Identification of a Neisseria gonorrhoeae Histone Deacetylase: Epigenetic Impact on Host Gene Expression

Abstract

Epigenetic reprogramming in macrophages is termed trained innate immunity, which regulates immune tolerance and limits tissue damage during infection. Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. Here, we report that this pathogen harbors a gene that encodes a histone deacetylase-like enzyme (Gc-HDAC) that shares high 3D-homology to human HDAC1, HDAC2 and HDAC8. A Gc-HDAC null mutant was constructed to determine the biologic significance of this gene. The results showed that WT gonococci reduced the expression of host defense peptides LL-37, HBD-1 and SLPI in macrophages when compared to its Gc-HDAC-deficient isogenic strain. The enrichment of epigenetic marks in histone tails control gene expression and are known to change during bacterial infections. To investigate whether gonococci exert epigenetic modifications on host chromatin, the enrichment of acetylated lysine 9 in histone 3 (H3K9ac) was investigated using the TLR-focused ChIP array system. The data showed that infection with WT gonococci led to higher H3K9ac enrichment at the promoters of pro-inflammatory mediators' genes, many TLRs, adaptor proteins and transcription factors, suggesting gene activation when compared to infection with the Gc-HDAC-deficient mutant. Taken together, the data suggest that gonococci can exert epigenetic modifications on host cells to modulate certain macrophage defense genes, leading to a maladaptive state of trained immunity.

Keywords: H3K9ac; HDAC; Neisseria gonorrhoeae; chemokines; cytokines; epigenetic; gonorrhea; infection; macrophage; survival.

Figure 1

Gonococcal infection downregulates LL-37 and HBD-1 expression in monocytes. (A): Antimicrobial host defense peptide (AMP) gene expression in THP-1 cells infected with Gc strain FA19 overnight at multiplicity of infection (MOI) of 1, 5 and 25 measured by qRT-PCR. (B): LL-37 gene expression in primary human monocytes infected with Gc strain FA19 overnight at MOI of 10 measured by qRT-PCR. Peripheral monocytes were obtained from four different healthy donors. (C): Overexpression of LL-37 gene in human THP-1 monocytes treated with 10 nM of 1,25 dihydroxyvitamin D3 (VitD3) overnight prior to infection with Gc strain FA19 at MOI of 25. Downregulation of LL-37 gene expression was assessed at 5h and 18h post-infection. p values were calculated using a Student’s t-test in reference to noninfected cells (**). p values in reference to infection at MOI 1 for LL-37 expression (*), HBD1 (#) and SLPI ($). These data are representative of three independent experiments.

Figure 2

Evolution of Neisseria gonorrhoeae Gc-HDAC-like protein in human monocytes. Evolution of Neisseria Gc-HDAC-like protein compared to human HDACs. The multiple sequence alignment tool Clustal Omega was used to build the phylogenic tree (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Figure 3

Computational analysis and in silico modeling of Gc-HDAC-like protein in Neisseria gonorrhoeae. (A): Amino acid sequence alignment of Gc-HDAC from gonococcal strains FA19 and FA1090 and their 3D structural alignment. (B): In silico modeling of Gc-HDAC-like protein: (i) predicted Gc-HDAC-like protein 3D structure revealing the catalytic pocket with the conserved metal binding constellation (green); (ii) predicted Gc-HDAC-like protein 3D structure (brown) is superimposed on human HDAC1 protein (blue) and (iii) predicted dockings of HDAC inhibitors CRI, trichostatin A and CF3 in Gc-HDAC catalytic pocket. Computational Gc-HDAC 3D protein structure and HDAC inhibitors dockings were predicted using I-TASSER, and PDB files were viewed using Chimera. The BS scores of top predictions for HDAC inhibitors CRI, TSA and CF3 are 1.51, 1.09 and 1.4, respectively. BS score definition by I-TASSER is a measure of local similarity (sequence and structure) between template binding site and predicted binding site in the query structure. Based on large-scale benchmarking analysis, a BS score >1 reflects a significant local match between the predicted and template binding site.

Figure 4

Expression of Neisseria gonorrhoeae Gc-HDAC-like protein during infection in peripheral human monocytes. Gc-HDAC gene expression during infection compared to human HDAC1 expression in peripheral human monocytes (PMNC) obtained from four healthy donors and infected with live gonococci at MOI 25 overnight. Gc-HDAC and hHDAC1 expression is assessed by quantitative RT-PCR normalized to β-actin gene expression and compared to noninfected PMNC (n = 4). p values were calculated using a Student’s t-test in reference to noninfected PMNC.

Figure 5

Expression of Neisseria gonorrhoeae AMPs gene expression in infected monocytes in the presence and absence of Gc-HDAC. (A): Expression of LL-37, HBD-1 and SLPI genes in THP-1 monocytes infected with WT strain FA19 or its isogenic Gc-HDAC-deficient mutant or the complemented H’C strain at MOI of 1 overnight (n = 3) was assessed using qRT-PCR. (B): H3K9ac epigenetic mark enrichment at the promoters of LL-37 and HBD-1 in THP-1 monocytes infected with WT strain FA19 or its isogenic Gc-HDAC-deficient mutant at MOI of 25 overnight (n = 3) was assessed using a ChIP assay. p values were calculated using a Student’s t-test in reference to cells infected with the WT FA19 strain for LL-37 expression (*), HBD1 (#) and SLPI ($).

Figure 6

Gonococci exert epigenetic modifications in THP-1 monocytes. H3K9ac epigenetic mark enrichment at the promoters of genes involved in the TLRs signaling pathways. WT parent strain FA19: blue bars and isogenic HDAC-deficient mutant: red bars. Data are average of three independent ChIP experiments. p values were > 0.05 and were calculated using a Student’s t-test comparing WT FA19 to the HDAC-deficient mutant.