Introduction

Brucellaceae are gram-negative, facultative, intracellular bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. They cause an acute or chronic disease with a wide range of symptoms, and difficult to diagnose and treat [1]. The species with the highest zoonotic potential are B. abortus, responsible for bovine brucellosis, B. melitensis, the main etiologic agent of ovine and caprine brucellosis, and B. suis, responsible for swine brucellosis [2, 3].

The lipopolysaccharide (LPS) is considered to be a major virulence factor of Brucella and it induces a strong antibody response during infection or when whole cell vaccines are used [4, 5]. LPSs are exposed on the cell surface and present three different regions with different chemical and biological properties: the lipid A, the core oligosaccharide, and the polysaccharide, which in most cases represents the O-specific polysaccharide (O-PS, O-antigen) [6, 7]. The LPS of Brucella shows very low endotoxicity, which illustrates poor detection by the innate immune system and is considered to be one of the virulence factors that allows the pathogen to escape detection by the host immune system [4, 8].

The structure of the polysaccharide chain of the LPS of B. abortus, B. melitensis, and B. suis has been studied [9,10,11]. In all cases it was described as a homopolymer of 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine, Rha4NFo), comprising two antigenic determinants named A (from abortus) and M (from melitensis). Epitope A was defined as a polymer of 1-2-linked N-formylperosamine (Rha4NFo), whereas epitope M was ascribed to the presence of some amounts of 1-3-linkages between perosamine residues. The highest concentration of M-epitope was found in the O-chain of B. melitensis strain 16 M and the following pentasaccharide repeating unit was suggested for epitope M: [–2-α-D-Rha4NFo-2-α-D-Rha4NFo-3-α-D-Rha4NFo-2-α-D-Rha4NFo-2-α-DRha4NFo–] [12]. More recently, a reinvestigation of both types of structures by 2D NMR showed that the M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo compared with the previously proposed structure. It was also determined that the polysaccharide from B. melitensis 16 M contains a fragment of 1-2- linked polymer, capped with M-type polymer and other strains containing one or two M-type units at the nonreducing end of the 1-2-linked O-chain [13]. Regarding the core oligosaccharide, the analysis of a wadC mutant suggested that the B. abortus LPS core presents a branched structure [14]. In a more recent structural study, the first chemical and spectroscopic analysis of its structure [15] and the identification of a new glycosyltransferase gene [16] confirmed the branched structure. Furthermore, a structural study of the LPS purified from B. melitensis defective mutants also identified a branched core oligosaccharide critical in virulence [17].

It is known that the hydrophobic lipid A region constitutes most of the outer leaflet of the outer membrane and is responsible for many of the endotoxic properties attributed to LPSs [18]. The first studies by thermotropic phase behavior [19] and immunochemical analysis [20] of B. abortus and B. melitensis lipid A suggested the presence of a disaccharide backbone molecule linked in a β1–6 configuration. Ethanolamine, neutral sugars, and ester-linked acyl-oxyacyl fatty acids were not detected, and phosphate was described as absent or present in reduced quantities [21]. Later, a structural analysis of the lipid A from B. abortus LPS, purified by HPLC was performed. Mono- and bisphosphoryl species containing a β-1-6-linked 2,3-diamino-2,3-dideoxy-(diaminoglucose) disaccharide backbone (diGlcN3N), substituted mainly with hydroxylated C16, C14, and C12 fatty acids and C16 or C18 in acyloxyacyl linkages, were characterized [22]. More recently, the lipid A profiles of R and S Brucella abortus strains were compared with Escherichia coli lipid A by mass spectrometry analysis, and the differences detected were ascribed to the presence mainly of hepta-acylated species in Brucella and hexa-acylated residues in E. coli lipid A [23].

It has been claimed that determination of the intrinsic heterogeneity in Brucella LPS is important to explain in a realistic perspective its chemical nature and biological behavior. This is significant, since the LPS is the most relevant antigen during infection and vaccination, and LPS/LPS-related molecules are extensively used in immunological studies as well as in the diagnosis of brucellosis [2]. Therefore, in this work we have reinvestigated the lipid A structure isolated from B. suis and B. abortus LPSs. Interestingly, a detailed study by MALDI-TOF mass spectrometry of the lipid A moieties purified from both species allowed detection of a new, interesting feature: the presence of a mainly hexa-acylated and penta-acylated diGlcN3N backbone substituted with a pyrophosphorylethanolamine residue.

Experimental

Bacterial Strains

Brucella abortus 2308 [24] and Brucella suis 1330 [25] were used as the strains to purify the LPS. Work with Brucella was performed at the Biosafety Level 3 laboratory facility at the Universidad Nacional de San Martín.

Growth of Bacteria and Isolation of the LPS

All Brucella abortus strains were cultured in tryptic soy broth (TSB) for 16–20 h at 37 °C with orbital shaking (250 rpm) or in solid media plates with the addition of nalidixic acid at 5 μg/mL.

LPS was extracted from B. abortus strains using a modification of the phenol-hot water method [26]; 250 mL of stationary cultures were washed twice with PBS pH 7.4 and resuspended at 100 mg of bacteria per mL of PBS. The suspension was autoclaved at 121 °C for 30 min and centrifuged 15 min at 5000 rpm. Supernatant was ultracentrifuged for 6 h at 40,000 rpm, and the pellet was resuspended in 100 mM Tris-HCl pH 7 with 20 μg/mL of DNase and RNase and incubated for 30 min at 37 °C. The sample was treated with 50 μg/mL Proteinase K for 3 h at 37 °C and 50 μg/mL Proteinase K was further added and incubated overnight at room temperature. The last two steps were repeated and the preparation was precipitated with 2 volumes of methanol with 1% sodium acetate-saturated methanol and incubated at least 1 h at –20 °C before centrifugation for 15 min at 5000 × g (4 °C). The pellet was resuspended in MilliQ water and dialyzed on MilliQ water at least overnight before ultracentrifugation for 6 h at 40,000 rpm. Pellet was resuspended in 1.5 mL of MilliQ water and extracted with PCP (phenol:chloroform:petroleum ether) as described. LPS was obtained from the phenol phase and the water-phenol interphase. Both phases were combined, washed with methanol, and resuspended in 0.1 mM MgCl2 in MilliQ water. LPS was ultracentrifuged for 16 h at 15 °C and the pellet was resuspended in 1 mL of MilliQ water. LPS concentration was determined by the 3-deoxy-D-manno-2-octulosonic acid method [27]. Purified LPS was analyzed on 12% SDS-PAGE gels and stained by silver nitrate.

Isolation and Purification of Lipid A

LPS was hydrolyzed with 2% acetic acid for 3 h at 100 °C. Precipitated lipid A was recovered by ultracentrifugation at 4 °C, 10,000 g for 60 min. The solution containing the sugar moiety was separated and lyophilized rendering the oligosaccharide. The precipitated lipid A was washed with water, lyophilized, and further extracted with CHCl3/MeOH 3:1 (v/v). The organic phase was dried to obtain purified lipid A.

UV-MALDI-TOF Mass Spectrometry Analysis

Matrices and calibrating chemicals were purchased from Sigma-Aldrich. Measurements were performed using an Ultraflex II TOF/TOF mass spectrometer equipped with a high performance solid-state laser (λ = 355 nm) and a reflector. The system was operated by the Flexcontrol 3.3 software package (Bruker Daltonics GmbH, Bremen, Germany).

The MALDI-TOF MS spectra were the result of 1000–1500 laser shots. All samples were measured in the linear and the reflectron mode, and as routine in both positive and negative polarity. Laser power was typically 40%–60% of its maximum intensity for mass spectra, and the accelerating voltage 20 kV.

For all experiments using the tandem-time-of-flight LIFT mode, the ion source voltage was set at 8.0 kV with a precursor ion mass window of 3 Da. Precursor ions generated by LID (laser-induced dissociation) were accelerated at 19.0 kV in the LIFT cell. The reflector voltage was set at 29.5 kV.

The samples were loaded onto a ground steel sample plate (MTP 384 ground steel; Bruker Daltonics GmbH) using the sandwich method or by the classic dried-droplet method: a sample/matrix solution mixture (1 mL, 1:1, v/v) was deposited on the target plate and allowed to dry under ambient conditions. In addition, different matrices were assayed. Best spectra were obtained using gentisic acid.

Results and Discussion

First, to elucidate the structures the LPS from Brucella suis wild type in order to compare it with Brucella abortus wild type, acid hydrolysis with acetic acid was performed on the corresponding LPSs to split the lipid A from the oligosaccharide. The corresponding lipids A were purified and analyzed by UV-MALDI-TOF mass spectrometry.

Mass spectrum of the lipid A obtained from B. suis LPS was acquired in the positive ion mode using GA as matrix (Figure 1a). The ions at m/z 2225.10 (C112H216N5Na3O27P3 +), m/z 2209.05 (C114H222N5NaO27P3 +), m/z 2136.99 (C110H214N5NaO26P3 +), and m/z 2037.12 (C104H202N5NaO25P3 +) were attributed to the hexa-acylated diGlcN3N backbone substituted with one phosphoryl (P) and one pyrophosphorylethanolamine (PPEA) group. Furthermore, the ion at m/z 2123.08 (C112H216N5Na2O24P2 +) corresponds to m/z 2225.10 minus a sodium phosphoryl group. In the region m/z 1850–1700, three main signals were detected. The signals at m/z 1820.79 (C94H182N4NaO23P2 +) and m/z 1752.69 (C96H185N4NaO19P+) correspond to penta-acylated species carrying a diphosphoryl and monophosphoryl diGlcN3N backbone, respectively. The ion at m/z 1707.62 (C82H160N4Na2O23P3 +) was attributed to a tetra-acylated diGlcN3N backbone substituted with a pyrophosphoryl and a phosphoryl group, and the major ion at m/z 1398.16 (C74H143N4NaO16P+) corresponds to a monophosphoryl tetra-acylated species (Table 1). When the mass spectrum of the lipid A obtained from B. abortus LPS was acquired in the positive ion mode, a similar pattern of signals was detected (Figure 1b).

Figure 1
figure 1

MALDI-TOF mass spectra acquired in the positive ion mode using gentisic acid as matrix. (a) Mass spectrum of B. suis lipid A. (b) Mass spectrum of B. abortus lipid A

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Table 1 Proposed structures for lipid A obtained from Brucella suis LPS after MALDI-TOF mass spectrometry analysis in the positive ion mode. Sugar backbone: [GlcN3N-GlcN3N]
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To further confirm the structure of the lipid A constituents, LID-MS/MS analysis of some ions were performed. Among them, LID-MS/MS spectrum of m/z 2225.10 (Figure 2a) showed ions at m/z 2165.28 (calc. m/z 2165.4139; C110H210N4Na3O26P3 +.), m/z 2123.03 (calc. m/z 2123.5106; C112H216N5Na2O24P2 +), and m/z 2057.77 (calc. m/z 2058.4786; C110H211N4NaO24P2 +.) that correspond to the loss of an ethanolamine group, a sodium phosphate group, and a sodium phosphorylethanolamine group, respectively. Accordingly, fragment ions ascribed to tetra-, tri-, and di-acylated species substituted with one P and one PPEA group were detected (Escherichia coli 1). In the low mass range, diagnostic ions generated after laser-induced cleavage of the glycosidic linkage between the two GlcN3N units within the lipid backbone [28,29,30] confirmed the structure. Thus, the oxonium ion at m/z 827.99 (calc. m/z 827.5521; C42H81N2NaO10P+) is compatible with the GlcN3N II unit acylated with C16:0 (3-OH) and C20:0 (3-OH) fatty acids carrying one sodium phosphoryl group. The complement fragment ion at m/z 940.05 (calc. m/z 940.5599; C42H85N3NaO14P2 +) indicates the presence of a PPEA group substituting the GlcN3N I unit, acylated with C16:0 (3-OH) and C18:0 (3-OH) fatty acids.

Figure 2
figure 2

(a) LID-MS/MS spectrum of parent ion m/z 2225.10 from Figure 1. (b) LID-MS/MS spectrum of parent ion m/z 2037.12 from Figure 1

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The LID-MS/MS spectrum of the parent ion m/z 2037.12 (Figure 2b, Table 2) showed a signal at m/z 1978.39 in agreement with the loss of an ethanolamine group. The signal at m/z 1918.63 matches the loss of a sodium PPEA group, and the ion at m/z 1494.67 corresponds to the loss of a PEA group concomitant with a C14:0 (3-OC16:0) as ketene. Similarly, the ion at m/z 1240.35 agrees with the loss of a sodium PEA group, a C16:0 (3-OC12:0) and a C16:0, both of them as ketenes.

Table 2 Proposed structures for the ion m/z 2037 and fragment ions obtained from Brucella suis Lipid A after MALDI- LID-MS/MS analysis in the positive ion mode. Sugar backbone: [GlcN3N-GlcN3N]
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Further on, the MALDI-TOF mass spectrum acquired in the negative ion mode of B. suis lipid A using gentisic acid as matrix was performed (Figure 3a, Table 3). The spectrum showed in the high mass region, the ion at m/z 2223.32 analogous to the signal at m/z 2225.10 obtained in the positive ion mode. In addition, the loss of a sodium phosphate group gave rise to the ion at m/z 2121.65. Also, a main cluster of ions ranging from m/z 1950 to 2040 indicates the presence of hexa-acylated species containing the diGlcN3N backbone attached to a phosphate group with Δ 14 Da, attributable to differences in the acyl groups. Thus, among them, the most intense peak at m/z 1994.54 (calc. m/z 1994.5908; C114H218N4O20P-) bears three C16:0 (3-OH), one C18:0 (3-OH), one C20:0 (3-OH), and one C16:0 fatty acids. Accordingly, a penta-acylated species at m/z 1756.03 (calc. m/z 1756.3611; C98H188N4O19P–) was detected. In the low mass range, the ion at m/z 1394.46 (calc. m/z 1394.8242; C64H127N5O21P3 -) is consistent with the tri-acylated diGlcN3N backbone carrying one P group and one PPEA group.

Figure 3
figure 3

MALDI-TOF mass spectra acquired in the negative ion mode using gentisic acid as matrix. (a) Mass spectrum of B. suis lipid A. (b) Mass spectrum of B. abortus lipid A. Inset: magnification of region m/z 1700–2300

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Table 3 Proposed structures for the ion m/z 2223.32 and fragment ions obtained from Brucella suis lipid A after MALDI- LID-MS/MS analysis in the negative ion mode. Sugar backbone: [GlcN3N-GlcN3N]
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In order to assure the new feature, MALDI-LID-MS/MS analysis in the negative ion mode of the parent ion m/z 2223.32 (Figure 4a, Table 3) was performed. Thus, the fragment ions at m/z 2157.52, m/z 2120.58, and m/z 2056.01 correspond to the loss of NH2CH2CH2 ., of a NaP, and a sodium PEA, respectively, from the parent ion. Also, losses of acyl groups were detected. Thus, m/z 1473.02 was ascribed to the loss of C18:0 (3-OH) as ketene, a C12:0 fatty acid, a PEA, and one P from m/z 2223.32. In accordance, the loss of C16:0 (3-OC16OH) and sodium PEA group from m/z 2223.32 yielded m/z 1429.08. On the other hand, the ion at m/z 1392.11 shows the loss of a P from ion m/z 1473.02. Ions at m/z 1147.80 and m/z 1129.64 (ΔΗ2O) correspond to the loss of a C16:0 (3-OH) as ketene/acid, concomitant with the loss of a C16:0 (3-OC12OH) and a C18:0 (3-OH) as ketenes and a NaP from the parent ion.

Figure 4
figure 4

(a) MALDI-LID-MS/MS spectrum of parent ion m/z 2223.32 from Figure 3. (b) MALDI-LID-MS/MS spectrum of parent ion m/z 2120.58 from Figure 3

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In addition, the fragment ions at m/z 727.33 (calc. m/z 727.4264; C32H64N4O12P-) and m/z 647.21 (calc. m/z 647.4595; C32H63N4O9 -) (Δ 80 Da) consist of the diGlcN3N backbone carrying one C20:0 (3-OH) and a phosphate group; and m/z 682.34 (calc. m/z 682.3685; C30H57N3O12P-) consists of the diGlcN3N backbone after the loss of ammonia carrying C18:0 (3-OH) and a phosphate group. These last ions indicate the presence of C20:0 (3-OH) and C18:0 (3-OH), both linked to the diGlcN3N backbone. In the low mass range, diagnostic ions at m/z 78.65 and 96.65 account for the presence of phosphate groups and ions at m/z 282.37 and 327.44 confirm the presence of 3-hydroxyoctadecanoic acid and 3-hydroxyeicosanoic acid, respectively.

As expected, MALDI-LID-MS/MS spectrum of the precursor ion m/z 2120.58 (Figure 4b, Table 4) showed below m/z 1500 a similar fragmentation profile as m/z 2223.32. In accordance, the fragment ions at m/z 2061.52 and m/z 1953.62 correspond to the loss of ethanolamine and PEA, respectively, from the parent ion. In addition, the fragment ion m/z 1568.94 attributed to the loss of C16:0 (3-OC16OH) as ketene also from the parent ion. Further loss of C16:0 (3-OC12) as ketene from the latter gave rise to the fragment ion m/z 1177.48. Moreover, the ion at m/z 622.28 (calc. m/z 621.2562; C24H46N3NaO10P2 -.) corresponds to a GlcN3N residue carrying a PPEA group and anhydroC16:0 (3-OH). In addition, diagnostic fragment ions corresponding to phosphate (80 Da; 96 Da) and a pyrophosphate group (H3O6P2 – ; 160 Da) were detected [31].

Table 4 Proposed structures for the ion m/z 2121.65 and fragment ions obtained from Brucella suis Lipid A after MALDI- LID-MS/MS analysis in the negative ion mode. Sugar backbone: [GlcN3N-GlcN3N]
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It has been reported that B. abortus lipid A presents mono- and diphosphorylated acylated diGlcN3N structures [22, 23]. However, when the mass spectrum of B. abortus lipid A acquired in the negative ion mode was analyzed, the ion at m/z 2223.62 (calc. m/z 2223.4438, C112H214N5Na3O27P3 –) although in low intensity, was also detected (Figure 3b). As described above, this ion confirms the presence of one P group and one PPEA group in the structure. In accordance, the ion at m/z 1916.56 (1917.3465; C100H193N5NaO23P2 -) corresponds to a penta-acylated diGlcN3N backbone also carrying a PPEA group. In addition, a cluster of ions ranging from m/z 1680 to 1595 with main ion at m/z 1627.39 (1628.2410, C90H172N4O18P-) corresponds to penta-acylated species carrying one phosphate group. Another cluster of ions in the range m/z 1350–1280 with main ion at m/z 1322.02 (1321.8289; C64H124N4NaO18P2 -) was ascribed to the tri-acylated diGlcN3N backbone substituted with one pyrophosphate group or two phosphate groups. Di-acylated species with hexadecanoyl 3-hydroxy and octadecanoyl 3-hydroxy fatty acids carrying one phosphate and one pyrophosphate group at m/z 1113.81 (calc. m/z 1113.5523; C46H92N4O20P3 -), and two phosphate groups at m/z 1033.73 (m/z 1033.5860; C46H91N4O17P2 -), were also detected.

Conclusions

Brucella harbors a peculiar nonclassic LPS in comparison to classic LPS from enterobacteria such as E. coli [4], and it has been described that the lipid A, linked to the core oligosaccharide, contains 2,3 diamino-2,3,-dideoxy-D-glucose (diaminoglucose) as backbone substituted with amide and ester linked long chain saturated and hydroxylated fatty acids [21]. The hydrophobic lipid A region constitutes mostly the outer coating of the outer membrane and is responsible for many of the endotoxic properties attributed to LPS [18]. Dramatic differences on the lipid A profiles of R and S Brucella abortus strains compared with E. coli have already been reported [23]. MALDI-TOF mass spectra of B. abortus lipid A showed a cluster of ions at high mass range that was ascribed to hepta-acylated species not detected before. However, under the light of our results obtained by MALDI-TOF MS and confirmed by MALDI-LID-MS/MS analysis, these signals correspond to hexa-acylated species substituted with one P and one PPEA group.

It is known that one mechanism of antimicrobial peptide resistance is to modify the cell surface with positively charged moieties, which results in electrostatic repulsion of the positively charged antimicrobial peptides [32]. Frequently, gram-negative bacteria modify their lipopolysaccharide with positively charged aminoarabinose or PEA and these modifications affect virulence [33, 34]. For example, it has been reported that the modification of lipid A with PEA increases resistance of N. gonorrhoeae to complement killing and survival of both gonococci and meningococci in vivo [35]. PEA modification of Lipid A in Salmonella enterica serovar Typhimurium and E. coli strains has also been well documented [36].

The canine pathogen Capnocytophaga canimorsus and plant pathogens Xanthomonas campestris and X. axonopodis also modify their lipid A with PEA [37,38,39], which alters the innate immune responses they trigger, altering the disease manifestations. Thus, modification of lipid A by PEA as a means to evade the effects of antimicrobial agents by altering the electrostatic charge on the bacterial membrane is prevalent.

Furthermore, it has been reported that modifications of the lipid A can drastically alter its immunostimulatory ability as well as resistance to antibiotics. For example, the addition of positively charged residues, including ethanolamine, amino-arabinose, or glucosamine to lipid A, modulates CAMP resistance [40,41,42]. Colistin resistance has been linked to modifications of the lipid A of the LPS moiety from many bacterial species, including A. Baumanii strains [43, 44]. In Helicobacter pylori modification of the lipid A is performed by a two-step process, removal of the 1-phosphate group by a phosphatase followed by the addition of a PEA residue, resulting in increased resistance to polymyxin [45].

Regarding pyrophosphorylethanolamine substitutions, the lipid A of Campylobacter jejuni is characterized by the presence of PEA linked to the phosphate group attached to the 1 and 4′ positions of the disaccharide backbone [46]. Conversely, the lipid A from A. baumanniss is both bisphosphorylated and pyrophosphorylated and substituted with a GalN unit at the 1-phosphate position and a PEA at the 4′phosphate position. Also, the PEA group was found to be attached to the lipid A backbone via a phosphodiester bond at either the C-4′monophosphate or the C-1 monophosphate position [47].

The finding that B. abortus and B. suis contain one PPEA on their Lipid A moieties may open new possibilities of exploring the poorly elucidated set of mechanisms involved in Brucellae virulence that include the ability to escape prompt detection by innate immunity during the initial stages of the infectious process.