Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)

Masoud, Hussein; Sadovskaya, Irina; Kievit, Teresa de; Altman, Eleonora; Richards, James C.; Lam, Joseph S.

doi:10.1128/jb.177.23.6718-6726.1995

Cited by 38 publications

(48 citation statements)

References 41 publications

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“…The outer core is composed of four D-glucose (D-Glc) residues, one L-rhamnose (L-Rha) residue, and one N-(L-alanyl)-D-galactosamine residue, and its biosynthesis is not fully understood. The structures of the core OS of several different serotypes, clinical isolates, and rough mutants of P. aeruginosa have been reported previously (2,3,12,17,24). The results of the previous studies showed that there are minor variations in phosphate and O-acetyl substituents in some of the core sugars.…”

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confidence: 76%

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

et al. 2008

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Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an ␣-1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an ␣-1,6-linked L-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of L-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked ␣-1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked ␣-1,6-L-Rha. These results provide evidence that WapR is the ␣-1,3-rhamnosyltransferase, while MigA is the ␣-1,6-rhamnosyltransferase.Pseudomonas aeruginosa is an important gram-negative opportunistic pathogen capable of producing virulence determinants, including secreted toxins and enzymes, as well as cell surface structures, such as adhesins, flagella, and lipopolysaccharide (LPS) (18). LPS is a glycolipid composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. P. aeruginosa produces two forms of O antigen, which are the homopolymeric A band and the heteropolymeric B band (15,21). The gene clusters associated with the biosynthesis of Aband and B-band O antigen in P. aeruginosa have been well characterized (for reviews, see references 18 and 22). In contrast, relatively little is known about the biosynthesis of the core oligosaccharide (OS).The P. aeruginosa core OS, like that of other gram-negative bacteria, can be conceptually subdivided into the inner core and the outer core (Fig. 1). The inner core is highly conserved and contains two L-glycero-D-manno-heptose (Hep) residues and two 3-deoxy-D-manno-octulosonic acid (Kdo) residues; the genetics and enzymology of this region are the best understood to date (14). The outer core is composed of four D-glucose (D-Glc) residues, one L-rhamnose (L-Rha) residue, and one N-(L-alanyl)-D-galactosamine residue, and its biosynthesis is not fully understood. The structures of the core OS of several different serotypes, clinical isolates, and rough mutants of P. a...

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confidence: 76%

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

et al. 2008

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“…Finally, a fourth transferase, WbpR, likely adds the fourth residue, -Rha, to the B-band unit. -Rha is found both in the O antigen and in the core oligosaccharide of serotype O6 (Knirel, 1990 ;Masoud et al, 1995). In bacteria, -Rha synthesis is encoded by the highly conserved rmlABCD genes (Macpherson et al, 1994 ;Reeves, 1993).…”

Section: Discussionmentioning

confidence: 99%

Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide The GenBank accession number for the sequence reported in this paper is AF035937.

1999

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This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype O6. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL O6 is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and Aband synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp O6 genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either O5 or O6 ; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.

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“…These procedures have accomplished a striking reduction of infection and diseases. Purification of microbial components and molecular biology techniques improved our knowledge of the immune system and of the mechanisms of virulence of pathogens, allowing the production of attenuated mutants, the expression of vaccine proteins in live vectors, the purification and synthesis of microbial antigens, and the induction of immune responses through nucleic acids, proteins, and polysaccharides (8,22,25,30,37).…”

Section: Discussionmentioning

confidence: 99%

Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

et al. 2010

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So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a ⌬ 12 -desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (M⌽) (A. Porta et al., J. Bacteriol. 192:1988Bacteriol. 192: -1998Bacteriol. 192: , 2010). Here, we show that overexpression of a homologous ⌬ 9 -desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine M⌽ along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in M⌽ of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.So far, a methodology to obtain live attenuated strains has not been generated for fungal pathogens. With respect to parasites, a live attenuated strain of Toxoplasma has been produced. However, this modified strain was used to develop a vaccine that was considered expensive, caused side effects, and had a short shelf life. Furthermore, the authors emphasized that this vaccine might revert to a pathogenic strain and, therefore, was not considered suitable for human use (17). In Leishmania and other parasites, as yet none of the current candidate subunit vaccines has achieved complete protection reproducibly. Attempts to develop an effective vaccine to control leishmaniasis have been shown to be feasible, but no vaccine is in active clinical use (18). The ability to create genetically modified pathogens by eliminating virulence or essential genes is considered a powerful alternative in the development of an effective protective vaccine. The current genetic procedures are based on the use of specific (virulence) genes that may cause attenuation and are species specific. In other words, a given method of attenuation may be effective in a specific organism but not in other pathogens.We have previously shown in several prokaryotic and eukaryotic cells, including pathogens such as Salmonella (34) and in the human pathogenic fu...

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Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)

Cited by 38 publications

References 41 publications

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide The GenBank accession number for the sequence reported in this paper is AF035937.

Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

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