Use of a Bacteriophage Lysin to Identify a Novel Target for Antimicrobial Development

Schuch, Raymond; Pelzek, Adam J.; Raz, Assaf; Euler, Chad W.; Ryan, Patricia; Winer, Benjamin Y.; Farnsworth, Andrew; Bhaskaran, Shyam S.; Stebbins, C. Erec; Yong, Xu; Clifford, Adrienne; Bearss, David J.; Vankayalapati, Hariprasad; Goldberg, Andrew V.; Fischetti, Vincent A.

doi:10.1371/journal.pone.0060754

Cited by 42 publications

(50 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

mentioning

confidence: 99%

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

View full text Add to dashboard Cite

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.

show abstract

mentioning

confidence: 99%

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…PatAB1/2-mediated acetylation of SCWP molecules affects the assembly of EA1 and of some but not all BSLs (20). Recent studies have begun to identify genes for SCWP synthesis, which, unlike pyruvyl or acetyl modifications of SCWP, appears to be essential for B. anthracis growth (23,24). Nevertheless, most of the genetic determinants for SCWP synthesis, including genes whose products catalyze attachment of the polysaccharide to the peptidoglycan scaffold of B. anthracis, remain unknown (19).…”

mentioning

confidence: 99%

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

et al. 2014

View full text Add to dashboard Cite

bBacillus 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 the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant 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. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan. Bacillus anthracis, the causative agent of anthrax, is a sporeforming, Gram-positive bacterium that germinates in host infected tissues and replicates as elongated chains of vegetative forms (1, 2). This unique growth pattern appears to be caused by the regulated separation of septal peptidoglycan, which generates bacterial chains whose mere size precludes clearance by host phagocytes (3-5). The genetic determinants for B. anthracis chain formation are conserved among pathogenic species of the Bacillus cereus group and, as demonstrated with B. cereus G9241, likely contribute to disease pathogenesis (6, 7). Hallmarks of pathogenic Bacillus species are virulence plasmids, pXO-1 and pXO-2 in B. anthracis (8, 9), providing for toxin production and capsulation (10, 11) as well as the chromosomally encoded surface (S)-layer locus (12). Two S-layer proteins of B. anthracis, Sap and EA1, are endowed with S-layer homology (SLH) domains, which retain these proteins in the bacterial envelope by binding to the secondary cell wall polysaccharide (SCWP) (13-15). S-layer protein crystallization domains, responsible for the spontaneous assembly of these polypeptides into a paracrystalline array (16), form a twodimensional lattice that can be thought of as bacterial integument (17)(18)(19).The structural genes of S-layer proteins, sap and eag, are flanked by genes encoding determinants for S-layer protein secretion (secA2 and slaP) (12) or pyruvylation (csaB) (14) as well as acetylation of the SCWP (patA1/2 and patB1/2) (20). CsaB-mediated pyruvylation of the terminal N-acetylmannosamine (ManNAc) of the SCWP (21), with the repeat struc- (22), is a prerequisite for the assembly of S-layer proteins and 22 B. anthracis S-layer-associated proteins (BSLs) (14). PatAB1/2-mediated acetylation of SCWP molecules affects the assembly of EA1 and of some but not all BSLs (20). Recent studies have begun to identify genes for SCWP synthesis, which, unlike py...

show abstract

“…Further, the ⌬BA5436 mutant assembled reduced amounts of S-layer proteins (Sap and EA1) in the bacterial envelope, as determined by urea extraction and SDS-PAGE analysis of these polypeptides (22). Nevertheless, ⌬BA5436 mutant bacilli did not display defects in growth, which was surprising, as studies of gneZ, gneY (12,14), and tagO (this report) suggest that SCWP synthesis is essential for B. anthracis growth. Of note, the structural gene for BA5436 (BAS5051 in B. anthracis Sterne) is located immediately adjacent to tagO (BAS5050).…”

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

confidence: 66%

“…The enzymes that synthesize the trisaccharide repeats and galactosyl modifications of the SCWP are not yet known (10,11). Comparative genome analysis identified the surface polysaccharide synthesis locus sps (BAS5116-BAS5127) (12). The gneZ gene (BAS5117), a member of this locus, encodes a UDP-GlcNAc 2-epimerase and catalyzes the conversion of GlcNAc into ManNAc (13).…”

mentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

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...

show abstract

Use of a Bacteriophage Lysin to Identify a Novel Target for Antimicrobial Development

Cited by 42 publications

References 38 publications

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

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

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

Contact Info

Product

Resources

About