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Bordetella bronchiseptica is a respiratory pathogen in mammal species. Its endotoxin is a powerful virulency factor. The lipoid A mediety of lipopolysaccharides is responsible for most of the toxic belongingss while the polyose mediety carries the antigenic determiners of the molecule. We have compared the lipoid A constructions of two human and one coney isolates in their virulency and non-virulence stages. The happening of palmitate in these constructions at one or two sites was correlated with virulency stage, the late identified glucosamine alterations of Bordetella lipoids A were non.

Introduction

Lipopolysaccharide ( LPS ) , a complex glycolipid, is the major structural constituent of the Gram-negative bacterial outer membrane. The general construction of the bacterial LPSs consists of three distinguishable spheres: a hydrophobic mediety called lipid A, a nucleus oligosaccharide incorporating 2-keto-3-deoxy-octulosonic acid ( Kdo ) , and a serospecific O polyose composed of reiterating oligosaccharide units ( Caroff and Karibian, 2003 ) . Lipids A ground tackle LPS molecules in the outer cusp of the external bacterial membrane. The nucleus has merely limited structural variableness compared to the O antigen, which is present in the LPS of most, but non all, bacterial species. It comprises the most variable portion of LPSs and confers serotype specificity on the bacterium.

LPS forms a ligand with the TLR4-MD2-CD14 composite, which is present on many cell types including macrophages and dendritic cells ( Poltorak et al. , 1998 ; Hoshino et al. , 1999 ; Qureshi et al. , 1999 ) . The stimulation of this receptor complex leads to activation of signaling tracts, ensuing in initiation of antimicrobic cistrons and release of cytokines, thereby originating inflammatory and immune defence responses ( Miller et al. , 2005 ) . Most of the biological activities of LPS have been associated with their lipoid A mediety. Lipid A itself can be modified via mechanisms such as acylation, deacylation, fatty acerb hydroxylation, and phosphate group permutation with aminoarabinose, galactosamine, or phosphoethanolamine ( Dixon and Darveau, 2005 ; Trent et Al. 2006 ; Geurtsen et al. , 2007 ) . These alterations can play a important function in modulating host responses to infection. We showed late that bacterium of the Bordetella genus such as B. bronchiseptica and B. whooping cough are capable of modifying their lipoids A by permutation of both phosphate groups with glucosamine ( GlcN ) ( Marr et al. , 2008 ) . Up to now this permutation had non been observed in any other bacterial genus. Its presence was shown to strongly increase the biological activities of purified LPS and of the whole bacterium ( Marr et al. , 2010 ) .

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The Bordetella genus presently contains nine species, most of which are respiratory piece of land pathogens, and the most extensively studied 1s include B. whooping cough, B. parapertussis, and B. bronchiseptica. Although these three pathogens are really closely related genetically ( Parkhill et al. , 2003 ) , their LPS molecules have important differences ( Caroff et al. , 2002 ) . B. petrii is the lone known facultative anaerobiotic species of the genus with an environmental beginning ; it seems to be more closely related to a common, putative ascendant than the other infective Bordetella species ( von Wintzingerode et al. , 2001 ) . Interestingly it was late isolated from worlds holding different infections, including respiratory 1s ( Fry et al. , 2005 ; Stark et al. , 2007 ; Spilker et al. , 2008 ; A. Le Coustumier et al. , unpublished ) . Even though other Bordetella species portion many facets of pathogenicity, they have distinguishable host scopes and do different pathologies, which could be related to their surface constituents ( Mattoo and Cherry, 2005 ) . Experiment in the sphere is expected to take to of import information on infective procedures specific to bacterial niches.

B. whooping cough has long been recognised as the pathogen that infects merely worlds and is the causative agent of whooping cough in babies and in grownups ( Mattoo and Cherry, 2005 ) . Some B. parapertussis strains are adapted to the human host and cause whooping cough, while others are adapted to the ovine host doing chronic pneumonia ( Mattoo and Cherry, 2005 ) . B. bronchiseptica, on the other manus colonizes the respiratory piece of land of a big figure of carnal hosts and can be diagnostic ( Kennel cough, atrophic coryza, bronchial pneumonia etc. ) or symptomless and chronic. Although B. bronchiseptica most frequently infects animate beings, it can be collected from worlds, most of the clip contaminated by septic animate beings. B. whooping cough and B. parapertussis are assumed to hold evolved independently from a B. bronchiseptica-like ascendant ( Diavatopoulos et al. , 2005 ) . The nucleus oligosaccharides from different isolates of B. whooping cough and B. bronchiseptica display an about indistinguishable construction of a bifurcate nonasaccharide with several free amino and carboxyl groups ( Caroff et al. , 2001 ) . This nucleus may transport an antigenic distal trisaccharide, ( Caroff et al. , 2000 ) which was shown to be of import for bacterial opposition to pulmonary surfactant proteins ( Schaeffer et al. , 2004 ) . The latter was non found in B. parapertussis “ free ” nucleus ( Caroff et al. , 2001 ) . However, nil is known about LPS polymorphism within the species. The familial determiners of host specificity and virulency across the Bordetella genus still stay to be explored. Genome decrease, accompanied by proliferation of Insertion Sequence ( IS ) elements, has played a cardinal function in the development of the infective Bordetella species ( Parkhill et al. , 2003 ; Cummings et al. , 2004 ; Bouchez et al. , 2009 ) .

Bordetella lipid A constructions have a common bisphosphorylated I?-1,6 glucosamine disaccharide anchor with two amide-linked 3-OH C14 substituents. The nature and distribution of ester-linked fatty acids have so far proved to be species- or strain-specific and extremely variable ( Caroff et al. , 1994 ; Zarrouk et al. , 1997 ) . One of the unusual characteristics of Bordetella lipid A compared to those of most other lipoids A is the absence of symmetricalness at the C-3 and C-3 ‘ places. The Bordetella genus shows a singular ability to modify lipid A constructions by late-steps in their biogenesis. It has long been known that lipid A constructions differ non merely between different genera but besides frequently between species of a genus, every bit good as among different isolates of a species ( Aussel et al. , 2000 ; Therisod et al. , 2001 ) . Furthermore, a given isolate may show different LPS molecular species at the same time with changing copiousness.

Zarrouk et al. , ( 1997 ) earlier demonstrated that the lipoid A constructions of three B. bronchiseptica isolates were extremely heterogenous and different ; the differences were found largely in the nature of the fatty acids. The ability to modify the construction of its lipid A constituents may let Bordetella to get away or change TLR4-dependent host defence mechanisms every bit good as to diminish the susceptibleness to incursion of the bacterial cell wall by antibiotics ( Dixon and Darveau, 2005 ; Miller et al. , 2005 ) . It may lend to version of B. bronchiseptica, a spontaneously mutating carnal pathogen, to diverse niches or hosts, taking to the comparatively recent visual aspect of the human pathogens ( Parkhill et al. , 2003 ) .

B. bronchiseptica, one time thought to infect lone animate beings is presently geting increased importance as a human pathogen ; it colonizes the ciliated epithelial tissue of the respiratory piece of land of the host and establishes chronic infections ( Woolfrey and Moody, 1991 ; Gueirard et al. , 1995 ) . The development of such chronic infections may partly depend on the ability of bacteriums to accommodate phenotypicaly to variable stimulations.

In order to better qualify construction to virulence relationships, particularly in the context of new structural elements found in Bordetella lipids A ( Marr et al. , 2008 ) we studied the lipoid A constructions of B. bronchiseptica isolates from coney and human beginning as a map of their virulency stage. To our cognition, this is the first structural comparing of the sort refering Bordetella clinical isolates.

Consequences and treatment

Entire fatty acid composing

Entire fatty acid analysis revealed the presence of 3-hydroxytetradecanoic acid ( 3-OH C14 ) , tetradecanoic acid ( C14 ) , 2-hydroxydodecanoic acid ( 2-OH C12 ) and dodecanoic acid ( C12 ) in lipids A extracted from all isolates in their non-virulent ( H- ) and virulent ( H+ ) phases. In add-on to these fatty acids, hexadecanoic acid ( C16 ) was merely detected in lipoids A extracted from all virulent ( H+ ) isolates.

MALDI-MS analysis of untreated lipoids A

The negative-ion matrix-assisted laser-desorption/ionization-mass spectra ( MALDI-MS ) of 9.73 H- and 9.73 H+ B. bronchiseptica coney isolates are shown in Figures 1A and B, while the spectra corresponding to DANG H- and DANG H+ human isolates are shown in figures 1C and D, severally. The MALDI-MS spectra of ALI H- and ALI H+ human isolates are given as auxiliary Figures S1 A and B, severally. The lipoid A readyings of all the isolates examined except for ALI H- ( Fig. S1A ) were extremely heterogenous. Comparison of lipid A spectra from the isolates in their virulent and non-virulent stages demonstrated that all deadly stage ( H+ ) isolates express perceptibly more heterogenous lipoids A than their several non-virulent ( H- ) opposite numbers.

Major extremums matching to molecular-ions [ M-H ] – common to all isolates were observed at m/z 1345, 1361, 1571, and 1587. In conformity with the gas chromatography ( GC ) information, these extremums were attributed to tetra- and penta-acyl molecular species in which the di-phosphorylated di-glucosamine anchor was substituted with two 3-OH C14, one C14, and one C12 or 2-OH C12 fatso acids ( m/z 1345, 1361 ) with extra 3-OH C14 in the penta-acyl molecules ( m/z 1571 and 1587 ) .

In all spectra, except for that of ALI H- isolate, a series of extra extremums was observed at m/z 161 U or 322 U higher than the mentioned common extremums ( m/z 1522, 1732, 1748, 1893, and 1909 ) . These extremums were attributed to lipid A molecular species in which one or both phosphate groups were substituted with GlcN. This Bvg-regulated lipid A alteration was late unveiled in our group in B. bronchiseptica strain 4650 and B. whooping cough Tahoma I strain ( Marr et al. , 2008 ) . We and others have besides shown that this alteration affects LPS biological activity by strongly increasing pro-inflammatory cytokine production in cells showing the human but non the murine TLR4-CD14-MD2 composite ( Geurtsen et al. , 2009 ; Marr et al. , 2010 ) . We demonstrate here that the presence of these substituents is non correlated with bacterial virulency as it was present in both deadly and non-virulent isolates and was besides non related to the human or rabbit beginning of the isolate.

On the other manus, the fatty acerb composings revealed differences between LPS of isolates in virulent and non-virulent stages. In contrast to non-virulent stage, all the virulent 1s express an LPS with two series of molecular species incorporating palmitate ( C16 ) in their constructions. The first series is represented by molecular-ion extremums at m/z 1809, 1825, 1971, 1987, 2132, and 2148, i.e. , 238 U higher to the corresponding penta-acyl molecular species with and without extra GlcNs. The 2nd series of palmitate-containing molecular species was represented by extremums at m/z 1401, 1627, 1788, 1866, 1949, 2027, and 2188. All these extremums were shifted by add-on of 56 u with regard to the extremums at m/z 1345, 1571, 1732, 1809, 1893, 1971, and 2132, matching to molecular species incorporating a non-hydroxylated C12 fatso acid. Taking into history the GC analyses and the likeliness that the 56 u displacement corresponds to four CH2 units, we concluded that a C16 fatty acid replaced C12 or 2-OH C12. The presence of a C16 fatty acid in the lipoid A structures of this genus has been considered to be effected by a late biosynthetic measure, utilizing fatty acids taken from the outer membrane phospholipids ( Bishop et al. , 2000 ) . Bvg regulated palmitoylation at the secondary C-3 ‘ place of B. bronchiseptica lipid A by PagP enzyme was described before ( Preston et al. , 2003 ) . This alteration is required for continuity of B. bronchiseptica within the mouse respiratory piece of land and for opposition to antibody-mediated complement lysis during respiratory tract infection ( Pilione et al. , 2004 ) . PagP mutations in Salmonella typhimurium and Legionella pneumophila addition sensitiveness to cationic antimicrobic peptide-mediated violent death, proposing that, in these species, lipid A palmitoylation may increase opposition to these peptides ( Guo et al. , 1998 ; Robey et al. , 2001 ) .

In all the mass spectra, the major extremums ( stand foring major molecular species ) have smaller extremums, on both sides, matching to molecular species differing by 2 or 4 CH2 units ( plus or minus 28 or 56 u ) or by an O atom ( plus or minus 16 U ) demoing the capacity of the bacteriums to change their lipoid A constructions, perchance by the Bordetellae relaxed enzyme specificity already described ( Sweet et al. , 2002 ) or by the late biogenesis stairss. These spectra besides showed multiple extremums ensuing from a little loss of phosphate groups ( 80 U ) labeled by -P in the figures. This was due to some grade of dephosphorylation during the hydrolysis measure.

Lipids A constructions

Structures of all the lipid A molecular species mentioned above were established by atomization analysis of native samples in MALDI-MS ( positive ion-mode ) and by consecutive fatty-acid release utilizing alkalic interventions followed by MALDI-MS analysis ( negative-ion manner ) of treated samples and taking into history GC information of the released fatty acids.

The positive-ion MALDI mass spectra ( non shown ) of all samples had a outstanding fragment extremum at m/z 904 and a minor, but unambiguous one at m/z 678. The lipoid A samples extracted from virulent strains had an extra prominent fragment extremum at m/z 1142. In conformity with a good established atomization form ( Karibian et al. , 1999 ) these extremums were attributed to lipid A fragments composed by the distal glucosamine ( GlcN II ) substituted with one phosphate group and different Numberss of fatty acids: one 3-OH C14 and one C14 for tetraacyl molecular species, two 3-OH C14 and one C14 for pentaacyl 1s and two 3-OH C14, one C14 and one C16 for hexaacyl 1s, the latter being characteristic of virulent strains. Fragments incorporating extra GlcN replacing the phosphate group were non observed because of the loss of this component during the atomization procedure. Therefore, it was concluded that in all molecular species the cut downing glucosamine ( GlcN I ) was substituted with two fatty acids: one 3-OH C14 and one C12 or 2-OH C12 or C16.

The exact place of each fatty acid was determined by its release form upon alkalic interventions. The transmutation of native lipid A negative-ion MALDI mass spectra under different conditions of alkaline intervention is summarized in Table 1. Under the conditions of the method ( Tirsoaga et al. , 2007 ) all substituents at C-3 and C-3 ‘ places are liberated by ammonium hydrated oxide intervention. The B. whooping cough lipid A deacylation form was used as a mention ( non shown ) . In this instance, there was entire release of the fatty acid at place C-3 ( C10-OH ) in the first 15 min of intervention. The first 15 min of intervention applied to B. bronchiseptica lipids A did non alter the general facet of the spectrum, which could be explained by an unsubstituted C-3 place. After 2 H of intervention, C14-OH and C14-O-C16 were about wholly released, which clearly indicated that these fatty acids were at the C-3’position. Penta- ( m/z: 1587, 1748, 1909 ) and hexa-acylated ( m/z: 1825, 1986, 2148 ) molecular species, severally, released 3-OH C14 and C14-O-C16 fatty acids to bring forth tetra-acylated species at m/z 1361, 1522, and 1683 substituted with two 3-OH C14, one C14 and one 2-OH C12. Similarly, the 2nd series of penta- ( m/z: 1627, 1788, 1949 ) and hexa-acylated ( m/z: 1865, 2026, 12188 ) species released 3-OH C14 and C14-O-C16, severally, giving rise to tetra-acylated species with m/z 1401, 1562, and 1723 substituted with two 3-OH C14, one C14 and one C16. After 5 H of ammonium hydrated oxide intervention, these extremums were still present. After intervention with methylamine, aimed at emancipating secondary ester-linked fatty acids, all that remained were three extremums at m/z 952, 1113, and 1274 matching to diacyl molecules with and without extra GlcNs and substituted at the C-2 and C-2 ‘ places via amide bonds with 3-OH C14 fatty acids. Release of C14, 2-OH C12 and C16 fatty acids under these conditions showed that they substituted acyloxyacyl at C-2 or C-2 ‘ places. Harmonizing to the atomization informations, C-2 ‘ secondary place was ever substituted by C14 fatty acid. As for the C-2 secondary place it was substituted by C12 or 2-OH C12 or by C16.

Taken together, these informations set up the lipid A constructions presented in Figure 2. All the isolates studied shared a common structural footing consisting of a bis-phosphorylated di-glucosamine anchor substituted at the C-2 and C-2 ‘ places with 3-OH C14 and 3-C14-O-C14 fatty acids, severally. In the penta- and hexa-acyl molecular species, the C-3 ‘ place is substituted with a 3-OH C14 fatso acid. The C-3 place is ever free because of the activity of the PagL enzyme ( Geurtsen et al. , 2005 ) . This characteristic is common to the Pseudomonas aeruginosa ( Ernst et al. , 2006 ) and B. parapertussis lipid A constructions ( El Hamidi et al. , 2009 ) but non to that of B. whooping cough, in which PagL is non active and this place was ever found to be substituted by 3-OH C10 fatso acid in the strains analyzed ( Geurtsen et al. , 2005 ) . In lipid A structures common to deadly and non-virulent stages of isolates, the secondary C-2 place is substituted with either a C12 or a 2-OH C12 fatso acid. This permutation, which is most likely due to the action of a late acyltransferase, LpxL ortholog, adds one more structural similarity between lipoids A of B. bronchiseptica and P. aeruginosa ( Hancock et al. , 1970 ) . LpxL homologues were late discovered in the genome of B. whooping cough, one of which ( LpxL1 ) mediates the add-on of C12 to the secondary C-2 place. Increased LpxL1 look in B. whooping cough was shown to take to more endotoxic lipoid A constructions and favours infection of human macrophages ( Geurtsen et al. , 2007 ) . Hydroxylation of this secondary fatso acid is known to be due to the hydroxylase LpxO ( Gibbons et al. , 2000 ) .

Lipids A molecules specific to deadly stage of isolates are therefore characterized by the presence of one or two C16 fatty acid in their constructions. We demonstrate that this fatty acid can busy two different places: the secondary C-3 ‘ place acylated by the action of the PagP enzyme and before associated with B. bronchiseptica infection in a mouse theoretical account ( Preston et al. , 2003 ) . The other place is in the secondary C-2 permutation. The latter is demonstrated for the first clip in B. bronchiseptica. It was earlier found in B. parapertussis ( El Hamidi et al. , 2009 ) . The sequence of the biosynthetic stairss remains to be established. However it is interesting to observe that in Enterobacteria such as Escherichia coli and S. typhimurium, C16 is added at this place by the PagP enzyme ( Guo et al. , 1998 ; Bishop et al. , 2000 ) .

Finally, these informations confirmed that lipids A of all the B. bronchiseptica isolates tested ( except ALI H- ) had one or two excess GlcNs, a characteristic alteration due to the activity of the ArnT ortholog glycosyl transferase in Bordetellae ( Marr et al. , 2008 ) . The importance of phosphate groups in LPS biological activity interactions has been good documented in this group ( Caroff et al. , 1986 ; Lebbar et al. , 1986 ; Cavaillon et al. , 1989 ; Haeffner-Cavaillon et al. , 1989 ) . Lipid A alteration is non unusual in Bordetella: significant transcriptional and familial diverseness among different isolates of the same Bordetella species have been demonstrated late ( Cummings et al. , 2004 ; Diavatopoulos et al. , 2005 ; Cummings et al. , 2006 ) . Small is known on how this affects virulency, but neutralisation of phosphate groups is known to beef up bacterial opposition to antibacterial peptides ( Gunn et al. , 1998 ) . In this work, “ excess ” GlcN being present in lipid A constructions from isolates from both deadly and non-virulent stages, no correlativity was established with the virulency standards.

We demonstrate here that one of import alteration of lipid A associated with virulency stage was palmitoylation. We earlier hypothesized ( Preston et al. , 2003 ) that such a structural alteration is harbored by LPS of isolates in their virulent stage within the host and non by the isolation in their non-virulent stage adopted in the environment. In this study, lipid A palmitoylation would happen in clinical homo and coney isolates. The other difference is the grade of palmitoylation with the presence at C-2 of a 2nd palmitate to the one described in the mouse theoretical account. The molecular species presented in Fig. 2B is indistinguishable to the construction antecedently reported for one B. parapertussis lipid A isolate ( El Hamidi et al. , 2009 ) . Park et al. , ( 2009 ) late presented an interesting molecular theoretical account exemplifying the function of extra fatty acids on a lipid A construction for modifying the presentation of the LPS-MD2 composite to TLR4. The ground why some Bordetella species, but non others can modulate their LPS construction is at present unknown, but may be linked, in B. bronchiseptica, to its capacity to prevail several old ages inside its homo or animate being host ( Woolfrey and Moody, 1991 ; Gueirard et al. , 1995 ) . The molecular heterogeneousness of B. bronchiseptica lipids A may explicate how these pathogens adapt to new host scopes and to alterations in host kineticss by pull stringsing the host immunity-pathogen interactions. Again, lipid A construction can be considered as a good tool to exemplify development of the bacterial Bordetella species, the human pathogen B. whooping cough being assumed to be derived from a common ascendant of B. bronchiseptica.

Experimental processs

Bacterial isolates and growing conditions

Three B. bronchiseptica isolates were used in this survey: 9.73 H+ from coney beginning ( Gueirard and Guiso, 1993 ) and DANG H+ and ALI H+ from worlds ( Le Blay et al. , 1997 ) . From these three hemolytic isolates, self-generated non-hemolytic ( 9.73 H- , DANG H- and ALI H- ) discrepancies were obtained. These discrepancies were shown by western blotting and specific antibodies non to show adenylate cyclase-hemolysin ( AC-Hly ) , filiform hemagglutinin ( FHA ) , and pertactin ( PRN ) harmonizing to Khelef et Al. ( 1993 ) . These discrepancies are considered to be in non-virulent stages since they do non show the virulency factors and are non deadly in the murine theoretical account ( Gueirard et al. , 1995 ) while the parental isolates are considered to be deadly. Bacterias were grown as described in Gueirard and Guiso ( 1993 ) .

LPS and lipid A readying

L-p from B. bronchiseptica isolates were extracted by the enzyme-phenol-water method ( Johnson and Perry, 1976 ) and deposit by ultracentrifugation ( 105,000 g, 4 A°C, 12 H ) .

Lipid A was isolated from LPS utilizing mild, detergent-facilitated hydrolysis as described antecedently ( Caroff et al. , 1988 ) . Alternatively, when samples of less than 1mg were used, lipid A was obtained after hydrolysis of LPS in 1 % acetic acid at 100 A°C for 1 H at a concentration of 10 mg/ml. With both hydrolysis, applied to 1 milligram LPS samples, lipid A was recovered from the lyophilized residue by three extractions with 100 milliliters of a choloroform-methanol-water mixture ( 3:1.5:0.25, V: V: V ) and analyzed by MALDI-MS.

Consecutive release of ester-linked fatty acids by mild alkali intervention

Consecutive release of ester-linked fatty acids by mild alkali intervention was used to set up the lipid A acylation forms ( Tirsoaga et al. , 2007 ) . Briefly, for the first-step release of primary ester-linked fatty acids, lipid A ( 200 Aµg ) was suspended at 1 mg/ml in 28 % ammonium hydrated oxide solution in 1.5 milliliters Eppendorf tubing and stirred in a thermomixer ( Eppendorf, Germany ) for 5 H at 50 A°C and 1000 revolutions per minute. Dynamicss of fatty acerb release was followed after 15 min, 30 min, 1 H, 2 H and 5 h. To emancipate the secondary ester-linked fatty acids, 50 Aµg of lipid A was suspended in 50 Aµl of 41 % methylamine solution and stirred for 5 H at 37 A°C. At this phase all sample suspensions were dried under a watercourse of N, the residues taken up in a mixture of chloroform-methanol-water ( 3:1.5:0.25, V: V: V ) and analysed by MALDI-MS. B. whooping cough lipid A fatty acerb release dynamicss ( 15 min, 30 min, 1 H, 2 H, 5 H ) was used as a mention.

MALDI-MS

Matrix-assisted laser-desorption/ionization mass spectra were obtained in the additive manner with delayed extraction utilizing a Perseptive Voyager STR ( PE Biosystem, France ) time-of-flight mass spectrometer ( I.B.B.M.C. , University of Paris Sud, France ) . A suspension of native or treated lipid A in chloroform-methanol-water ( 3:1.5:0.25 ; V: V: V ) ( 1 mg/ml ) was desalted with a few grains of Dowex 50W-X8 ( H+ ) , one Aµl was deposited on the mark, assorted with one Aµl of the matrix solution and dried. Matrix: 2, 5-dihydroxybenzoic acid ( DHB ) was dissolved at 10 Aµg/Aµl in the same dissolver or in 0.1 M citric acid in chloroform-methanol-water ( 3:1.5:0.25 ; V: V: V ) ( Therisod et al. , 2001 ) . Analyte ions were desorbed with pulsations of a N optical maser ( 337 nanometer ) . Spectra were recorded in the negative- and positive-ion manners with ion acceleration set at 20 kilovolt.

Fatty acid analysis

To find the overall fatty acids content in lipoids A, all fatty acids were released after hydrolysis of the LPS or lipids A with 4 M HCl for 2 H at 100 A°C, extracted with ethyl ethanoate and methylated with a mixture of anhydrous methyl alcohols and acetyl chloride ( 10:1.5 ; V: V ) ( Wollenweber and Rietschel, 1990 ) . Fatty acids were identified by gas chromatography. An HP 5890 gas chromatograph was equipped with an HP5 capillary column ( 30 m x 0.32 millimeter ) and a temperature gradient from 150 to 300 A°C, 2 A°C/min was used. Fatty acids were characterized by comparing of their keeping times with mention samples ( methyl esters of 2-OH and 3-OH C10 to C16 fatty acids every bit good as of non-hydroxylated C10, C12, C14 and C16 fatty acids ) . C20 was used as an internal criterion.