Bacteria are one of the biggest threats to our medical system and an estimated 33,000 people die every year due to infections caused by antibiotic resistant bacteria. These bacteria can have inherent resistance to antibiotics or acquire genes for antibiotic resistance, which can affect how certain illnesses and infections are treated. Antibiotic resistance is one of the biggest challenges facing our medical system, and therefore it is important to study bacteria to understand their role and influence on our immune systems.
One species of the bacteria that is at the forefront of antibiotic resistance is Pseudomonas aeruginosa. P. aeruginosa is an encapsulated rod-shaped bacteria that is highly resistant to antibiotics and is an opportunistic pathogen that causes infections in immunocompromised individuals as well as cystic fibrosis patients. P. aeruginosa is an environmental bacteria and is most commonly found in water and soil. P. aeruginosa also forms biofilms, a collective of bacteria that grows together, that protect it from harmful environmental factors. These biofilms are also known to cause chronic opportunistic infections.
Many bacteria, like P. aeruginosa, are gram-negative, meaning they have two membranes. The typical peptidoglycan, a mesh-like barrier, layer is in between an inner and outer membrane. The outer membrane is composed of lipopolysaccharides. Lipopolysaccharides are large complex molecules that contain lipid and polysaccharide parts.
The outer membrane is asymmetric and is formed with the help of the lipopolysaccharide transport (Lpt) system. The system consists of seven essential proteins, LptABCDEFG which have been extensively characterized in Escherichia coli, but have not been thoroughly studied in other Gram negative bacteria.
In order to target bacteria that cause infections, it is important to understand the mechanisms underlying their virulence. A study from 2018, Lo Sciuto et al., investigated a system that is part of gram-negative bacteria and demonstrated that, in P. aeruginosa, disrupting this system greatly reduces virulence and antibiotic resistance. It also seems like LptE plays a role in the morphology and arrangement of P. aeruginosa.
This study attempted to generate a clean deletion in the lptE gene. However, this did not work, so they attempted to create a conditional mutant that had the lptE gene deleted from its original spot and inserted into a neutral part of the genome where its expression was under the control of an arabinose dependent regulatory element. This means that any gene that is under the control of this element will only be expressed in the presence of arabinose, which is a type of sugar. This prevents the gene from being reintegrated back into the genome in another location and allows the scientists to control the expression of the gene. This study found that although cell growth wasn’t affected by the loss of the gene, cell envelope integrity was severely impacted.
Figure 2. Effect of different detergent concentrations on cell wall integrity
Since the seven proteins mentioned are essential for Lpt, the presence of each protein must have a role in the role of lipopolysaccharide transport. In this study, it was hypothesized that LptE depletion could seriously impact a cell's cell envelope integrity. Therefore, in order to test this, SDS sensitivity assays were created to determine the effect in LptE-depleted cells.(Figure. 2). SDS is a common detergent and protein denaturant that disrupts cell membranes, and therefore a decrease in SDS resistance would affect the cell envelope. This experiment found a decrease in SDS resistance in the lptE conditional mutant when compared to the wild type, and was even more defective in LptE cells cultured in two subsequent passages. To further investigate this finding concerning the outer membrane, they conducted a Kirby-Bauer disk diffusion assay. This test uses disks infused with antibiotics to test how sensitive a bacteria is to a certain antibiotic. They found that the conditional lptE mutant with depleted LptE was much more sensitive to all of the antibiotics they tested than the wild type strain.
Figure 3. Images of P. aeruginosa wild type PAO1 and lptE conditional mutant cells grown in MH without arabinose and stained
To further investigate changes in the cells and LptE growth, microscopy was done to show the morphological changes caused by LptE-depleted cells. Microscopic analysis shows that the morphology of LptE-depleted cells at the first refresh is very similar to that of the wild type cells, PAO1. Nevertheless, a second subsequent passage caused the LptE-depleted cells to be slightly shorter and grow in shorter cell chains. The chaining grouping was more drastic in LptH-depleted cells, which formed very long chains. These morphological changes of the cells show that the integrity and development of the cell envelope and the outer membrane can be altered by the absences of LptE.
The previous experiments found that depletion of LptE resulted in morphological changes in P. aeruginosa and reduced resistance to antibiotics. So the next step for this study was to see if LptE depletion impacted the virulence of P. aeruginosa. In order to test this, they infected the larva of Galleria mellonella, also known as the great wax moth, with different doses of the wild type (PAO1) or conditional mutant cultured in arabinose (lptE) or in the absence of arabinose (lptE 2nd). G. mellonella larvae are typically used as an infection model as they have a complex innate immune system similar to mammals and are relatively cheap to use. After infection, the scientists monitored the larva for 4 days after initial infection and calculated the survival curves.
Figure 7. Survival curves of larvae infected with different doses of P. aeruginosa cells
As we can see from the graph (Figure 7), the wild type cells have a very low lethal dose, the lethal dose being the amount of cells required to kill 90% of the population. Only about 3 cells are required to kill 90% of the population which shows how acute P. aeruginosa infections can be. However, after the lptE gene has been knocked out, we can see that the lethal dose for both lptE mutants was 9,000-10,000 fold higher. This means that LptE plays an important role in the infection process of P. aeruginosa and suggests the LPS transport proteins play an important role in virulence.
These findings have significant implications in dealing with gram-negative pathogens such as P. aeruginosa. Bioinformatic data has found LptE homologs in many other bacteria and although this protein has only been characterized in E. coli, N. meningitidis and P. aeruginosa, it would be informative to investigate the role of this protein in other bacteria. If depletion of this protein has similar effects on cell integrity and virulence then understanding the role of LptE in virulence and antibiotic resistance is important in validating LptE as an antibiotic target.
About the authors:
Emily Kellogg ‘20 is a Neuroscience major and a Computer Science minor at Mount Holyoke College. She is currently in the Colodner Lab studying the toxicity of the six different isoforms of tau in a Drosophila model of glial tauopathy. After graduating, Emily plans on working as a research technician.
Genesis Lara Granados ‘21 is a rising senior majoring in Biology and works in the Brennan lab at Mount Holyoke College. Her thesis project is focusing on the intraspecific variation of the genital morphology of the diamondback watersnake, Nerodia rhombifer. She is also captain of the squash team and is a member of MEChA de Mount Holyoke. You can always find Genesis in lab with an iced hazelnut latte or in Old Blanch with a bag of hot Cheetos.
Emily Kellogg ‘20 is a Neuroscience major and a Computer Science minor at Mount Holyoke College. She is currently in the Colodner Lab studying the toxicity of the six different isoforms of tau in a Drosophila model of glial tauopathy. After graduating, Emily plans on working as a research technician.
Genesis Lara Granados ‘21 is a rising senior majoring in Biology and works in the Brennan lab at Mount Holyoke College. Her thesis project is focusing on the intraspecific variation of the genital morphology of the diamondback watersnake, Nerodia rhombifer. She is also captain of the squash team and is a member of MEChA de Mount Holyoke. You can always find Genesis in lab with an iced hazelnut latte or in Old Blanch with a bag of hot Cheetos.
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