- freely available
Int. J. Mol. Sci. 2017, 18(5), 1080; doi:10.3390/ijms18051080
2.1. Fatty Acids Composition
2.2. Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry Structural Analyses
2.2.1. Interpretation of the Main Molecular Species in the Different Lipid a Spectra
2.2.2. Distribution of the Fatty Acids on the d-Glucosamine Residues of the Clinical Isolates
2.2.3. Distribution of the Fatty Acids on the d-Glucosamine Residues of the Isolates and the Reference Strain
2.2.4. Linkage of Fatty Acids in B. holmesii Lipids A
2.3. Genomic Analyses of Genes Involved in Lipid A Biosynthesis
- An orthologous sequence corresponding to the lgm locus was identified in the genome of the three clinical isolates as in the ATCC 51541 reference strain (position 1966909..1970398). We observe no sequence difference for this locus comprising ArnT (position 1967952..1969541) between ATCC type strain and the three clinical isolates.
- Bho1 and FR4020 lpxA sequence is identical to that of ATCC 51541 reference strain (position 3231726..3232520) but FR4101 lpxA sequence displays a non-synonymous SNP in position 508 leading to an amino acid modification (S instead of G in position 170 of LpxA protein). We also sequenced lpxA gene from 16 additional B. holmesii isolates collected from blood or from respiratory samples and found that this SNP was not linked to the origin of the isolates (unpublished data).
- pagL sequence is identical for the three clinical isolates and the ATCC 51541 reference strain (position 2144078..2144623). We did not observe any sequence difference between the three tested isolates for pagP but a difference was observed in ATCC 51541 reference strain pagL sequence (position 630144..630686) corresponding to an additional G leading to a frameshift.
- We found two homologs of lpxO in the genome of the three isolates of B. holmesii as in the ATCC 51541 reference strain (position 2899145..2900041 for the first one and 2402394..2403293 for the second). They respectively displayed 84% nucleotidic identity with the KF214918 lpxO1 of B. avium ATCC 35086 and 84% nucleotidic identity with the KF214919 LpxO2 of B. avium ATCC 35086. We then identified a lpxL1 homolog in the three isolates as in ATCC 51541 reference strain (position 1194726..1195580) with no sequence difference between them.
- The differences in length of the carbon FA at C3’, 14:0(3-OH) for Bho1, FR4020 and ATCC 51541 or 10:0(3-OH) for FR4101. LpxA is the first enzyme of the lipid A biosynthesis pathway catalyzing the addition of an acyl chain onto the C3’ carbon . We identified lpxA B. holmesii gene and observed a non-synonymous SNP in FR4101, leading to an amino acid change, as compared to ATCC 51541, FR4020 and Bho1 lpxA sequences. Thus, we concluded that the difference observed in the length of the carbon FA at C3’ between FR4101 and the other isolates could be due to this difference within lpxA gene. Analysis of additional French B. holmesii isolates also led to the conclusion that this was not related to the blood or respiratory origin of the sample which was also confirmed by the lpxA sequence analysis of isolates with available genomes on NCBI.
- The presence of 10:0(3-OH) at C-3, like in B. pertussis  and B. petrii  is also a common trait between the three clinical isolates. In B. pertussis, this is the consequence of the lack of activity of the C-3 de-O-acylase PagL, which has been shown to result in a lower cytokine induction capacity of the Bordetella human pathogens [29,42]. The presence of this fatty acid was interpreted as resulting in the facility of such bacteria, with short-chain fatty acid structures, and low acylation pattern, to escape the human host defense through the MD2–TLR4 complex [42,43]. We found that the pagL gene sequence is the same for the three isolates and the ATCC 51541 strain. Both H and S residues in the C-terminal part of PagL protein, described as essential for catalytic activity are present in B. holmesii . In B. pertussis, pagL is a pseudogene because of a frameshift due to a deletion of “CA” bases. Such a deletion is not found in the B. holmesii sequence. Further investigations are necessary to understand why PagL is inactive in these isolates.
- The presence of a secondary palmitoyl chain at two positions of lipid A (3′ and 2). We previously described the presence of such palmitoyl chains at C-3′ in B. avium, and in both C3′ and C2 in B. parapertussis [27,45] as a result of PagP action. In the present study, we observed no pagP sequence difference between the three tested isolates. They nevertheless display an additional G insertion as compared to ATCC 51541 reference strain, leading to a frameshift that probably explains why no palmitoyl acid is observed in these isolates.
- The hydroxylation of 12:0 and 14:0 at position 2 of the two secondary acylated residues was observed in the lipid A of the three isolates (Figure 5). In Novikov et al. , such hydroxylations have been observed in B. avium, B. hinzii and B. trematum and were attributed to two homologs of the LpxO enzyme. We found two homologs of lpxO in the genome of the three isolates of B. holmesii as in the ATCC 51541 type strain that support the degree of hydroxylation observed.
- The presence of a 12:0(2-OH) residue in a secondary position at C-2 on the amide-linked 14:0(3-OH) is a common trait between all the B. holmesii structures. LpxL is a lipid A lauroyl acyltransferase. Geurtsen et al.  showed that LpxL1, a homolog of the classical LpxL2 lauroyl acytransferase, is present but usually poorly expressed in B. pertussis, compared to LpxL2 [40,46]. However, through overexpressing it in B. pertussis B213 strain, they showed that this enzyme led to the presence of an extra secondary 12:0(2-OH) chain at the C-2 position, like we describe here in the B. holmesii isolates, and they reported that this type of acylation was required for efficient infection in human macrophages and could help host infection. We found a lpxL1 homolog in the three isolates as in ATCC 51541 type strain supporting this substitution.
4. Materials and Methods
4.1. Strains and Isolates
4.2. LPS Preparation
4.3. Lipid A Isolation from Whole Cells
4.4. Lipid A Isolation from LPS as Performed by the Triethylamine Citrate (TEA) Method
4.5. Thin-Layer Chromatography
4.6. Fatty Acid
4.8. Mass Spectrometry
4.9. Genomic Analysis
- Presence of free amino GlcN on the phosphate groups of lipid A structure: in Bho1 as in ATCC 51541, the failure of BvgA to function leads to a weaker substitution than in the two other isolates.
- Presence of 10:0(3-OH) at C-3, which we already observed in other Bordetella species such as B. pertussis , is thought to be the consequence of the lack of activity of the 3 O-deacylase PagL, even if genomic explanation could not be found to support this hypothesis.
- The 14:0(2-OH) and 12:0(2-OH) fatty acids found in all the presented structures were previously shown to be important structural features for the infection of human macrophages .
Conflicts of Interest
|MALDI||Matrix-ssisted laser desorption|
References and Notes
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|Treatment and Time Point||[M-H]− Ions m/z||Fatty Acid Released|
|NH4OH, 15 min||1376||1603||1603||10:0(3-OH)||C3|
|NH4OH, 2 h||1376||1376||1376||14:0(3-OH)||C3′|
|NH4OH, 5 h||1178||1178||1178||12:0(2-OH)||secondary C2 partial release|
|1150||1150||1150||14:0(2-OH)||secondary C2′ partial release|
|CH3NH2, 5 h||952||952||952||14:0(2-OH)||secondary C2′|
|NH4OH, 15 min||1376.6||1547||1547||10:0(3-OH)||C3|
|NH4OH, 2 h||1376.6||1376.6||1376.6||10:0(3-OH)||C3′|
|NH4OH, 5 h||1178||1178||1178||12:0(2-OH)||secondary C2 partial release|
|1150||1150||1150||14:0(2-OH)||secondary C2′ partial release|
|CH3NH2, 5 h||952||952||952||14:0(2-OH)||secondary C2′|
|Genes of Interest||Position within ATCC 51541 Reference Strain (Accession Number: CP007494.1)||Sequences Differences Observed Between Isolates|
|lpxA||3231726..3232520||No sequence differences between Bho1, FR4020 and ATCC 51541|
|Non-synonymous SNP in position 508 for FR4101 (*)|
|pagP||630144..630686||No sequence differences between Bho1, FR4020 and FR4101|
|Additional G compared to ATCC51541 leading to a frameshift|
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