Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae

Abstract

The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

FIG. 1.

Mapping and phenotypic characterization of pptA-linked transposon insertion mutants. (A) Physical map of the gonococcal pptA locus with transposon insertions indicated by circles. •, transposon insertions that could not be recovered in N. gonorrhoeae; ○, insertions with wt growth phenotype. The red circles represent transposon insertions that resulted in aberrant cell septation and coccal morphology. The precise sites of transposon insertion are shown in Fig. S1 in the supplemental material. The striated box defines the Pfam PF00884 sulfatase domain of PptA. (B) Cell septations and coccal morphologies of transposon mutants detected by immunofluorescence microscopy. Gonococci were detected using fluorescein-labeled MAbs (green). (C) Gonococcal colonies on conventional medium showing the different growth phenotypes of transposon mutants. The strains used were wt (N400), pptA::kan (KS9), Tn22 (pptA::Tn22; KS8), Tn5 (pptA::Tn5; KS5), Tn9 (pptA::Tn9; KS6), and Tn11 (pptA::Tn11; KS7).

FIG. 2.

PptA-linked transposon insertion mutations perturb murF expression and PptA activity. (A) Real-time RT-PCR analysis of murF mRNA levels in mutants carrying transposon insertions. Amplification of tbpA (5) served as an internal reference and control. Negative controls were performed without reverse transcriptase treatment (not shown). The values are means ± standard errors of the mean; n = 3. The strains used were wt (N400), pptA::kan (KS9), Tn22 (pptA::Tn22; KS8), Tn5 (pptA::Tn5; KS5), Tn9 (pptA::Tn9; KS6), and Tn11 (pptA::Tn11; KS7). The Tn22 mutation reduces PilE phospho-form modification. (B and C) Intact mass analysis of PilE N400 (wt) (B) and KS8 (pptA::Tn22) (C). Species labeled as bearing PE/PC modifications also carry the hexose-2,4-diacetamido-2,4,6-trideoxyhexose (HexDATDH) dissacharide in its acetylated form. Peaks in the MS spectra that are not labeled are listed in Table S3 in the supplemental material.

FIG. 3.

Relative pptA mRNA levels in defined isogenic backgrounds. Real-time RT-PCR amplification of tbpA mRNA (5) served as an internal reference/control. Negative controls included reactions performed without reverse transcriptase; no products were detected under these conditions (not shown). The values are means ± standard errors of the mean; n = 3. Bars: 1, wt (N400); 2, pilV(Fs) (GV1); 3, pilV(Fs) pptA::kan (KS10); 4, pilV(Fs) pptA::kan iga::pptA (KS27); 5, pilV(Fs) iga::pptA (KS26); 6, pilV(Fs) pptA::kan iga::pptA(Ind) (KS11) induced with 0.25 mM IPTG. pptA_wt and pptA_iga refer to pptA allele status, with the gene at either the endogenous locus (wt) or the ectopic locus (in iga). ind., ectopic, inducible pptA in its derepressed state.

FIG. 4.

PilE PC modification levels parallel regulated expression of PptA. (A) Lane 1 (from left), KS9 (pptA::kan); lane 2, KS11 without IPTG [pilV(Fs pptA::kan iga::pptA(Ind)]. Lanes 3 to 9, KS11 with IPTG in the following concentrations: lane 3, 0.01 mM; lane 4, 0.015 mM; lane 5, 0.02 mM; lane 6, 0.03 mM; lane 7, 0.04 mM; lane 8, 0.05 mM; lane 9, 0.1 mM; lane 10, control (GV1). (B to D) Deconvoluted molecular weight spectra from intact PilE ESI MS analyses in strains KS11 without IPTG [pilV(Fs) pptA::kan iga::pptA(Ind)] (B), KS11 with IPTG (0.015 mM) (C), and KS11 with IPTG (0.250 mM) (D). Species labeled as bearing PE/PC modifications also carry the hexose-2,4-diacetamido-2,4,6-trideoxyhexose (HexDATDH) dissacharide in its acetylated (ac) form. A complete list of m/z forms and corresponding species of PilE can be found in Table S3 in the supplemental material. Asterisks indicate samples in which pptA was induced with 0.015 mM IPTG (A and C).

FIG. 5.

PilE PC modification occurs in a wt background when propagated in defined, choline-free medium. (Top) Immunoblot of whole-cell lysates using polyclonal antibodies specific for PilE. (Middle) MAb TEPC-15 specific for PC. (Bottom) Polyclonal antibodies specific for PilV. N. gonorrhoeae strains were grown either on solid conventional medium (C) or on solid defined, choline-free medium (D). Lanes: 1 and 2, wt (N400); 3 and 4, pilV(Fs) (GV1); 5 and 6, pptA::kan (KS9); 7 and 8, pilE(S68A) (GE68).

FIG. 6.

MS analysis of intact PilE from N. gonorrhoeae strains grown in defined, choline-free medium. (A) Wt (N400) grown on solid conventional medium. (B) Wt (N400) grown on solid defined, choline-free medium. (C) pilE(S68A) (GE68) grown on solid defined, choline-free medium. The species labeled as bearing PE/PC modifications also carry the hexose-2,4-diacetamido-2,4,6-trideoxyhexose dissacharide (HexDATDH) in its acetylated (ac) form. The peaks in the MS spectra that are not labeled are listed in Table S3 in the supplemental material.

FIG. 7.

Comparative modeling of the catalytic domain of PptA. (A) (Top) Crystallographic structure of human N-acetylgalactosamine-4-sulfatase (residues 43 to 533; Protein Data Bank [PDB] no. 1fsu). (Bottom) Three-dimensional structure of the sulfatase domain of PptA (residues 230 to 540) based on comparative modeling with 1fsu (sequence identity, 15%; E value, 1e-16; model score, 1.00). (B) (Top) Crystallographic structure of human arylsulfatase A (residues 19 to 503; PDB no. 1auk). (Bottom) Three-dimensional structure of the sulfatase domain of PptA (residues 230 to 547) based on comparative modeling with 1auk (sequence identity, 14%; E-value, 6e-42; model score, 0.90). Color coding of the protein backbone is as follows: red, α-helices; blue, β-strands; gray, loop regions. Color coding for residues is as follows: yellow, residues involved in metal ion coordination (human N-acetylgalactosamine-4-sulfatase, D⁵³, D⁵⁴, D³⁰⁰, and N³⁰¹; arylsulfatase A, D²⁹, D³⁰, D²⁸¹, and N²⁸²); cyan, active-site residues (human N-acetylgalactosamine-4-sulfatase, C⁹¹; arylsulfatase A, C⁶⁹). Yellow, analogous residues in PptA, i.e., putative residues involved in metal ion coordination (H³⁷⁴, H³⁷⁹, D⁴³⁵, and H⁴³⁶); cyan, putative active-site residues (S²⁴¹ and T²⁷⁸).

FIG. 8.

Alignment of sulfatase domains from the PptA/LPS PE transferase protein family. (A) Graphical overview of the domain structure of PptA. Color codes: orange, signal peptide (residues 1 to 33) with a hidden transmembrane region (residues 13 to 32); red, transmembrane regions (residues 42 to 61, 68 to 90, 122 to 144, and 156 to 175); green, sulfatase domain PF00884 (residues 230 to 516); blue, Prodom families PD005703 and PD461453; pink, Prodom families PD883089 and PD144483 (found in Lpt6); yellow, Prodom families PD605628 and PD698194 (found in MdoB). The domain structures are similar for all the proteins belonging to Prodom families PD005703 and PD461453. (B) Juxtaposed alignment of the sulfatase domain-containing proteins from Prodom family PD005703. Color codes: red, absolutely conserved residues; green, either S or T; blue, residues conserved only between characterized proteins. Lpt6 is part of the Prodom families PD883089 and PD144483, as indicated by the pink coloring of the residues. MdoB is part of the Prodom families PD605628 and PD698194, as indicated by the yellow coloring of the residues. (C) Alignment of characterized proteins from Prodom family PD461453. The color coding is the same as in panel B. The aligned proteins are as follows: PptA, Q9RMJ3 NEIGO; LptA, Q7DD94 NEIMB; Lpt3, Q9JXJ7 NEIMB; EptB, Q38J80 ECOLI (formerly YhjW); PmrC, YjdB SALTY; CptA, Q7CPC0 SALTY; Lpt6, Q9JWE8 NEIMA; MdoB, P39401 (OPGB) ECOLI.

FIG. 9.

PptA activities in structure-based, site-directed PptA mutants. (Top) Immunoblot of whole-cell lysates using polyclonal antibodies specific for PptA. (Middle) Polyclonal antibodies specific for PilE. (Bottom) MAb TEPC-15 specific for PC. Lanes: control, pilV(Fs) (GV1); pptA::kan, pilV(Fs) pptA::kan (KS10); G239A, G239A (KS18); S241A, S241A (KS16); ΔS241, ΔS241 (KS17); H374A, H374A (KS19); G377A, G377A (KS20); S378A, S378A (KS21); H379A, H379A (KS22); S434A, S434A (KS23); D435A, D435A (KS24); H436A, H436A (KS25). As controls for all mutants, immunoblotting was used to ensure that reduced or absent phospho-form modification did not result from decreased PptA stability.

FIG. 10.

PptA-mediated PilE modification in P. aeruginosa and E. coli. (A) MS analysis of intact PilE expressed in P. aeruginosa. (B) MS analysis of intact PilE coexpressed with PptA in P. aeruginosa. (C) Intact mass analysis of a PilE(S68A) mutant coexpressed with PptA in P. aeruginosa. (D) Immunoblot of whole-cell lysates using polyclonal antibodies specific for PilE. Lanes 1 to 3 (from left), P. aeruginosa pilA pilT expressing N. gonorrhoeae proteins: lane 1, PilE; lane 2, PilE(S68A); lane 3, PilE and PptA. Lanes 4 and 5, E. coli DH5α expressing the proteins: lane 4, PilE; lane 5, PilE and PptA. Peaks in the MS spectra that are not labeled are listed in Table S3 in the supplemental material.