Attribute-Based Access Control

Literature Review

Shigellae are rod-shaped gram-negative bacteria, that belong to the enterobacteria family. These bacteria possess pathogenicity within a human host, primarily targeting the GI tract of the human body. Shigellae obtain multiple properties that cooperate to its operation of survival. These bacteria are non-motile, facultative anaerobes, which allow Shigellae to utilize oxygen when oxygen is present to generate ATP, while also possessing the capability to make ATP when oxygen is not present, via fermentation (Anaerobic Respiration) . The genus Shigella, is classified into four separate species.

These species are known as, S. sonnei, S. boydii, S. dysenteriae, and S. flexneri. (Gentle, et al 2016) Species of Shigella are highly virulent and can cause infection with an amount as low as 10 microorganisms. (Gentle, et al. 2016) Once this bacterium enters the body and proliferates, the victim can then be exposed to rapid growth and infection, causing bascillary dysentery, also known as shigellosis.

Shigellosis, being one of the most communicable diseases of the body’s enteric system, is recorded as the world’s leading cause of death from dysenteric diseases, initially affecting populations of poor sanitation.

(Gao, et al 2018) In fact, recorded by the CDC, up to 80-165 million cases of shigellosis occur around the world every year, and of those cases 0.6 million are fatal, affecting mostly children. (Killackey, et al. 2016) This dysenteric disease was named from a highly virulent bacteria, called Shigella.

In 1930 the genus name, Shigella, was discovered by Dr. Kiyoshi Shiga, upon a case of bacterial research. (Trofa, et al. 1999) After surpassing medical school, Dr.

Get quality help now

WriterBelle

Verified

Proficient in: Biology

4.7 (657)

“ Really polite, and a great writer! Task done as described and better, responded to all my questions promptly too! ”

+84 relevant experts are online

Hire writer

Shiga further expanded his studies in the Institute for Infectious Diseases where he continued to work as a research assistant, under Japanese scientist, Dr. Kitasato. (Trofa, et al. 1999) During his time dedicated to research, frequent outbreaks of dysentery, which later discovered to be shigellosis, became an epidemic in the land of Japan as well as other international regions. This illness included severe diarrhea, bloody diarrhea, nausea, fatigue, fever, vomiting and dehydration. (World Health Organization 2005) At the time there were no resolutions to the illness beside natural immune recovery. In fact, in 1897, the Japanese society became victims of the disease with up to a 20% mortality rate, involving approximately 91,000 individuals. (Trofa, et al. 1999) With children being the most susceptible to death from this disease, this outbreak escalated with no known cure.

In approaching the 1900s, Dr. Kitasato shifted his focus upon microbiological research to further discover the main cause of the population’s death. (Trofa, et al. 1999) As Dr. Shiga began to study this disease, he discovered that its causation was influenced directly by bacteria, specifically Shigella dysenteriae. In furthering Shiga’s research, he discovered that there were different species of Shigella, today named S. sonnei, S. boydii, S. dysenteriae, and S. flexneri, all which are capable of causing dysentery. (Trofa, et al. 1999)

Found in infected waters and food, all species of Shigella access the human body primarily through gestation. (Carayol, et al. 2013) Other ways of access occur via transmission from person-to-person, as well as the fecal-oral route. (Killackey, et al. 2016) Capable to withstand extreme low levels of pH, this bacterium can survive and surpass the environment of the stomach, upon periplasmic proteins, in continuation to the large intestine. (Carayol, et al. 2013) Once Shigella encounter cells of the intestinal epithelium, they exit the lumen via small endocytic cells called M-Cells, invading the colonic mucosa.

M-cells, also known as microfold cells, are located in dome-shaped regions of the intestinal wall called Peyer’s patches. (Jung, et al. 2010) Described by Marco Severino in 1645, and Johann Peyer in 1677, Peyer’s patches are composed of gut associated lymphoid tissue (GALT) while being also composed of follicle associated epithelium, where M-cells are found. (Jung, et al. 2010) Peyer’s patches function to house immune cells, ready to attack any foreign material processed through the GI tract. The ability for M-cells to identify which molecules and pathogens will enter, is by recognition of glycosylated receptors, as well as immune receptors (IgA) found specifically on the luminal surface of the M-cell. (Jung, et al. 2010) When M-cells target desired molecules or pathogens, they are engulfed into the Peyer’s patch to be utilized or degraded. (Jung, et al. 2010) Shigella species, Shigella flexneri, are found passing through M-cells in the same fashion. When S. flexneri travels through the M-cells, it will undergo the same process to be targeted, engulfed, and destroyed.

Although, when additional S. flexneri is channeled through, this bacterium can surpass immune responses from white blood cells, allowing freedom of invasion. If S. flexneri overcomes attack, it will invade neighboring host epithelial cells, robbing the cell of its contents to replicate and move laterally to neighboring cells. After host cells have been infected, Ca2+ within the cytosol will increase in concentration to signal apoptosis. (Carayol, et al. 2013) As cell death occurs due to severe inflammation, the surface of the intestines will form lesions and the cells will lose their function to absorb nutrients and process waste. (Headley and Payne 1990) Victims suffering from invasion of this bacteria become starved of nutrients and endure severe diarrhea and dehydration.

Microbiological tests from Shiga’s research unraveled the infection process of Shigella on a chemical level. Through his tests, Shiga, along with other scientists, discovered that Shigella in fact produced a toxin within the body, known as the Shiga toxin. (Melton-Celsa 2014) Overtime, the components of this toxin were discovered to be phage genes, encoded by a large virulence plasmid, to produce viral components such as bacteriophages. (Wagner, et al. 2002) The Shiga toxin unquestionably found within serotypes of Shigella species Shigella dysenteriae, is also found in certain serotypes of Shigella flexneri, Shigella sonnei, and E. coli. (Melton-Celsa 2014)

Though found within these species, the Shiga toxin is classified by two different groups, Stx1 and Stx2 with many variants of each. The difference between each group is depended upon its genetic makeup, according to structure and function. Shiga toxin 1 (Stx1) is found within species S. dysenteraie, while Stx2 can be found in S. flexneri and S. sonnei, as well as STEC, (Stx-producing Escherichia coli) (Melton-Celsa 2014) In order for this toxin to enter into body cells, it requires an apparatus, known as the Type III Secretion System (T3SS). When temperatures within the body shift after ingestion of Shigella, the VirF master transcriptional regulator will work to induce another transcriptional regulator, known as VirB, to impact the production of virulence genes. (Carayol, et al. 2013) This process includes the production of the T3SS. (Carayol, et al 2013)

Encoded by mxi and spa genes, this system allows for multiple translocator proteins, known as effector proteins, to enter the host cells. (Pieper, et al. 2013) These effector proteins, are composed of the virulence factors that work to rearrange and reform the inner contents of host cells for proliferation. (Burgess. et al. 2016) Because T3SS is found in gram-negative bacteria, all species of Shigella acquire it. (Coburn, et al. 2007) In Shigellae, the basal body of the T3SS is found embedded within the inner and outer membranes, protruding through the peptidoglycan layer in between. T3SS is formed by the advantage of utilizing the products of homologous genes to create a nanosyringe, which will inject itself through the plasma membrane of host cells. (Notti and Stebbins, 2016)

The proteins important for the process of promoting uptake and invasion into the cell is due to genes located on the 220-kb virulence plasmid (Gore and Payne 2010) Importantly found on this plasmid are coded invasion plasma antigens, or Ipa genes. Ipa genes are responsible for producing the Ipa proteins capable of utilizing the T3SS system for effector protein injection. Once the bacterium enters into a cell, it is taken up into a vacuole where it will penetrate and free itself with the assistance of effector proteins. (Waligora, et al. 2014)

As the bacterium exit the vacuole, it must utilize the cell’s metabolic contents for intracellular growth and spreading to other cells. Because S. flexneri is nonmotile, it is dependent on actin filaments formulated from the host’s cytoskeleton for motility. (Ruetz, et al. 2012) This process begins when effector proteins rearrange the actin filaments to form actin tails due to polymerization by the icsA protein and allow propulsion through the cell’s intracellular fluid as well into surrounding cells. (Bliven and Maurelli 2012) Now exposed to the cytosol, the host cell will perform a defense mechanism to protect itself from rapid bacterial growth and infection.

This process will occur via phagolysosome, which will operate to phagocytize the bacteria in hopes to destroy it by degradation. (Coburn, et al. 2007) Unfortunately for the cell, the effector proteins IpaB and IpaH7.8, assist in the bacteria’s ability to bypass degradation developing a mode of survival (Coburn, et al. 2007)

In a research study conducted by Pieper, et al., the proteome of Shigella flexneri was observed within three environments of medium, that being; grown in vitro in broth, grown intracellularly in epithelial cell cytoplasm, and grown extracellularly, cultured with Henle cells. (Pieper, et al. 2013) Mutations impacting metabolic pathways, such as glycolysis and carbon storage regulator protein (CsrA), affect the growth of S. flexneri as well as the production of aromatic amino acids, nucleotides, diaminopimelic acid, and iron transport. (Pieper, et. al 2013)

Although proteins were observed, metabolic pathways were also viewed. Within the intracellular medium, the growth of Shigella flexneri was impacted by an increase in the production of glycogen enzymes, as well as a change in the expression of sugar transporters, and a decrease in the CsrA. (Pieper, et al. 2013) Some of many proteins involved in taking up and metabolizing sugars were also elevated in the intracellular medium. In the process where S.flexneri desire to grow, it will metabolize pyruvate from glycolysis and undergo mixed-acid fermentation (Anaerobic Respiration). (Pieper, et al. 2013) In cells measured via intracellular medium, the presence of proteins involved in mixed-acid fermentation were more abundant than in cells measured via in vitro. (Pieper, et al. 2013) Also compared with in vitro cells, extracellular samples were shown to possess enzymes, AdhE of acetate fermentation, and pflB of pyruvate fermentation, identifying that mixed-acid fermentation could also occur in the outside cellular environment. (Pieper, et al. 2013)

There are many contributors involved in the growth of Shigellae once in the cell. In research conducted by Gore and Payne, Carbon regulators, Cra and CsrA were tested against the virulence factors of S. flexneri to determine how these factors assist in the breakdown of glucose. (Gore and Payne 2010) Through their experiments, both carbon regulators revealed to show a direct relationship with the production and breakdown of glucose. (Gore and Payne 2010) Mutations of the CsrA gene decreased the ability for Shigella flexneri to attach to its host and invade into its cell. Meanwhile, mutations of the Cra gene showed opposite effects, and increased glycolysis. (Gore and Payne 2010) Another protein responsible for the breakdown of glucose is the ATP-dependent 6-phosphofructokinase isozyme 1, (pfkA) protein.

When pfkA is mutated, glycolysis is inhibited causing a reduction in attachment and invasion (Gore and Payne 2010) Mutation in these three genes in reducing cellular invasion showed a correlation to a reduction in the expression of effector proteins, virF and virB, which are needed for invasion. (Gore and Payne 2010)

When glucose is exposed to Shigella flexneri in vitro, fermentation (Anaerobic Respiration) occurs increasing the pH and lowering the acidity of solution. When tested with IMvic methyl red test, this solution containing bacteria turns red, indicating that the pH has reached a certain level of acidity to alter its color. In order for this microorganism to grow in the presence of glucose while in absence of oxygen, it desires three specific pathways. These pathways are known as the Embden-Meyerhof-Parnas (EMP) pathway, pentose-phosphate pathway (PPP) and the Entner-Doudoroff (ED) pathway utilized to breakdown glucose forming pyruvate. (Killackey, et al. 2016)

To understand how S. flexneri utilizes glucose, pathways of glycolysis are measured according to its plaque formation. In the presence of plaque, researchers can determine important factors that cause the growth of bacteria. This can lead to an indication that glucose plays an important role in plaque formation as it signifies an increase in growth. (Waligora, et al. 2014) In a study by Waligora, et al., S. flexneri was tested using a plaque assay to determine how each pathway of glycolysis played a vital role in bacterial growth of Henle cells.

Each pathway was mutated in relation to the bacteria. When the EMP pathway was inhibited, due to mutation of enzymes phosphofructokinase (pfkAB) or pyruvate kinase (pykAF), signs of small plaque formation occurred. (Waligora, et al. 2014) In the ED pathway, eda enzymes are responsible for converting the intermediate 2-keto-3-deoxy-6-phosphogluconic acid (KDPG) into pyruvate. (Waligora, et al. 2014) When eda enzymes were mutated, the conversion of pyruvate could not directly take place, leaving small plaques and toxicity of the KDPG intermediate as a result. (Waligora, et al. 2014) Also along the ED pathway, the edd enzymes are responsible for converting gluconate-6-phosphate into KDPG. (Waligora, et al. 2014)

When both eda and edd enzymes were mutated, normal plaques formed, indicating a possibility that gluconate-6-phosphate of this pathway could convert into pyruvate, through various steps, in absence of these enzymes. During PP pathway alone, the signs of plaque formation were not evident. (Waligora, et al. 2014) This indicated that the EMP and ED pathways are both utilized by the bacteria to invade host cells, grow, and spread into neighboring cells. (Waligora, et al. 2014) To determine if the process to metabolize pyruvate was inhibited, pyruvate was supplemented to the same mutated bacteria to remove all chances that formation of various sized plaques was due to a fault in the breakdown of pyruvate. (Waligora, et al. 2014) The results proved larger plaque formation, indicating that the bacteria’s ability to utilize pyruvate, was not inhibited. (Waligora, et al. 2014) When wild-type strain was supplemented with pyruvate, larger plaques also formed. This data supports evidence that pyruvate is a preferably chosen carbon source for the growth of bacteria. (Waligora, et al. 2014)

Pyruvate carries a lot of importance in the growth of Shigella. Because this microorganism is classified as a facultative anaerobe, S. flexneri can utilize fermentation (Anaerobic Respiration) for ATP as oxygen is not present. This is understood when glycolysis mutated bacteria were not able to breakdown glucose but were able to utilize supplemented pyruvate when pyruvate was present. This indicates that initially, glucose is an important sugar molecule and carbon source molecule for intracellular growth.

In a 2014 study by Kentner, et al. the utilization of glucose upon Shigella-infected HeLa cells and uninfected cells were examined to determine how this bacterium utilizes the cells metabolic pathways, Kentner, et al. used an isotope tracking method by labeling the path of glucose using 13C. (Kentner, et al. 2016) In using NMR spectrometry, the products after following 13C could be determined. (Kentner, et al. 2016) HeLa cells that were not infected by S. flexneri produced lactate and pyruvate from glucose, while inside infected cells neither lactate nor pyruvate were excreted. (Kentner, et al. 2016) Additional data revealed that acetate was utilized from pyruvate at 81%, meaning that, S. flexneri began to use pyruvate from the additional intermediate steps to form acetyl-CoA to be converted into acetate. This is known as acetate fermentation (Anaerobic Respiration). (Kentner, et al. 2016)

This signifies that by the invasion of S.flexneri into infected HeLa cells, S. flexneri reroutes the behavior of glycolysis to form acetate rather than enter into the TCA cycle. From pyruvate, acetyl CoA becomes phosphorylated by enzyme phosphotransacetylase to make acetyl phosphate and acetyl kinase, which is the enzyme to convert acetyl phosphate and ADP to acetate and ATP, leaving acetate, or acetic acid as the product as ATP is used. (Kentner, et al. 2014) To prove the acetate pathway is primely used by S. flexneri within HeLa cells, enzyme encoded genes, phoshphotransacetylase (pta) and acetate kinase (ackA), were mutated. (Kentner, et al. 2014) These enzymes are responsible for the intermediate conversion of pyruvate into acetate.

The results proved that acetate indeed was chosen as a pathway of metabolism for the infected HeLa cells of mutated bacteria. (Kentner, et al. 2014) This data showed no products of acetate, and some product of lactate. This indicates the enzymes for this process are needed for S. flexneri to rapidly proliferate. (Kentner, et al. 2016) Although this pathway yields only one ATP per pyruvate, it is used as a pathway of mixed-acid fermentation (Anaerobic Respiration). Other anaerobic pathways chosen to generate ATP can require lactate, succinate, ethanol, as well as formate. (Pieper, et al. 2013)