Characterization of Silver-Associated SilE Folding and Structural Information Retrieval

Abstract
The increase in bacterial resistance to antibiotics has encouraged augmented usage of
antimicrobials like silver in dressing wounds. This in turn had resulted in the subsequent
isolation of bacteria that are silver resistant and generate two periplasmic silver binding
proteins and an efflux pump, which are thought to act in a way that allows expulsion of silver
from periplasm, thus stopping silver from disturbing the cytoplasmic membrane. The
particulars of silver resistance mechanism have not been completely explored yet. SilE is a
major silver binding protein, which is considered to display native unfolded configuration.
The protein generates tertiary fold upon binding with silver and has also been found to confer
resistance. The purpose of this assignment is to characterize silver-associated SilE folding
and retrieve adequate structural information related to the silver bound and folded form. The
assignment will also involve SilE overexpression in E. coli cells and subsequent protein
purification. It will then involve NMR spectroscopy for determining the protein folding, in
relation to silver. The assembly of the folded, silver-associated protein will be then assessed
with the use of NMR spectroscopy and/or X-ray crystallography. The findings of the research
will significantly contribute gaining a sound understanding of bacterial resistance to silver.
By gaining an awareness of the resistance mechanisms, antibiotic resistance can be prevented
or circumvented in future.
Key words: Structural biology, NMR spectroscopy, protein purification
Hypothesis and Aims
It is hypothesised that bacterial resistance to antibiotics has prompted increased This
has led to the isolation of silver resistant bacteria that produce an efflux pump and two
periplasmic silver binding proteins that are thought to act to expel silver from the periplasm,
preventing silver from perturbing the cytoplasmic membrane.

Therefore, the main aims of this project are to:
• overexpression of SilE in E. coli and protein purification
• Characterise silver induced folding
• Obtain structural information on the folded, silver bound form
Background
Internationally, the wide spread of bacteria resistance made a significant impact on
the efficacy of the antibiotic which is saved many lives from this organism. The antibiotic
resistance situation happened due to the overuse of this drug and the lack of developing new
medication by the pharmaceutical companies (Ventola, 2015). Silver is considered an
antimicrobial agent a long time before the antibiotic was discovered, as well as silver
consider the most effective antibacterial and less toxicity out of all metals that have
antimicrobial properties. The usage of silver decreased by the time the antibiotic discovered.
However, due to the antibiotic resistance bacteria endanger our lives, an interest of silver as
an antimicrobial agent begun to re-use. Silver resistance in Gram-negative bacteria was first
encountered in a strain of Salmonella typhimurium that caused an outbreak on a burns ward
in 1975 and resulted in the death of three people (WHO. int, 2019). Silver resistance bacteria
develop a technique to expel the silver from the cell membrane using an efflux pump and two
periplasmic silver binding proteins. There have been studies that show the significant role of
SilE in the occurrence of bacteria silver resistance. However, there is a poor understanding of
the genetic mechanism that underlies the susceptibility of the resistance gene that contributes
to the resistance. Owing to the high antimicrobial activity of ionic silver against a plethora of
microorganisms, silver has been recognized as a key component for numerous commercially
accessible healthcare products (Zheng et al., 2018).

Figure 1- Silver resistance operon
Source- (Asiani et al., 2016)
The usage of silver is snowballing speedily in the arena of wound care, and a
widespread diversity of silver-impregnated wound dressings are currently commonplace such
as, polyurethane foams, hydrofiber dressing, and gauzes. The primary benefit of silver-
containing wound dressings can be accredited to the fact that they provide a moist wound
environment, thus hastening the process of wound recovery, with proven effectiveness in
local infection or wounds having bioburden (Wu et al., 2014). In addition, efflux pumps
provides the opportunity to microorganisms for regulating the internal environment, which in
turn is facilitates removal of toxic substances, together with metabolites, antimicrobial agents,
and quorum sensing signal molecules (Chacón et al., 2014).