2.2. Genetic Manipulations

PCRs were performed using High Fidelity Polymerase (Roche Diagnostics GmbH, Mannheim, Germany). PCR mixes consisted of 1 μL of genomic DNA as a template, 200 µM dNTPs (ThermoFisher Scientific, Vilnius, Lithuania), 0.25 µM of different primer combinations, 0.5 U DNA polymerase, and PCR buffer. All primers used are listed in Table S1. The mixtures were incubated for 10 min at 95 °C for DNA denaturation, followed by 30 cycles of 1 min at 95 °C, 0.5 min at 58 °C and elongation at 72 °C for 1 min per kb of expected amplicon size. The reactions were terminated with an extended elongation step for 10 min at 72 °C. The resulting products were separated on 1% agarose gels by electrophoresis and visualized using ethidium bromide.

Genes encoding LPS-biosynthesis enzymes of different bacteria were amplified by PCR and cloned into broad host-range expression vector pMMB67EH [30] by exchanging them for the pagL gene in pMMB67EH-PagL_Pa [8] (Table 1). To this end, the PCR products and plasmid pMMB67EH-PagL_Pa were purified using the Clean-Up System and Plasmid Extraction kit (both from Promega, Madison, USA), respectively. Purified plasmid and PCR products were digested for 2 h at 37 °C with NdeI and HindIII (ThermoFisher Scientific) for which recognition sites are included in the PCR primers (Table S1), and subsequently ligated together overnight at room temperature using T4 DNA ligase (ThermoFisher Scientific). The resulting products were used to transform E. coli strain DH5α, and clones containing the target gene inserted in the pMMB67EH backbone were elected by restriction enzyme digestions and DNA sequencing at Macrogen (Amsterdam, The Netherlands).

The lpxL₂ gene is located in an operon of three genes in between the lpxL₁ and dapF genes. To inactivate lpxL₂ by allele exchange, we made use of the suicide vector pKAS32 [31] (Table 1). Initially, plasmid pKA32-EFGH-lpxL₂::gm was used (Table 1). This plasmid contains a 750-bp sequence of the 3′ end of the lpxL₁ gene and a 753-bp sequence immediately downstream of lpxL₂ separated by a gentamicin-resistance cassette. Attempts to knock out the lpxL₂ gene with this construct failed. We considered the possibility that the rigorous deletion of the lpxL₂ gene might affect the expression of the downstream-located dapF gene. Therefore, constructs with a more subtle deletion, affecting only the first two codons of lpxL₂, were generated. The downstream sequence in pKA32-EFGH-lpxL₂::gm was replaced by a PCR product obtained with primers listed in Table S1 and encompassing the lpxL₂ gene but without the first six nucleotides and, in addition, 866 bp downstream of lpxL₂. Then, a gentamicin-resistance cassette, also obtained by PCR, was re-introduced in the resulting plasmid, and plasmids pKOlpxL₂::gm (a) and pKOlpxL₂::gm (b), which contain the resistance cassette in two different orientations, were selected (Table 1).

Plasmid pRT669 was used to knock out the B. pertussis lpxA gene. P. aeruginosa lpxA and lpxD genes were used to replace the B. pertussis counterparts by homologous recombination as follows. Synthetic DNA fragments constituting the fabZ-lpxA-lpxB region (1599 nucleotides) and the skp-lpxD-fabZ region (1881 nucleotides) were designed, such that the lpxA and lpxD open reading frames were replaced by the P. aeruginosa sequence while the flanking regions were derived for B. pertussis. The synthetic DNAs were obtained from Genscript and cloned into the HindIII site of the suicide shuttle vector pSS1129 [32]. The resulting plasmids were called pClpxA_Pa and pClpxD_Pa.

E. coli DH5α was transformed with ligation products or plasmids following standard protocols. Correct clones were elected by PCR, and plasmids were purified and sequenced at the Macrogen sequencing service (Amsterdam). Then, plasmids were transferred to E. coli strain SM10λpir [33] by transformation and subsequently to B. pertussis strain B213 by conjugation using ampicillin and nalidixic acid for selection and counterselection, respectively. To generate chromosomal mutations, various knockout plasmids (Table 1), which contain an rpsL gene conferring streptomycin sensitivity [31], were integrated into the chromosome via a single crossover by selecting for kanamycin- or gentamicin-resistant transconjugants; the resulting bacteria had lost streptomycin resistance. Subsequently, to select for plasmid loss by a second crossover, bacteria were grown in liquid medium and mutants were selected on plates with streptomycin and kanamycin or gentamicin. The presence of the plasmids in B. pertussis transconjugants and the proper generation of knockout mutants were verified by PCR.

2.3. RNA Extraction and Reverse Transcriptase (RT)-PCR

To obtain RNA, cells from exponentially growing cultures were collected by centrifugation for 10 min at 5000 rpm in an Eppendorf Centrifuge 5424 (Eppendorf, Nijmegen, The Netherlands), adjusted to an OD₅₅₀ of 4, and resuspended in trizol (Invitrogen, Carlsbad, USA). Then, 200 μL of chloroform were added per ml of trizol, followed by centrifugation at 5000 rpm for 30 min. The resulting upper layer was mixed with an equal volume of ice-cold 75% ethanol. Next, RNA was isolated using the Nucleospin RNA II kit (Macherey-Nagel GmbH, Düren, Germany) according to the manufacturer’s instructions. The resulting solution was treated with Turbo DNA free (Ambion, Berlin, Germany) for 1 h at 37 °C to remove genomic DNA followed by DNase inactivation according to recommendations of the manufacturer to generate pure RNA. This was used immediately to generate cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Amsterdam, The Netherlands). RNA, cDNA and genomic DNA were used as templates in PCRs with primers listed in Table S1 to determine the generation of specific transcripts following previously described protocols [34].

2.4. Electrophoresis and Western Blotting

Whole-cell lysates from bacteria adjusted to an OD₆₀₀ of 5.0 were mixed 1:1 with double-strength sample buffer, and heated for 10 min at 100 °C. For the detection of LPS, whole-cell lysates were boiled in sample buffer as above and then treated with proteinase K for 1 h at 37 °C. Proteins and LPS were separated by SDS-PAGE on gels containing 13% and 16% acrylamide, respectively, after which they were stained with Coomassie brilliant blue G250 or silver, respectively. For Western blotting, proteins separated on gels were transferred to nitrocellulose membranes as described [35] and anti-6xHis tag antibodies (Invitrogen) were used for protein detection.

2.5. LPS Purification and Analysis

For LPS isolation, bacteria were grown to the exponential phase, i.e., for 12 h for wild type and chromosomal mutant derivatives and for 24 h for strains B213 ΔlpxA-pLpxA_Pa, B213-pLpxD_Pa, and B213-pLpxL_Nm. LPS were extracted with hot phenol-water [36] and purified further by solid-phase extraction on C8 reversed-phase cartridges as described [26]. Negative-ion ESI Fourier transform (FT) mass spectrometry (MS) of purified LPS was performed on an LTQ Orbitrap XL instrument (ThermoFisher Scientific) following the described methods [26]. LPS samples were dissolved in a mixture of 2-propanol, water, and triethylamine (50:50:0.001, v/v/v) (pH 8.5) and infused into the mass spectrometer by nano-electrospray ionization (ESI)-mass spectrometry (MS) using gold-coated pulled glass capillaries. The spray voltage was set to from −1.3 to −1.85 kV and the temperature of the heated capillary to 250 °C. Then, nano-ESI-FT-MS was performed with in-source collision-induced dissociation [26]. This fragmentation technique produced intense fragment ions corresponding to intact lipid A domain, which originated from the rupture of the labile linkage between the nonreducing lipid A glucosamine and 3-deoxy-D-manno-oct-2-ulosonic acid. Lipid A compositions proposed are based on the chemical structure of the LPS from B. pertussis reported previously [37]. Mass-to-charge ratios given refer to mono-isotopic molecular masses.

2.6. Eukaryotic Cell Lines and TLR4 Signalling

Human NF-κB/SEAP reporter HEK293-Blue cells stably transfected with human TLR4 (hTLR4), mouse TLR4 (mTLR4) in combination with the corresponding MD-2 and CD14 or human TLR2 (hTLR2) were purchased from InvivoGen. These cell lines contain an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. The cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg mL⁻¹ streptomycin, 300 ng mL⁻¹ L-glutamine, 100 µg mL⁻¹ normocin, and 1× HEK Blue selection at 37 °C in a 5% saturated CO₂ atmosphere. For TLR4 signaling, HEK-Blue cells (2.5 × 10⁴) were incubated with serial dilutions of purified LPS or heat-inactivated bacterial cell preparations adjusted to an OD₅₉₀ of 0.1 in a 96-well plate as before [26]. As a positive control, ultrapure LPS from E. coli K-12 (InvivoGen, Toulouse, France) was used. After 16–24 h incubation at 37 °C, supernatants were collected and incubated for 2 h with the SEAP substrate Quanti-Blue (InvivoGen), and the OD₆₃₀ was measured using an absorbance microplate reader.

2.7. Animal Experiments

The animal experiments of this study were performed at Triskelion B.V., Zeist, The Netherlands. The welfare of the animals was maintained in accordance with the general principles governing the use of animals in experiments of the European Community and Dutch legislation. This included licensing of the project by the Central Committee on Animal Experimentation (project license 2016602) and approval of the study by the Triskelion Animal Welfare Body (AWB number TRIS-333). Five male New Zealand rabbits per group were housed and manipulated according to standard operating procedures of Triskelion facilities. The body temperature of each animal was recorded three times a day for five days before the first injection, just before the administration, and about 0.5, 1, 2, 4, 6, 24, and 48 h after the injection. The body temperature was measured using an external scanner from subcutaneously implanted transponders (Plexx, IPTT-300 transponder, Elst, The Netherlands). The rabbits were inoculated intramuscularly with a saline solution or 10 µg of pure LPS in saline solution. All rabbits were considered in the final data set.

3.2. Analysis of Recombinant Lipid A Structures

The lipid A structures were analyzed by nano-ESI-MS analysis using purified LPS extracted from exponentially growing bacteria in the presence of IPTG. For the wild-type strain, a major peak was observed at m/z 1557.97, which corresponds with the expected bis-phosphorylated penta-acylated lipid A (Figure 2A). In the strain expressing LpxA_Pa, the spectrum revealed, besides the ion at m/z 1557.97, two additional prominent ions at m/z 1501.91 and 1529.94 (Figure 2B). The ion at m/z 1501.91 corresponds with the expected substitution of the primary 3OH-C14 acyl chain at position 3′ by 3OH-C10, whilst the m/z 1529.94 ion indicates its substitution by a hydroxylated fatty acid with an intermediary C12 chain length. The relative abundance of the two new species was only 75 and 48%, respectively, relative to the wild-type structure at m/z 1557.97, which could be due to the expression of the endogenous lpxA on the chromosome. Therefore, we decided to knock out the chromosomal lpxA copy. This inactivation caused a growth defect (Figure S1D), perhaps due to a polar effect on the expression of the downstream located lpxB gene. MS analysis of the lipid A of the resulting strain showed the complete loss of the m/z 1557.97 ion and a drastic decrease in the abundance of the m/z 1529.94 ion leaving a major peak of m/z 1501.91 corresponding to the expected substitution (Figure 2C).

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Figure 2

Structural analysis of lipid A by ESI-MS. Negative-ion lipid A mass spectra were obtained by in-source collision-induced dissociation nano-ESI-MS of intact LPS isolated from cells of (A) B213, (B) B213 expressing lpxA_Pa (B213-pLpxA_Pa), (C) ΔlpxA mutant of B213 expressing lpxA_Pa (B213 ΔlpxA-pLpxA_Pa), (D) B213 expressing lpxL_Nm (B213-pLpxL_Nm), (E) B213 expressing lpxL_Pg (B213-pLpxL_Pg), (F) B213 expressing lpxD_Pa (B213-pLpxD_Pa), (G) B1917 with the chromosomal lpxA replaced by lpxA_Pa (B1917 lpxA_Pa), and (H) B1917 with the chromosomal lpxD replaced by lpxD_Pa (B1917 lpxD_Pa). A major singly-deprotonated ion at m/z 1557.97 was interpreted as the typical B. pertussis lipid A structure: a diglucosamine (2 GlcN), penta-acylated (three 3OH-C14, one 3OH-C10, and one C14) with two phosphates residues (2 P) as illustrated in Figure 1A. Additional singly-deprotonated lipid A ions were detected in different derivatives and their interpretations are also indicated. Only the m/z range covering lipid A ions is shown.

MS analysis of lipid A from B213-pLpxL_Nm showed a drastic reduction of the wild-type m/z 1557.97 ion, whilst the major peak at m/z 1529.94 corresponds with the expected substitution of the secondary C14 acyl chain by C12 (Figure 2D). Attempts to delete the chromosomal lpxL failed. B. pertussis contains two adjacent lpxL homologs on the chromosome, but only one of them, called lpxL₂, is active under laboratory growth conditions [38]. Different constructs were used to delete the lpxL₂ gene partially or completely; however, despite considerable efforts, we could not obtain the desired knockout. This is not due to a polar effect of lpxL₂ disruption on the expression of the downstream gene, dapF, which encodes an essential enzyme involved in the synthesis of meso-diaminopimelate (DAP), a precursor of the cell wall, and lysine, since also attempts to inactivate the lpxL₂ gene in the presence of meso-DAP were unsuccessful. Thus, whilst the partial substitution of wild-type LPS in B213 expressing lpxL_Nm already causes a considerable growth defect (Figure S1D), the complete substitution of this LPS by the altered form is possibly lethal.

MS analysis of lipid A from B213-pLpxL_Pg also revealed a drastic reduction in the abundance of the m/z 1557.98 ion and a new peak was detected in this case at m/z 1586.01, which corresponds with the expected substitution of the C14 by a C16 chain (Figure 2E). Peculiarly, although the vast majority of lipid A was substituted in this strain without causing any appreciable growth defect (Figure S1D), inactivation of the chromosomal lpxL₂ gene appeared also not possible in this strain.

Analysis of the lipid A structure from B213-pLpxD_Pa revealed, besides the ion at m/z 1557.97 corresponding with the wild-type structure, two abundant ions at m/z 1529.94 and m/z 1501.91 (Figure 2F) corresponding with the reduction of the length of one or two acyl chains, respectively, from 3OH-C14 to 3OH-C12. These results show that indeed the production of LpxD_Pa modified the structure of lipid A as predicted, although not completely. In summary, the heterologous production of LpxA_Pa, LpxL_Nm, LpxL_Pg, and LpxD_Pa in B. pertussis resulted in the expected LPS alterations.

3.3. Differential Activation of TLR4 by the LPS Variants

We next investigated whether the altered structures of the LPS affect TLR4 signaling. To this end, purified LPS preparations were added to HEK293-Blue cells expressing hTLR4. After exposure, the activation of the receptor was evaluated by measuring the expression of a reporter gene encoding SEAP. Interestingly, LPS from B213-pLpxA_Pa stimulated hTLR4 much less than did LPS from the wild-type strain (Figure 3A). The residual activation observed was due to the expression of the chromosomal lpxA gene, as it was eliminated after the inactivation of this gene (Figure 3A). Thus, the length of the primary acyl chain at position 3′ is relevant for the activation of hTLR4 by B. pertussis LPS. Furthermore, we tested whether modification of the acyl chains at positions 2 and 2′ would have a similar outcome. HEK-Blue cells expressing hTLR4 showed no induction of SEAP activity after incubation with purified LPS from B213-pLpxD_Pa (Figure 3A). Thus, also reduction of the length of the acyl chains at positions 2 and 2′ of B. pertussis lipid A has a drastic impact on the activation of hTLR4. Finally, LPS from B213-pLpxL_Nm and B213-pLpxL_Pg reduced and increased hTLR4 activation, respectively (Figure 3A). Hence, stimulation of hTLR4 also correlates with the length of the secondary acyl chain in the order C16 > C14 > C12.

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Figure 3

Stimulation of HEK293-Blue cells expressing hTLR4 (A,C) or mTLR4 (B,D) with purified LPS (A,B) or whole-cell preparations of B213 and derivatives (C,D). LPS preparations and bacterial suspensions were serially diluted. After incubation for 2 h with HEK293-Blue cells expressing mTLR4 or for 4 h with HEK293-Blue cells expressing hTLR4, alkaline phosphatase activity was determined. Graphs show the mean and standard deviation from a representative experiment of three repeats in duplicate.

For comparison, we also measured the activity of some of the LPS preparations in stimulating the mTLR4. Stimulation of HEK293-Blue cells expressing mTLR4 with LPS preparations from wild-type strain B213 resulted in a stronger response than observed in the cells expressing hTLR4 (compare panels A and B in Figure 3). LPS preparations from B213 cells expressing the heterologous enzymes, including those expressing LpxL_Pg resulting in a secondary C16 chain, appeared slightly less effective in stimulating these cells (Figure 3B). Thus, activation of the human and mouse TLR4 is differently affected by the LPS modifications. However, when the chromosomal lpxA gene was inactivated in B213 expressing LpxA_Pa, the resulting LPS was severely affected in its capacity to activate mTLR4 (Figure 3B). Apparently, the length of the primary acyl chain at position 3′ is critical in stimulating both hTLR4 and mTLR4.

Previously, we reported that the decreased toxicity of B. pertussis LPS that had lost the primary acyl chain at the 3 position due to the expression of a pagL gene encoding a lipid A de-acylase was nullified in whole-cell preparations by its increased release from the membranes [8]. As our goal is to generate a new cellular vaccine, we wished to determine also the biological activity of whole-cell preparations from cells producing the LPS variants with altered acyl-chain length. Synthesis of heterologous LPS biosynthetic enzymes in strain B213 affected the stimulation of HEK293-Blue cells expressing hTLR4 similarly in whole-cell and purified LPS preparations (compare panels A and C in Figure 3). Stimulation of HEK293-Blue cells expressing mTLR4 by whole-cell preparations was barely affected by the expression of the heterologous enzymes in B213 (Figure 3D). However, whole-cell preparations of the ΔlpxA mutant of B213 producing LpxA_Pa failed also to activate these cells (Figure 3D).

3.4. Construction of Stable LPS Mutants in a Vaccine Strain

To generate stable strains with lipid A modifications, the lpxA and lpxD genes of B. pertussis were replaced on the chromosome by the corresponding genes from P. aeruginosa. The replacement of both genes was performed in strain B1917, a worldwide predominant strain associated with the resurgence of pertussis since the 1990s [28]. MS spectra of lipid A of B1917 indicated a similar structure as that of B213. The correct replacement of both genes was confirmed in PCR assays. The mutant strains showed growth defects similar to those of strain B213 lpxA-pLpxA_Pa and B213-LpxD_Pa (data not shown). In both cases, the gene substitution resulted in the synthesis of lipid A species with the expected shortened acyl chains as evidenced by MS (Figure 2G,H). Both mutants were grown for up to 10 passages in a liquid medium, and subsequent MS analysis revealed no alteration of the lipid A structure (data not shown), thus evidencing the stable production of modified lipid A. Interestingly, the growth rate of the B1917 lpxA_Pa mutant was restored to wild-type levels after the consecutive passages, and the resulting strain is, thus, more suitable for vaccine production. In contrast, the growth defect of mutant B1917 lpxD_Pa persisted after successive passages. The bioactivity of LPS preparations from the mutants was determined in HEK293-Blue cells expressing mTLR4 or hTLR4. In accordance with the results obtained with the B213 derivatives (Figure 3A,B), LPS preparations from B1917 lpxA_Pa did barely activate hTLR4 or mTLR4, whilst LPS from B1917 lpxD_Pa showed drastically lower activity than wild-type LPS (Figure S2). As a control, we also stimulated the reporter cell lines with hexa-acylated E. coli LPS and found that, as expected, it is a more potent hTLR4 agonist than the penta-acylated B. pertussis LPS, whilst mTLR4 was stimulated similarly by both LPS preparations (Figure S3).

We wish to thank Arjen Sloots (Intravacc, Bilthoven) and Hendrik Jan Hamstra (RIVM, Bilthoven) for logistics and plasmids, Anastasios Tsiavos (Utrecht University) for technical support, Naomi van Vlies (Intravacc, Bilthoven) for assistance with the pyrogenicity test, and Wim Crielaard (ACTA, Amsterdam) for kindly providing P. gingivalis.