Resistance of Neisseria gonorrhoeae to nutritional immunity protein-mediated zinc limitation during epithelial infection

Neisseria gonorrhoeae (Gc) is an obligate human pathogen that infects mucosal epithelia to cause the common sexually-transmitted infection (STI) gonorrhea, and antimicrobial-resistant Gc is an urgent public health threat. Gonorrhea has the potential to cause serious sequelae when untreated, especially in women, who may be asymptomatic and unaware that they are infected. The initial site of infection of the female reproductive tract is usually the cervix. In order to establish infection, Gc must attach to the epithelium and acquire essential nutrients such as iron and zinc. The host innate immune system can withhold these nutrients through a process called nutritional immunity, where trace metals are bound by host metal binding proteins. Zinc-binding proteins, including the S100-family proteins calprotectin (S100A8/S100A9) and psoriasin (S100A7), are produced by epithelial cells in response to inflammatory cytokines. These proteins sequester free zinc and make it unavailable to pathogens, and can kill multiple species of bacteria and fungi through zinc starvation. Studies of suspension-grown Gc revealed that expression of the outer membrane transporters TdfH and TdfJ which bind calprotectin and psoriasin, respectively, can pirate zinc from the host. In contrast to Gc in suspension, the role of zinc acquisition in Gc that are adherent to mucosal epithelial cells was unknown. In studying adherent Gc I found that, under zinc sequestration conditions that reduced viability of suspension-grown Gc, adherent Gc were able to survive and replicate. In suspension-grown bacteria, TdfH and TdfJ were necessary to survive zinc sequestration by human calprotectin and psoriasin. TdfH and TdfJ deletion mutant (ΔtdfH ΔtdfJ) Gc adherent to Ect1 immortalized ectocervical cells survived in spite of this zinc sequestration. I examined this further by using murine calprotectin or Site 1 KO calprotectin (neither of which WT Gc can use as a zinc source), as well as the membrane- permeable zinc-chelator TPEN. In each of these conditions, the negative effect of zinc sequestration on viable CFU was abrogated in the adherent compared to suspension Gc. This protection from zinc sequestration occurred along with a significant change in gene expression between adherent and suspension Gc during zinc sequestration, as assessed by RNAseq. The contribution of the periplasmic zinc shuttle protein ZnuA was assessed after znuA was found to be more greatly expressed in adherent vs. suspension Gc when zinc was sequestered. ZnuA was necessary but not sufficient for enhanced survival of adherent Gc under zinc sequestration. Suspension Gc that overexpressed ZnuA did not exhibit increased survival when zinc was sequestered compared to those with wildtype ZnuA expression.
In addition to ZnuA, numerous other candidate genes emerged as potential actors in the defense of adherent Gc from zinc sequestration. Additionally, new questions can be raised about the effect of adherence on Gc biology, signaling mechanisms, control of gene expression, zinc stores in Gc, and the role of thus far uncharacterized proteins in zinc uptake. These findings may open new avenues in understanding Gc biology, as well as lend insight to the development of new drugs and vaccines to combat the global public health threat of Gc.