Neisseria gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular characterization of the genetic locus involved

Abstract

In an effort to identify potential cytotoxins expressed by Neisseria gonorrhoeae, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus, gly1, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in Escherichia coli and N. gonorrhoeae. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of E. coli expressing gly1 and in outer membrane preparations of wild-type N. gonorrhoeae. The gly1 locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a gly1 null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in trans for the gly1 mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the gly1 locus in the toxicity of N. gonorrhoeae to human fallopian tubes.

FIG. 1

SDS-PAGE analysis of OM preparations and culture CF of E. coli strains expressing gly1. Lanes: 1, CA201(pHSS6) (vector); 2, CA201(pGly1); 3, JM109(pBluescript II SK−); 4, JM109(pBluGly31; pBluescript II SK− plus 1.4 kb EcoRI fragment of pGly1). Eight micrograms of protein was loaded onto each lane of 15% polyacrylamide gels. The gels were stained with Coomassie brilliant blue. MW, molecular weight standards in thousands. Arrows indicate the position of the 15.6-kDa band encoded by ORF1 that was excised and sequenced.

FIG. 2

Nucleotide sequence of the 2,245-bp insert from pGly1. Important restriction sites are underlined and labelled. The predicted amino acid sequence is shown below the DNA sequence. Putative promoter (−10 and −35) and ribosome binding sites (rbs) are double underlined. The transcriptional start point is the bold-faced A at position 708 and is indicated as +1. The amino-terminal 20-amino-acid residues determined by protein sequencing are shown in bold type. The boxed region of ORF2 denotes the region of homology with hemD (see text). Neisserial uptake sequences (15) are indicated by broken underline. This sequence has been deposited in GenBank with accession no. AF003941.

FIG. 3

Immunoblot analysis with antibodies raised against the Gly1ORF1 polypeptide. Samples were run on 15% acrylamide SDS-PAGE gels and transferred to nitrocellulose electrophoretically. Immunoblotting was carried out as described in Materials and Methods. (A) Culture filtrates from E. coli strains. Lanes: 1, JM109(pGlyBssErm) (2 μg); 2, JM109(pBluGly31) (2 μg); 3, JM109(pBluescript II SK−) (2 μg). (B) OM blebs from N. gonorrhoeae strains. Lanes: (E. coli) JM109(pBluGly31) (2 μg) (CF); 2, 120 (Δgly1) blebs (9 μg); 3, MS11A (wild-type) blebs (9 μg).

FIG. 4

Interaction of N. gonorrhoeae gly1 mutants with epithelial cells in tissue culture. MS11A: P⁺gly1⁺; 120: P⁺gly1; 307: P⁻gly1⁺; 320: P⁻gly1. (A) Adherence to A431, HEC-1-B, and T₈₄ cells. Adhesion data are presented as a percentage of the wild-type control (MS11A). (B) Invasion of A431, Hec-1-B, and T₈₄ cells. Invasion data are presented as the ratio of GM-resistant (Gm^r) bacteria per number of cell associated bacteria. (C) Survival within A431 cells. Survival is expressed as the number of GM-resistant (Gm^r) CFU at each time point relative to the number of Gm^r CFU immediately following GM treatment. Error bars indicate average deviations.

FIG. 5

Scanning electron micrographs of human fallopian tubes infected for 24 h with wild-type N. gonorrhoeae strains and deleted for the gly1 locus. Tissues were infected with bacteria and examined 24 h postinfection as described in the text. (A) Uninfected tissue (magnification, ×2,925). (B) Tissue infected with the wild-type parent, MS11A (magnification, ×3,250). (C) Tissue infected with the gly1 mutant strain 120 (magnification, ×5,200).

FIG. 6

Scanning electron micrographs of human fallopian tubes infected for 24 h with gly1 mutant N. gonorrhoeae strains expressing gly1 in trans. Tissues were infected and examined as in Fig. 6. (A) Tissue infected with the gly1 mutant strain 120 (magnification, ×6,636). (B) Tissue infected with the complemented mutant, 120(pGly18.2.2) (magnification, ×3,634). (C) Tissue infected with MS11A(pGly18.2.2) (magnification, ×3,634).