The Characterization of a Gonococcal HicAB Toxin-Antitoxin System Capable of Causing Bacteriostatic Growth Arrest

Abstract

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin-antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae.

Keywords: HicA; HicAB; HicB; Neisseria gonorrhoeae; bacteriophage; biofilm; growth arrest; toxin–antitoxin.

Figure 1

Sequence alignments of the N. gonorrhoeae HicA and HicB proteins with the related characterized homologs. The 100% conserved residues are highlighted in dark blue; the 67% conserved residues are highlighted in mid-blue; the 50% conserved residues are highlighted in light blue.

Figure 2

Growth analysis of E. coli DH5α expressing gonococcal HicA, HicB, or HicAB. Cultures were equilibrated to OD₆₀₀ of ~0.3 and grown for 4 h following induction with (+) or without (−) arabinose at 0 h. Growth was monitored by OD₆₀₀ measurement and compared to the negative control strain E. coli DH5α (pBAD24). The gonococcal HicA toxin inhibited growth, but not in the absence of an inducing agent or when co-expressed with the HicB antitoxin. Data are expressed as the mean ± SD of ≥3 independent experiments.

Figure 3

Delayed expression of gonococcal HicB rescues HicA-mediated growth arrest in E. coli. Following induction (A) or not (B) with arabinose for up to 4 h, dilutions of E. coli DH5α cultures containing co-resident arabinose-inducible pMS1 (HicA), and IPTG-inducible pJTM7 (HicB) or empty pME6032 plasmids were plated on selective LB plates with (+) or without (−) IPTG and incubated at 37 °C overnight for the determination of colony-forming units. Data are expressed as the mean ± SD of ≥3 independent experiments. For clarity, values below the limit of detection in panel A are plotted at 1 × 10⁵ cfu mL⁻¹.

Figure 4

Site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, or His29) abolishes HicA-mediated E. coli growth arrest. (A) Predicted structure of the N. gonorrhoeae FA1090 HicA protein (AF-Q5F6D1-F1) with the five residues (Gly22, His24, His29, His40, and Gly58) selected for mutagenesis highlighted. The N- and C-termini and secondary structure elements are also indicated. (B) Growth analysis of E. coli DH5α expressing HicA-G22C or HicA-G58C. (C) Growth analysis of E. coli DH5α expressing HicA-H24A, HicA-H29A, or HicA-H40A. For growth analysis, cultures were equilibrated to OD₆₀₀ ~0.3 and expression induced at 0 h by the addition of L-arabinose. Growth, as judged by OD₆₀₀ measurement, was compared to the negative control strain E. coli DH5α (pBAD24). Data are expressed as the mean ± SD of ≥3 independent experiments.

Figure 5

Over-expression of HicA results in gonococcal growth arrest. (A) Growth analysis of FA1090∆hicAB derivatives expressing HicA, HicB, or both proteins. Cultures were equilibrated to OD₆₀₀ ~0.2. Growth, as judged by OD₆₀₀ measurement, was compared to the wild-type FA1090 control strain following induction with (+) or without (−) IPTG at 0 h. (B) Following growth with (+) or without (−) IPTG for 0, 3, or 7 h, dilutions of relevant FA1090 derivatives were plated on Thayer-Martin agar and incubated at 37 °C for 48 h for the determination of colony-forming units. Differences in viable count between FA1090ΔhicAB::hicA(+) and the other strains were statistically significant at 7 h (Student’s t-test; p < 0.05). Data in both panels are expressed as the mean ± SD of ≥3 independent experiments.

Figure 6

Gonococcal HicAB does not influence biofilm formation. Biofilm formation of FA1090 and derivatives in the presence (+) or absence (−) of IPTG inducer was assessed by crystal violet staining after static growth at 37 °C. No statistically significant differences were observed compared to the wild type grown under matching conditions (Student’s t-test; p > 0.05). Data are expressed as the mean ± SEM of ≥3 independent experiments.