A Study On the Role Of PA14_54520 Gene In Chronic Pseudomonas aeruginosa Catheter Associated Urinary Tract Infections (CAUTI)
Katherine Miller, Christina Ambrose, Keefe Bautista and Frederick Difiore
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Important Background Information on Pseudomonas aeruginosa
Important Background Information on Pseudomonas aeruginosa
Pseudomonas aeruginosa is a bacterial species that is ubiquitously found in the environment. P. aeruginosa is also an opportunistic pathogen in which patients that have a compromised immune system have an increased risk of contracting an infection as it is a common cause of hospital acquired infections (HAI) in the United States. HAI are infections that develop in admitted hospital patients unrelated to their admittance. One type of HAI is catheter associated Urinary Tract Infections (CAUTI) which are caused by P. aeruginosa from the environment, that come into contact with the catheter that has been implanted in the urinary tract of admitted patients. The infection can travel from the urinary tract to the kidneys and if untreated, the P. aeruginosa infection can lead to sepsis which can be fatal.
(Flores et al. 2015) (Cole et al. 2014)
Click for background by Keefe Bautista
Gene Introduction
Gene Introduction
Figure 1. Outer membrane porin in a Gram negative bacterial cell wall P. aeruginosa is a gram negative bacteria. Figure 1 demonstrates the function of a general porin that mediates molecular movement across the cellular membrane.
The gene PA14_54520 which encodes for the OpdH protein, was selected for our research due to its overexpression in urine (Cole et al. 2014). Through scholarly research, we found that OpdH functions as the cis-aconitate uptake porin (Tamber et al. 2006). Cis-aconitate is a crucial tricarboxylate intermediate for the Krebs cycle, which turns carbon molecules into energy sources for the bacterial cell to sustain growth (Krebs and Holzack 1952). Figure 1 demonstrates the function of porins which are transport channels that allow molecules, like tri carboxylates, to move through the membrane into the cell. This is distinctive in selective outer membranes as they only allow certain proteins beyond the outer membrane. This type of porin is typically unique to Gram negative bacteria like P. aeruginosa, as the single layer of peptidoglycan is much easier to pass through than multi- layered Gram positive bacteria.
Click for Gene Introduction Audio by Frederick Difiore
Figure 2. Sucrose specific porin in S. typhimurium.
https://en.wikipedia.org/wiki/Porin_(protein)
https://en.wikipedia.org/wiki/Porin_(protein)
Figure 3. A β barrel. Bacterial sucrose-specific porin from S. typhimurium.
https://en.wikipedia.org/wiki/Beta_barrel
https://en.wikipedia.org/wiki/Beta_barrel
Figure 2 is representative of the three dimensional structure of a porin and can be used to explain their complex function and structure. The OpdH protein makes up the particular selective porin that allows for the transport of cis-aconitate into the cell. This is possible due to the amino acid residue found on the tricarboxylate which the porin recognizes and allows to pass through. Porins are formed from beta sheets which are proteins that are connected in a lateral fashion and form a pleated sheet in Figure 3. The beta barrel represented in that figure shows the direction of the protein chain. Figure 2 represents three beta barrels as shown in Figure 3.
Background on the PA14_54520 gene and its role in P. aeruginosa CAUTI
Background on the PA14_54520 gene and its role in P. aeruginosa CAUTI
by Katherine Miller
Current research suggests that there is no tri-carboxylate specific binding site within OpdH but it has been shown to be the cis-aconitate uptake porin (Tamber et al. 2006). It is also not yet determined if OpdH has extracellular binding sites contributing to the uptake of certain molecules. This study suggested it functions as a large general porin with tricarboxylate characteristics.
Biological Rationale
Biological Rationale
Our Research Hypothesis
Our Research Hypothesis
Our biological rationale connects what is already know about the PA_54520 gene to the context of why it is being overexpressed when the bacteria is grown under CAUTI conditions. We rationalize that the PA14_54520 gene is essential for bacterial growth. We rationalize the overexpression of PA14_54520 during growth in urine is to increase the uptake of cis-aconitate which can then be used as a carbon energy source for the Krebs cycle. This will help promote survival of bacteria like Pseudomonas aeruginosa in a nutrient limiting environment like the urinary tract. Thus, if the expression of the PA14_54520 gene is knocked down using CRISPR/dCas9, transport of cis-aconitate as an energy source will be inefficient and result in poor growth of the bacteria.
Click for Biological Rationale and Research Hypothesis audio by Christina Ambrose
" If we knockdown the expression of the PA14_54520 gene, then there will be decreased growth of the P. aeruginosa bacteria in artificial urine media because the OpdH protein functions as a large transporter channel that mediates the uptake of cis-aconitate, a crucial tricarboxylate for the Krebs cycle that is present in human urine."
" If we knockdown the expression of the PA14_54520 gene, then there will be decreased growth of the P. aeruginosa bacteria in artificial urine media because the OpdH protein functions as a large transporter channel that mediates the uptake of cis-aconitate, a crucial tricarboxylate for the Krebs cycle that is present in human urine."
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