scholarly journals Galactosylation of the Secondary Cell Wall Polysaccharide ofBacillus anthracisand Its Contribution to Anthrax Pathogenesis

2017 ◽  
Vol 200 (5) ◽  
Author(s):  
Alice Chateau ◽  
Justin Mark Lunderberg ◽  
So Young Oh ◽  
Teresa Abshire ◽  
Arthur Friedlander ◽  
...  
Keyword(s):  
Cell Wall ◽  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Secondary Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Wild Type ◽  
Content Type ◽  
Bacterial Inoculum ◽  
Associated Proteins

ABSTRACTBacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division.B. anthracisSCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal,O4-β-Gal)-(1→]6-12. The genes whose products promote the galactosylation ofB. anthracisSCWP are not yet known. We show here that the expression ofgalE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required forB. anthracisSCWP galactosylation. ThegalE1mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-typeB. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion ofgalE1diminishes the capsulation ofB. anthraciswith poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of theB. anthraciscell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCEUnlike virulentBacillus anthracisisolates,B. anthracisstrain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growthin vitroin broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants ofB. anthracisstrains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genesgalE1andgalE2. We identifiedgalE1as necessary for SCWP galactosylation. Deletion ofgalE1decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form ofB. anthracisand increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

scholarly journals LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

2014 ◽  
Vol 197 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Megan Liszewski Zilla ◽  
Yvonne G. Y. Chan ◽  
Justin Mark Lunderberg ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Chain Length ◽  
Secondary Cell Wall ◽  
Teichoic Acid ◽  
Cell Wall Polysaccharide ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Associated Proteins ◽  
Layer Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified theB. anthracislcpDmutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, theB. anthracislcpB3variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan.B. anthracisdoes not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed inS. aureus, promote WTA attachment. We propose a model wherebyB. anthracisLCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.

scholarly journals Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis

2020 ◽  
Vol 202 (15) ◽  
Author(s):  
Alice Chateau ◽  
So Young Oh ◽  
Anastasia Tomatsidou ◽  
Inka Brockhausen ◽  
Olaf Schneewind ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Secondary Cell Wall ◽  
Turgor Pressure ◽  
Cell Wall Polysaccharide ◽  
Bacterial Cells ◽  
Content Type ◽  
Repeat Units ◽  
Undecaprenyl Phosphate

ABSTRACT Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3–α-Gal and O-4–β-Gal substitutions. UDP-Gal, synthesized by GalE1, is the substrate of galactosyltransferases that modify the SCWP repeat. Here, we show that the gtsE gene, which encodes a predicted glycosyltransferase with a GT-A fold, is required for O-4–β-Gal modification of trisaccharide repeats. We identify a DXD motif critical for GtsE activity. Three distinct genes, gtsA, gtsB, and gtsC, are required for O-3–α-Gal modification of trisaccharide repeats. Based on the similarity with other three-component glycosyltransferase systems, we propose that GtsA transfers Gal from cytosolic UDP-Gal to undecaprenyl phosphate (C55-P), GtsB flips the C55-P-Gal intermediate to the trans side of the membrane, and GtsC transfers Gal onto trisaccharide repeats. The deletion of galE1 does not affect growth in vitro, suggesting that galactosyl modifications are dispensable for the function of SCWP. The deletion of gtsA, gtsB, or gtsC leads to a loss of viability, yet gtsA and gtsC can be deleted in strains lacking galE1 or gtsE. We propose that the loss of viability is caused by the accumulation of undecaprenol-bound precursors and present an updated model for SCWP assembly in B. anthracis to account for the galactosylation of repeat units. IMPORTANCE Peptidoglycan is a conserved extracellular macromolecule that protects bacterial cells from turgor pressure. Peptidoglycan of Gram-positive bacteria serves as a scaffold for the attachment of polymers that provide defined bacterial interactions with their environment. One such polymer, B. anthracis SCWP, is pyruvylated at its distal end to serve as a receptor for secreted proteins bearing the S-layer homology domain. Repeat units of SCWP carry three galactoses in B. anthracis. Glycosylation is a recurring theme in nature and often represents a means to mask or alter conserved molecular signatures from intruders such as bacteriophages. Several glycosyltransferase families have been described based on bioinformatics prediction, but few have been studied. Here, we describe the glycosyltransferases that mediate the galactosylation of B. anthracis SCWP.

scholarly journals Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
So-Young Oh ◽  
J. Mark Lunderberg ◽  
Alice Chateau ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Vegetative Growth ◽  
Cell Shape ◽  
Secondary Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Genetic Determinants ◽  
Content Type ◽  
Repeat Structure ◽  
Layer Assembly

ABSTRACT The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis. Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.

scholarly journals Bacillus anthracistagOIs Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

2015 ◽  
Vol 197 (22) ◽  
pp. 3511-3520 ◽  
Author(s):  
J. Mark Lunderberg ◽  
Megan Liszewski Zilla ◽  
Dominique Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Vegetative Growth ◽  
Cell Shape ◽  
Secondary Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Content Type ◽  
Phosphodiester Bonds ◽  
Acid Labile ◽  
Layer Assembly

ABSTRACTBacillus anthraciselaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→] and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show thattagO(BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-PN-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required forB. anthracisSCWP synthesis and S-layer assembly. Similar togneY-gneZmutants,B. anthracisstrains lackingtagOcannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affectB. anthraciscell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further,B. anthracisvariants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth.IMPORTANCEBacillus anthraciselaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent ontagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes.

scholarly journals Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

2015 ◽  
Vol 197 (19) ◽  
pp. 3216-3227 ◽  
Author(s):  
Sao-Mai Nguyen-Mau ◽  
So-Young Oh ◽  
Daphne I. Schneewind ◽  
Dominique Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Bacillus Anthracis ◽  
Self Assembly ◽  
Protein Assembly ◽  
Crystalline Lattice ◽  
Cell Wall Polysaccharide ◽  
Cytoplasmic Protein ◽  
Content Type ◽  
Bacterial Surface

ABSTRACTBacillus anthracisvegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show thatslaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes thein vitroformation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly inB. anthracis.IMPORTANCES-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identifiedBacillus anthracisSlaQ, a small cytoplasmic protein that facilitates S-layer protein assemblyin vivoandin vitro.

scholarly journals An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Richard E. Wiemels ◽  
Stephanie M. Cech ◽  
Nikki M. Meyer ◽  
Caleb A. Burke ◽  
Andy Weiss ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Active Form ◽  
Ppiase Activity ◽  
Content Type ◽  
Associated Proteins

ABSTRACT Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus. IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

scholarly journals Bacillus anthracis Acetyltransferases PatA1 and PatA2 Modify the Secondary Cell Wall Polysaccharide and Affect the Assembly of S-Layer Proteins

2012 ◽  
Vol 195 (5) ◽  
pp. 977-989 ◽  
Author(s):  
J. M. Lunderberg ◽  
S.-M. Nguyen-Mau ◽  
G. S. Richter ◽  
Y.-T. Wang ◽  
J. Dworkin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Secondary Cell Wall ◽  
Cell Wall Polysaccharide

scholarly journals Bacillus anthracis lcpGenes Support Vegetative Growth, Envelope Assembly, and Spore Formation

2015 ◽  
Vol 197 (23) ◽  
pp. 3731-3741 ◽  
Author(s):  
Megan Liszewski Zilla ◽  
J. Mark Lunderberg ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Cell Size ◽  
Chain Length ◽  
Vegetative Growth ◽  
Secondary Cell Wall ◽  
Spore Formation ◽  
Content Type ◽  
Size And Shape ◽  
Layer Assembly

ABSTRACTBacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins andB. anthracisS-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP).B. anthracisexpresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1to -4,lcpC, andlcpD), which when expressed inStaphylococcus aureuspromote attachment of wall teichoic acid to peptidoglycan. Mutations inB. anthracislcpB3andlcpDcause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the otherlcpgenes is not known. By deleting combinations oflcpgenes from theB. anthracisgenome, we generated variants with singlelcpgenes.B. anthracisexpressinglcpB3alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressinglcpB1orlcpB4displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, thelcpB2strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2++), and variants expressinglcpCorlcpDdisplayed severe defects in growth and cell shape. ThelcpB2++,lcpC, orlcpDstrains supported neither S-layer assembly nor spore formation. We propose a model whereby LCP enzymes fulfill partially overlapping functions in transferring SCWP molecules to discrete sites within the bacterial envelope.IMPORTANCEProducts of genes essential for bacterial envelope assembly represent targets for antibiotic development. The LytR-CpsA-Psr (LCP) enzymes tether bactoprenol-linked intermediates of secondary cell wall polymers to the C6 hydroxyl ofN-acetylmuramic acid in peptidoglycan; however, the role of LCPs as a target for antibiotic therapy is not defined. We show here that LCP enzymes are essential for the cell cycle, vegetative growth, and spore formation ofBacillus anthracis, the causative agent of anthrax disease. Furthermore, we assign functions for each of the six LCP enzymes, including cell size and shape, vegetative growth and sporulation, and S-layer and S-layer-associated protein assembly.

Sign in / Sign up

Export Citation Format

Share Document