Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

Abstract

The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62⌬LgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62⌬LgtA, producing strain F62⌬LgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62⌬LgtA indicated that this strain contained two PEA modifications on its LOS. F62⌬LgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62⌬LgtAlpt3::Tn5.

Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis

“…This is the first report of the functional characterization of Lpt6, and while the activity of Lpt3 from N. gonorrhoeae has been demonstrated previously (24), this is the first report of the purification of bacterial PEtn transferases from solubilized membranes in a stable and active form as illustrated by specific MAb reactivities and MS analyses of enzymatic reactions.…”

“…The localization of Lpt3 and Lpt6 to the inner membrane was not unexpected, since (i) both proteins were previously predicted to be membrane proteins (24,27), (ii) the majority of enzymes involved in LOS assembly are located in the inner membrane (29), and (iii) the inner membrane is rich in PtdEtn (25), which is required for the PEtn transferase activities of Lpt3 and Lpt6. This dependence is presumably due to PtdEtn acting as the substrate donor, as is the case for the E. coli Kdo PEtn transferase, EptB (32), although this has not been demonstrated conclusively for either Lpt3 or Lpt6.…”

“…Alternately, it may simply be that the submaximal rate of catalysis of these enzymes at 37°C is sufficient to fulfill their roles in LOS biosynthesis in vivo. This is the first study describing in vitro activity of a bacterial PEtn transferase purified in isolation of membrane components, since EptB was characterized by using EptB-containing membrane preparations (32) and reactions with Lpt3 Ng utilized enzyme preparations purified from crude cell lysates in the absence of detergent (24). This is an important distinction for several reasons.…”

“…Biochemical characterization of the proteins encoded by the N. meningitidis lpt3 and lpt6 genes is therefore warranted not only to confirm that Lpt3 and Lpt6 exhibit HepII 3-and 6-PEtn transferase activities, respectively, but also to gain insight into the substrate specificities of these enzymes that may explain these observations. The lpt3 gene of N. gonorrhoeae (lpt3 Ng ) was recently identified by O'Connor et al (24), based on homology to N. meningitidis lpt3, and they were able to demonstrate that Lpt3 Ng is capable of restoring MAb 2-1-L8 reactivity (3-PEtn dependent) to LOS from an N. gonorrhoeae lpt3 mutant. However, they were unable to perform more extensive characterization, due to low activity levels and instability of the purified Lpt3 Ng protein (24).…”

“…The lpt3 gene of N. gonorrhoeae (lpt3 Ng ) was recently identified by O'Connor et al (24), based on homology to N. meningitidis lpt3, and they were able to demonstrate that Lpt3 Ng is capable of restoring MAb 2-1-L8 reactivity (3-PEtn dependent) to LOS from an N. gonorrhoeae lpt3 mutant. However, they were unable to perform more extensive characterization, due to low activity levels and instability of the purified Lpt3 Ng protein (24).…”