Molecular and Functional Characterization of O Antigen Transfer inVibriocholerae

Schild, Stefan; Lamprecht, Anna-Karina; Reidl, Joachim

doi:10.1074/jbc.m501259200

Cited by 59 publications

(59 citation statements)

References 50 publications

(58 reference statements)

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“…During LPS assembly, Wzy and RfaL/WaaL share the same substrate, undecaprenol-O-antigen polymer, which is subsequently transferred onto the sugar acceptor of the lipid A-core intermediate. Both Wzy and RfaL/WaaL proteins are highly hydrophobic; however, topology and biochemical studies point to distinct features (33,34,47). Wzy proteins encompass 14 TM segments with 2 large periplasmic loops, PL3 and PL5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

“…and Pseudomonas aeruginosa (33,34). The topology of P. aeruginosa WaaL has been experimentally determined with translational hybrids, revealing 12 transmembrane (TM) and 5 periplasmic loops (PL1 to -5) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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“…Also evident is a homologue (71% identity) of wbaZ encoding mannosyltransferase that may be involved in a mannose-containing linkage region in the repeat unit of Klebsiella K57. The wzy gene family encodes an O-polysaccharide polymerase that recognizes and extends the O-antigen polysaccharide-repeating units (31). This protein was also postulated as a polymerase responsible for lipid-linked repeat unit polymerization in the capsular synthesis process (36).…”

Section: Vol 46 2008 K Pneumoniae K57 and A New Capsular Serotype mentioning

confidence: 99%

Capsular Polysaccharide Synthesis Regions in Klebsiella pneumoniae Serotype K57 and a New Capsular Serotype

et al. 2008

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Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype.

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“…It has been reported that waaA (VC0233), wavC (VC0227), and waaC (VC0225) are highly conserved and putatively essential core OS genes in V. cholerae (34). Among all the wav genes, only mutations in waaL (VC0237), wavA (VC0223), and wavL (VC0239) affect O antigen attachment (34,41). Here, VC0229 and VC0231 were found to be relevant for N16961 sensitivity to phage VP3 in the process of phage attachment.…”

Section: Discussionmentioning

confidence: 87%

The Core Oligosaccharide and Thioredoxin of Vibrio cholerae Are Necessary for Binding and Propagation of Its Typing Phage VP3

Zhang

et al. 2009

J Bacteriol

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VP3 is a T7-like phage and was used as one of the typing phages in a phage-biotyping scheme that has been used for the typing of Vibrio cholerae O1 biotype El Tor. Here, we studied the receptor and other host genes of V. cholerae necessary for the lytic propagation of VP3. Six mutants resistant to VP3 infection were obtained from the random transposon insertion mutant bank of the sensitive strain N16961. The genes VC0229 and VC0231, which belong to the wav gene cluster encoding the core oligosaccharide (OS) region of lipopolysaccharide, were found to be interrupted by the transposon in five mutants, and the sixth mutant had the transposon inserted between the genes rhlB and trxA, which encode the ATP-dependent RNA helicase RhlB and thioredoxin, respectively. Gene complementation, transcription analysis, and the loss of VP3 sensitivity by the gene deletion mutants confirmed the relationship between VP3 resistance and VC0229, VC0231, and trxA mutation. The product of VP3 gene 44 (gp44) was predicted to be a tail fiber protein. gp44 could bind to the sensitive wild-type strain and the trxA mutant, but not to VC0229 and VC0231 mutants. The results showed that OS is a VP3 receptor on the surface of N16961, thioredoxin of the host strain is involved in the propagation of the phage, and gp44 is the tail fiber protein of VP3. This revealed the first step in the infection mechanism of the T7-like phage VP3 in V. cholerae.Vibrio cholerae is the pathogenic agent of cholera and has caused seven pandemics. Only the O1 and O139 serogroups of V. cholerae have been associated with cholera epidemics, and strains of the O1 serogroup are subdivided into two biotypes, classical and El Tor (12). V. cholerae is the host for many bacteriophages, such as the tailed phage CP-T1 (18), K139 (39), and many filamentous phages (3,24,49). Phages infect and destroy host strains with high specificity, so phage-typing schemes were developed to differentiate strains of the same serogroups or genera in many bacteria, such as Salmonella enterica serovar Typhi (28) and Staphylococcus aureus (22). In V. cholerae, phage-typing schemes were also developed for typing of the O1 biotype El Tor (7) and O139 (5). A phagebiotyping scheme has been used for nearly 40 years in China for the typing of O1 El Tor strains (14). Based on the lytic patterns of the five typing phages, El Tor strains can be clustered into 32 phage types.The first step of phage infection is the specific binding of phages to receptors on the host cell surface, triggering the ejection of phage DNA into the cell and initiating the propagation process (1). Some outer membrane proteins serve as receptors for phage adsorption, such as LamB (19), OmpA (8),and OmpC (52) of Escherichia coli and OmpK of Vibrio parahaemolyticus (23). Lipopolysaccharide (LPS) is a common phage receptor. Cytotoxin-converting ⌽CTX from Pseudomonas aeruginosa (51) and FC3-10 from Klebsiella spp. (4) bind to the core region of LPS, while vibriophage CP-T1 (17) and ⌽YeO3-12, specific for Yersinia enterocolitica serotype...

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Molecular and Functional Characterization of O Antigen Transfer inVibriocholerae

Cited by 59 publications

References 50 publications

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Capsular Polysaccharide Synthesis Regions in Klebsiella pneumoniae Serotype K57 and a New Capsular Serotype

The Core Oligosaccharide and Thioredoxin of Vibrio cholerae Are Necessary for Binding and Propagation of Its Typing Phage VP3

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