The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin

Abstract

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

Figure 1. Neisserial LptA::His_x6 transfers PEA to lipid A of E. coli LPS.

Lipid A profiles of LPS extracted from E. coli strains JCB571 expressing EcDsbA (CKEC272) (Panel A), E. coli JCB571 expressing LptA::His_x6 (CKEC543) (Panel B) and JCB571 expressing LptA::His_x6 and EcDsbA (CKEC564) (Panel C) as determined by MALDI-TOF MS. bis-Phosphorylated hexaacylated lipid A (m/z = 1796), the mono-phosphorylated derivative (m/z = 1716), and the heptaacylated version due to the addition of a palmitic acyl residue (m/z = 2034) were detected in all strains. bis-Phosphorylated tetraacylated lipid A (m/z = 1360) was found abundantly in the MALDI spectra of all three strains, which was likely produced from bis-phosphorylated hexaacylated lipid A (m/z = 1796) during the ionization step on MALDI. The lipid A preparations from CKEC543 expressing LptA (Panel B) and CKEC564 co-expressing LptA and EcDsbA (Panel C) also contained ions consistent with one PEA added to the bis-phosphorylated structure (such as m/z 1919; i.e. 1796+123) and the heptaacylated structure (such as m/z = 2157, i.e. 2034+123).

Figure 2. LptA::His_x6 stability is dependent upon oxidoreductase activity in E. coli.

Standardised whole cell lysates were separated by SDS-PAGE. A Western immunoblot was developed using anti-His tag antibody to detect the presence of LptA::His_x6 in the cellular extracts. Lanes were: Lane 1, ColorPlus pre-stained protein molecular weight marker (New England Biolabs); Lane 2: E. coli JCB571 expressing EcDsbA (CKEC272); Lane 3: E. coli JCB571 carrying pTrc99A (CKEC288); Lane 4: E. coli JCB571 expressing LptA::His_x6 (CKEC543); Lane 5: E. coli JCB571 expressing LptA::His_x6 and EcDsbA (CKEC564); Lane 6: CKEC564 treated with DTT and alkylated with AMS; and Lane 7: CKEC564 alkylated with AMS. Molecular weights (kDa) are indicated on the left.

Figure 3. Oxidation status of LptA::His_x6 in oxidoreductase mutants of N. meningitidis.

Standardised cell lysates were separated by SDS-PAGE, followed by transfer to a membrane and western immunoblot using anti-His_x6 HRP conjugate antibody to detect the presence of LptA::His_x6. Panel A. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMBΔdsbA1/dsbA2 expressing LptA::His_x6 from pCMK1001 (CKNM221) untreated; Lane 3: CKNM221 treated with DTT and alkylated with AMS; Lane 4: CKNM221 alkylated with AMS, Lane 5: NMBΔdsbA3 expressing LptA::His_x6 (CKNM222) untreated; Lane 6: CKNM222 treated with DTT and alkylated with AMS; Lane 7: CKNM222 alkylated with AMS. Panel B. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMB expressing LptA::His_x6 (CKNM216) untreated; Lane 3: CKNM216 treated with DTT and alkylated with AMS; Lane 4: CKNM216 alkylated with AMS; Lane 5: NMBΔdsbA1/NmdsbA2/dsbA3 expressing LptA::His_x6 (CKNM755); Lane 6: CKNM755 treated with DTT and alkylated; Lane 7: CKNM755 alkylated with AMS.

Figure 4. Lipid A substitution profiles of meningococcal oxidoreductase mutants.

Lipid A profiles of LOS extracted from N. meningitidis strain NMB (Panel A), NMBΔlptA::aadA (Panel B), NMBΔNmdsbA1/NmdsbA2 (Panel C), NMBΔNmdsbA3 (Panel D) and NMBΔdsbA1/dsbA2/dsbA3 (Panel E) as determined by MALDI-TOF MS. bis-Phosphorylated hexaacylated lipid A (m/z = 1712), the mono-phosphorylated (m/z = 1632) and the tri-phosphorylated derivative (m/z = 1792) were detected in all strains. Strain NMB and the oxidoreductase mutants all expressed the mono-phosphorylated, bis-phosphorylated and tri-phosphorylated hexaacylated lipid A with a single PEA addition (m/z = 1755, m/z = 1835 and m/z = 1915). Consistent with the loss of LptA activity, NMBΔlptA::aadA lacked these ions.