Abstract
Neisseria gonorrhoeae, also known as the gonococcus, or Gc, is a Gram-negative
diplococcus and an obligate human pathogen. Gc is the causative agent of the sexually
transmitted infection gonorrhea, which is the second most common bacterial STI in the
United States. Gc is considered an urgent public health threat due to increasing antibiotic
resistance and the lack of a licensed vaccine to prevent infection. In order to develop new
therapeutics to prevent Gc infection, it is crucial to understand the mechanisms by which
Gc persists during pathogenesis.
One of the challenges to Gc persistence in vivo is human nutritional immunity.
Nutritional immunity is a host defense against invading pathogens that consists of
induction of metal sequestration proteins at sites of infection. These host sequestration
proteins protect against invading pathogens by restricting the availability of free essential
metals, like iron and zinc. Interestingly, in evolving alongside its obligate human host, Gc
has developed outer membrane metal transporters that can mediate uptake from host
metal sequestration proteins and enable bacterial growth. These systems are well-
characterized in Gc, but less is known about how Gc maintains internal metal
homeostasis during infection, which is also crucial to prevent metal intoxication and
mismetallation of proteins and enzymes.
To uncover new factors involved in Gc resistance to zinc limitation, previous
work in our laboratory characterized the transcriptome of Gc under zinc sequestration.
The most upregulated open reading frame in zinc-limited Gc was rpmE2, which encodes
a 50S predicted non-zinc-binding ribosomal protein. In other bacteria, RpmE2 enables
growth under zinc limitation by replacing its zinc-binding paralog RpmE, on the
ribosome, which is thought to liberate RpmE-bound zinc for intracellular use. Alternation
of ribosomal protein paralogs has been shown to both maintain translation and alter
ribosome function under zinc limitation, as well as change the proteome. The goal of my
thesis work was to characterize the role of RpmE2, as well as another non-zinc-binding
ribosomal protein encoded by Gc, RpmJ2, in zinc-limited Gc. My hypothesis was that
induction of RpmE2 and RpmJ2 under zinc limitation would enable Gc growth by
liberating zinc for intracellular use or altering translation.
I found that rpmE2 and rpmJ2 are encoded in a zinc-repressed operon in Gc that
is regulated by the transcription factor Zur, which controls expression of Gc zinc uptake
factors. Interestingly, RpmE and RpmE2 were produced in zinc-limited Gc and
incorporated in ribosomes. Deletion of the operon encoding rpmE2 and rpmJ2 lead to a
growth advantage for Gc under zinc limitation rather than a growth deficit, which was
surprising and different from other bacterial species. Construction of Gc strains
expressing only RpmE or only RpmE2 revealed that RpmE2 cannot compensate for
RpmE in Gc growth, and RpmE2-only ribosomes exhibited reduced in vitro translation. I
hypothesize that induction of RpmE2 under zinc limitation in Gc slows growth and
reduces translation, enabling bacterial adaptation to zinc limitation. This work uncovers
an additional Gc adaptation to zinc limitation beyond production of zinc transporters and
opens the door for further investigation into how RpmE2 affects Gc translation and
growth.
