory pathway and share target proteins that are involved control of polar growth in S. coelicolor . Strains with a non-phosphorylatable mutant DivIVA were constructed and confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. Altogether, this study identifies a PPP-family protein phosphatase directly involved in the control of polar growth and cell shape determination in S. coelicolor and underscore the importance of eukaryotic-type Ser/Thr phosphorylation in regulation of growth and cell envelope biogenesis in Actinobacteria.

scholarly journals Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase

2007 ◽  
Vol 189 (11) ◽  
pp. 3987-3995 ◽  
Author(s):  
Mireille Hervé ◽  
Audrey Boniface ◽  
Stanislav Gobec ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
De Novo ◽  
Diaminopimelic Acid ◽  
Kinetic Properties ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Physiological Properties

ABSTRACT The UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide l-alanyl-γ-d-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide l-Ala-d-Glu and l-Ala very poorly. Replacement of meso-diaminopimelic acid by l-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the ld-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.

scholarly journals TheCryptococcus neoformansTitan cell is an inducible and regulated morphotype underlying pathogenesis

2017 ◽  
Author(s):  
Ivy M. Dambuza ◽  
Thomas Drake ◽  
Ambre Chapuis ◽  
Leanne Taylor-Smith ◽  
Nathalie LeGrave ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Size ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Lavage Fluid ◽  
Environmental Stimuli ◽  
Altered Cell ◽  
Bacterial Peptidoglycan

AbstractFungi undergo changes in cell shape in response to environmental stimuli that drive pathogenesis and niche adaptation, such as the yeast-to-hyphal transition of dimorphic fungi in response to changing temperature. The basidiomyceteCryptococcus neoformansundergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproduciblein vitroinduction system. Here we demonstrate reproduciblein vitroTitan cell induction in response to environmental stimuli consistent with the host lung.In vitroTitan cells exhibit all the properties ofin vivogenerated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify bacterial peptidoglycan as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid andE. colico-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established and previously undescribed regulators of Titanisationin vitroand investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-titan transition and establish an essentialin vitromodel for the future characterization of this important morphotype.Author SummaryChanges in cell shape underlie fungal pathogenesis by allowing immune evasion and dissemination.AspergillusandCandida albicanshyphae drive tissue penetration.Histoplasma capsulatumandC. albicansyeast growth allows evasion and dissemination. As major virulence determinates, morphogenic transitions are extensively studied in animal models andin vitro. The pathogenic fungusCryptococcus neoformansis a budding yeast that, in the host lung, switches to an unusual morphotype termed the Titan cell. Titans are large, polyploid, have altered cell wall and capsule, and produce haploid daughters. Their size prevents engulfment by phagocytes, yet they are linked to dissemination and altered immune response. Despite their important influence on disease, replicating the yeast-to-Titan switchin vitrohas proved challenging. Here we show that Titans are induced by host-relevant stimuli, including serum and bronchio-alveolar lavage fluid. We identify bacterial peptidoglycan as a relevant inducing compound and predict anin vivoTitan defect for a clinical isolate. Genes regulatingin vivoTitanisation also influencein vitroformation. Titanisation is a conserved morphogenic switch across theC. neoformansspecies complex. Together, we show that Titan cells are a regulated morphotype analogous to the yeast-to-hyphal transition and establish new ways to study Titans outside the host lung.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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