RESUMEN
BACKGROUND: Unique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed. METHODS: Lipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI-MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium. RESULTS: T. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2. CONCLUSION: The study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol. GENERAL SIGNIFICANCE: The effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets.
Asunto(s)
Bacteroidaceae/metabolismo , Ceramidas/análisis , Etanolaminas/análisis , Inositol/metabolismo , Bacteroidaceae/química , Radioisótopos de Carbono , Ceramidas/biosíntesis , Ceramidas/química , Cromatografía Líquida de Alta Presión , Deuterio , Etanolaminas/química , Etanolaminas/metabolismo , Glicerol/análisis , Glicerol/química , Marcaje Isotópico , Extracción Líquido-Líquido , Espectroscopía de Resonancia Magnética , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/metabolismoRESUMEN
We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.
Asunto(s)
Bacteriófagos/fisiología , Biopelículas , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/virología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , Genoma Viral , Genómica , Filogenia , Receptores ViralesRESUMEN
Enterobacterial common antigen (ECA) is a polysaccharide present in all members of Enterobacteriaceae anchored either via phosphatidylglycerol (PG) or LPS to the outer leaflet of the outer membrane (ECAPG and ECALPS, respectively). Only the latter form is ECA-immunogenic. We previously demonstrated that Yersinia enterocolitica Oâ:â3 and its rough (O-specific polysaccharide-negative) mutants were ECA-immunogenic, suggesting that they contained ECALPS; however, it was not known which part of the LPS core region was involved in ECA binding. To address this, we used a set of three deep-rough LPS mutants for rabbit immunization. The polyvalent antisera obtained were: (i) analysed for the presence of anti-LPS and anti-ECA antibodies; (ii) treated with caprylic acid (CA) to precipitate IgM antibodies and protein aggregates; and (iii) adsorbed with live ECA-negative bacteria to obtain specific anti-ECA antisera. We demonstrated the presence of antibodies specific for both ECAPG and ECALPS in all antisera obtained. Both CA treatment and adsorption with ECA-negative bacteria efficiently removed anti-LPS antibodies, resulting in specific anti-ECA sera. The LPS of the ECALPS-positive deepest-rough mutant contained only lipid A and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues of the inner core, suggesting that ECALPS was linked to the Kdo region of LPS in Y. enterocolitica Oâ:â3.
Asunto(s)
Antígenos Bacterianos/inmunología , Lipopolisacáridos/inmunología , Yersinia enterocolitica/inmunología , Animales , Anticuerpos/inmunología , Antígenos Bacterianos/química , Sueros Inmunes , Lipopolisacáridos/química , Mutación , Conejos , Serotipificación , Yersinia enterocolitica/clasificación , Yersinia enterocolitica/genéticaRESUMEN
The genus Yersinia represents Gram-negative bacteria that are widely distributed in the environment and possess lipopolysaccharide (LPS) as a major molecule on the surface of the bacterial envelope. LPS is composed of an anchoring lipid, termed lipid A, and a polysaccharide part which not only determines the bacterial serotype but may also serve as a bacteriophage receptor. Here we present the structure of the O-polysaccharide (O-PS) of Yersinia enterocolitica O:50 phage ΦR1-37-sensitive strain 3229. The structural characterization was performed utilizing 1D and 2D nuclear magnetic resonance spectroscopy and chemical analyses. The structure of the O-PS biological repeating unit was identified as â2)-α-L-Rhap-(1â3)-α-L-FucpNAc-(1â3)-α-L-FucpNAc-(1â3)-ß-D-GlcpNAc-(1â.
Asunto(s)
Antígenos O/química , Yersinia enterocolitica/química , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Antígenos O/aislamiento & purificaciónRESUMEN
Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.
Asunto(s)
Glicosiltransferasas/metabolismo , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Yersinia enterocolitica/enzimología , Anticuerpos Monoclonales/metabolismo , Bacteriófagos/metabolismo , Dominio Catalítico , Biología Computacional , Farmacorresistencia Bacteriana , Galactosa/química , Galactosa/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/genética , Modelos Moleculares , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Antígenos O/química , Antígenos O/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Polimixina B/farmacología , Yersinia enterocolitica/efectos de los fármacos , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
INTRODUCTION: Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. MATERIALS AND METHODS: Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA(PG)) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA(PG) is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA(LPS)) free of ECA(PG). The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. RESULTS: The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA(LPS), suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA(PG) could be demonstrated in the Rc mutant strain R4/O28. CONCLUSIONS: These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA(LPS). Since ECA(PG) is not immunogenic unless combined with some proteins, it is likely that ECA(PG)-protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.