Endolysins of <i>Bacillus anthracis</i> Bacteriophages Recognize Unique Carbohydrate Epitopes of Vegetative Cell Wall Polysaccharides with High Affinity and Selectivity

Mo, Kai‐For; Li, Xiuru; Li, Huiqing; Low, Lieh Yoon; Quinn, Conrad P.; Boons, Geert‐Jan

doi:10.1021/ja3069962

Cited by 46 publications

(32 citation statements)

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“…The SCWP promotes envelope attachment of murein hydrolases that specifically bind to the carbohydrate structure (18)(19)(20)(21). The ketal-pyruvyl-modified SCWP is a ligand for the S-layer homology (SLH) domains of native B. anthracis proteins.…”

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confidence: 99%

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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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)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡) with ␣-Gal and ␤-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.KEYWORDS Bacillus anthracis, S-layers, Wzy repeat polymerase, cell wall polysaccharide, envelope assembly T he peptidoglycan cell wall of Gram-positive microbes is a determinant of bacterial shape and integrity (1, 2). Peptidoglycan also serves as a scaffold for the attachment of secondary polymers, which support specific cell cycle functions of bacteria (3, 4). Some Gram-positive bacteria, for example, Bacillus subtilis and Staphylococcus aureus, attach wall teichoic acid (WTA) to peptidoglycan, thereby positioning peptidoglycan synthesis enzymes, division septa, and murein hydrolases for cell growth and division (5-7). Other microbes, including Streptococcus species and Bacillus cereus, attach a secondary cell wall polysaccharide (SCWP) to peptidoglycan, and these polymers presumably fulfill functions similar to that of WTA during the bacterial cell cycle (8, 9). While WTA synthesis has been studied in detail (10), little is known about the genetic determinants and assembly reactions for SCWP synthesis.

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confidence: 99%

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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“…Previous work demonstrated that bacteriophage lysins of B. anthracis, PlyG and PlyL, associate with the SCWP through their C-terminal cell wall binding domain (CBD) in a manner independent of csaB-mediated ketal-pyruvylation (38,(42)(43)(44). We generated purified PlyG CBD -mCherry, where the C-terminal CBD of PlyG (38) is fused to the mCherry reporter, to assess the overall abundance and subcellular distribution of the SCWP (with or without ketal-pyruvate) in wild-type and tagO::aad9(pts-tagO i ) vegetative forms.…”

Section: B Anthracis Tago::aad9(pts-tagomentioning

confidence: 99%

Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

et al. 2015

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Bacillus anthracis elaborates 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)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-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 that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell 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. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth. IMPORTANCEBacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-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.T he cell wall envelope of Bacillus anthracis, the causative agent of anthrax, is comprised of peptidoglycan and its attached secondary cell wall polysaccharide (SCWP) (1). The SCWP retains two surface (S)-layer proteins, surface array protein (Sap) and extractable antigen 1 (EA1) (2), as well as 22 S-layer-associated proteins whose S-layer homology (SLH) domains associate with ketal-pyruvylated SCWP (3, 4). Unlike nonpathogenic Bacillus spp., for example, Bacillus subtilis and Bacillus cereus AHU1030, B. anthracis does not synthesize wall teichoic acid (WTA) (5, 6). Nevertheless, B. anthracis expresses functional tagO and tagA genes, whose products provide for the synthesis of murein linkage units [P-GlcNAc-(4¡1)-␤-ManNAc-R] that are phosphodiester linked to the C 6 -hydroxyl of N-acetylmuramic acid in the repeating disaccharide of peptidoglycan [MurNAc(P-GlcNAc-ManNAc-R)-GlcNAc] (4). In B. subtilis, -R represents wall teichoic acid, i.e., either polyglycerol-phosphate or polyribitol-phosphate (7). Here, we test the hypothesis that in B. anthracis the SCWP is attached via the murine linkage unit and that the tagO gene is required for vegetati...

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“…ManNAc is a key residue of the repeating trisaccharide of the well-characterized SCWP, the only polymer known to contain ManNAc in B. anthracis (1). A library of oligosaccharides containing the core trisaccharide motif ␣-DGlcNAc-(1¡ 4)-␤-D-ManNAc-(1¡4)-␤-D-GlcNAc of the SCWP of B. anthracis was chemically synthesized with various patterns of ␣-DGal and ␤-D-Gal branching points (9). Measurement of dissociation constants for the cell wall binding domains of the endolysins PlyL and PlyG using surface plasmon resonance established a preferred interaction with a trisaccharide bearing the galactosyl moiety at C-4 of the nonreducing GlcNAc moiety (9).…”

Section: B Acillus Anthracis Elaborates the Secondary Cell Wall Polysmentioning

confidence: 99%

“…A library of oligosaccharides containing the core trisaccharide motif ␣-DGlcNAc-(1¡ 4)-␤-D-ManNAc-(1¡4)-␤-D-GlcNAc of the SCWP of B. anthracis was chemically synthesized with various patterns of ␣-DGal and ␤-D-Gal branching points (9). Measurement of dissociation constants for the cell wall binding domains of the endolysins PlyL and PlyG using surface plasmon resonance established a preferred interaction with a trisaccharide bearing the galactosyl moiety at C-4 of the nonreducing GlcNAc moiety (9). PlyL and PlyG were also found to interact with highly purified SCWP of several B. anthracis isolates via their C-terminal domains but not their Nterminal catalytic domains, thus corroborating the notion that the SCWP serves as a receptor for ␥ phage lysins (10).…”

Section: B Acillus Anthracis Elaborates the Secondary Cell Wall Polysmentioning

confidence: 99%

“…Variations in the galactosylation pattern in the SCWP core structure of bacilli of the Cereus group, but not the terminal pyruvylation, were found to govern specificity toward phage endolysins (7,9). By conducting a genomic comparison of the PlyGsensitive B. anthracis Ames and the related PlyG-resistant Bacillus cereus ATCC 10987 strains, Schuch et al noticed a gene cluster that could be responsible for the biosynthesis of the cell wall glycopolymer receptor of ␥ phage endolysins (7).…”

Section: B Acillus Anthracis Elaborates the Secondary Cell Wall Polysmentioning

confidence: 99%

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GneZ, a UDP-GlcNAc 2-Epimerase, Is Required for S-Layer Assembly and Vegetative Growth of Bacillus anthracis

2014

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Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡] n , where ␣-GlcNAc and ␤-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDPGlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.

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Endolysins of Bacillus anthracis Bacteriophages Recognize Unique Carbohydrate Epitopes of Vegetative Cell Wall Polysaccharides with High Affinity and Selectivity

Cited by 46 publications

References 46 publications

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

GneZ, a UDP-GlcNAc 2-Epimerase, Is Required for S-Layer Assembly and Vegetative Growth of Bacillus anthracis

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