Iron and the Motility of CFT073
Preface
After writing a first draft of this page, which came out to a couple thousand words, I decided to break this post up into multiple posts. In this first one, I will explain only one of the several projects I’ve been working on.
Iron and Motility
Introduction to Infection and Motility
In order to introduce the purpose of the project, one must first learn the necessary background information. The subject can be introduced by learning about the mechanism of colonization to induce infection.
CFT073 is a strain of Escherichia coli known widely to be the main cause of Urinary Tract Infections (UTIs). Infection is heavily dependent on the bacterial flagella, suggested to contribute to virulence by enabling UPEC to disseminate throughout urinary tract and escape host immune responses (Lane et al. 2005).
fliC, the cistron that codes for flagellin, is the main component of the bacterial flagella, as lesions in cistron F of region III result in loss of ability to produce flagellin (Silverman and Simon n.d.). Cistron F was later renamed fliC.
So when the deletion of fliC, the gene domain, is made, the absence of flagella is induced, so we expect to see a complete loss in motility, which is what was observed in the fliC deletion mutant (Lane et al. 2005). The deletion of the gene rendered the mutant completely non-motile, severely compromising virulence.
Naturally, it would follow that studying the inhibition of CFT073 motility could lead to novel ways of treating UTIs.
Purpose of Project
More than half of women in their lifetime will experience a urinary tract infection (UTI), with most cases caused by uropathogenic E. coli (Bacheller and Bernstein 1997).
Studying motility, a major virulence factor, makes sense when trying the understand mechanisms of infection for CFT073 and other infectious agents of the urinary tract
This is precisely why we turn to the role of iron in motility and its implications on the frequency and severity of infection. But why did we decide to study iron of all nutrients?
Why do we study Iron?
It has been known since the dawn of bacteriology that iron is an essential nutrient facilitating the survival of the E. coli cell.
The importance of iron is understood but the specific reason for its importance reveals itself in the role iron plays as a cofactor. There are specific enzymes within the citric acid cycle that require iron as a cofactor for proper catalysis and therefore, energy production. We expect depletion of iron to negatively impact growth.
We know how iron impacts growth, but we have not yet understood its impacts on motility.
The deletion of the Fur (ferric uptake repressor) expressed an excess of iron intracellularly (McHugh et al. 2003). Fur was proven to be a master regulator of iron dependent uptake systems and the synthesis of conjugated proteins with iron as its prosthetic group. Under iron repleted conditions, fur binds to iron, attaching itself to the furbox upstream of iron-related genes, restricting the amount of iron imported into the cell and allowing production of conjugated proteins. Conversely, under iron depleted conditions, fur falls off the furbox, allowing the uptake of iron and decreasing the production of conjugated proteins.
Motility happens to be one of the genes that is affected by the Fur repression. There is evidence that the Fe 2-Fur complex also represses genes (cyoA, flbB, fumC, gpmA, metH, nohB, purR, and sodA) involved in various noniron functions (respiration, flagella chemotaxis, the TCA cycle) (McHugh et al. 2003).
Why not explore other nutrients?
Iron concentration is, under normal conditions, very restricted in the urinary tract. It is therefore one of the limiting nutrients in the environment of infection.
Iron content of human urine is normally very low (∼0.1 μmol per liter) in the uninfected state. Even in the absence of inflammatory stimuli, iron is subject to tight physiologic control (Robinson, Heffernan, and Henderson 2018).
The idea behind understanding iron lies in the fact that iron concentration in the urinary tract is already very low. So when thinking of treatment options, a provider should be considering iron availability as a possible factor contributing to virulence.
Modes of Motility Expressed by CFT073
There are generally three modes of motility, hence avenues for virulence, expressed by CFT073 that we are concerned with: swimming, swarming, and biofilm formation. Inhibition of these virulence factors should decrease the likelihood, and if not, severity of infection.
Swimming
Independent movement of the cells. The kind of motility we are all familiar with; powered by rotating flagella. This mode of motility is required to swim up the urethra into the bladder, and in severe cases, required to travel up the ureter to the kidneys (Lane et al. 2005).
Swarming
Concerted, multicellular, and energy intense raft-like movement across semi-solid surfaces. Also powered by rotating flagella. Involves quorum sensing. In catheter associated UTIs (CAUTIs), swarming motility is oftentimes employed to travel up the catheter into the bladder, though most studies that looked at this possibility used Proteus mirabilis as the model uropathogen. (Jones et al. 2004).
Biofilm Formation
Matrix of extracellular polymeric substances protecting the pathogen from potentially harmful external conditions, facilitating attachment of the colony. Major causative agent of recurrent infections. Attachment is vital since the human bladder is emptied every couple hours, requiring the pathogen to attach itself to the bladder walls for successful infection (Yang et al. 2016).
In addition to modifying the availability of extracellular iron, we are also looking at 2 mutants which have different iron acquisition capabilities. ∆tonB has low intracellular iron since the import of iron is compromised with the deletion of a crucial component of the iron acquisition system. ∆fur has high intracellular iron because the repressor is lacking, hence, allowing for as much iron import via systems like TonB-ExbB-ExbD to occur.