Cytopathic Effects as a Result of Infection of E-coli

Analysis of cytopathic effects as a result of infection of Escherichia coli by T4 bacteriophage using spectrophotometry and plaque assay
Abstract
T4 bacteriophage is a double stranded DNA virus that infects Escherichia coli and is an important tool in research and study of genetics. It contains about 168,800 base pairs of DNA. It has an icosahedral capsid as its head that contains its nucleic acid and has a tail structure formed of tail fibres, sheath, base plates and coat. These different structures of T4-phage play important roles when invading the host cell (in this case E.coli). T4-bacteriophage infects the host cell by lytic pathway where the host cells are used to produce virus components and it results in the lysis of host cell releasing the virus components to the external membrane. This experiment plans to examine the cytopathic effects of the T4-phage on E.coli using a spectrophotometer. Also, plaque assay is carried out in order to determine the original concentration of phage in the solution. The spectrophotometer reading increased for both control and T4-infected cultures as the time of post infection increased. The highest reading was 0.192 for control at 120 min pi.

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Introduction
Viruses are small infectious agents and are about 10-30nm in size. Strelkauskas, et al (2010) describes viruses as “obligate intracellular parasites” which means they need a host cell in order to survive and replicate. The viruses are classified depending on their morphology, chemical composition and mode of replication. They are divided into two groups based on the nucleic acid they contain: DNA or RNA. They are sub-divided into further groups according to their symmetry of capsid where the genetic material is enclosed within an envelope or without an envelope (naked). Also, the configuration of the nucleic acid (single-stranded or double stranded and linear or circular) play a role in the classification of viruses (Gelderblom, 1996). A fully assembled infectious virus is known as a virion and its structure is based on two important things; the ability to survive in harsh conditions until it can infect the host cell and to be able to shed the protective barrier in order to enter a host cell.
Bacteriophages are viruses that infect bacterial cells and were first observed in 1915 and 1917 by Twort and d’Herelle. T4 bacteriophage is a member of T-even phages and infects Escherichia coli (E.coli) bacteria. It is considered as one of the complex viruses because of its genome which consist of 274 open reading frames and 40 of these reading frames encodes for structural proteins (Leiman, et al, 2003). T4- bacteriophage has been extensively studied since 1940s and plays a major role in advancement of modern genetics and molecular biology. Some of the early essential ideas of genetics including the basis of genetics code, mRNA, ribosome and the codon have came from studies using T4-bacteriophages (Miller et al, 2003). T4- bacteriophage contains double stranded DNA as its nucleic acid which is enclosed within a protective coat known as capsid. Its capsid is icosahedral in shape (shown in figure 1). It also has tail part which is made up of coat, sheath, tail fibres and base plates. The tail part is useful when recognising a host cell followed by penetration to the host cell thus causing infection.
Bacteriophages have two types of replication strategies: lytic cycle and lysogenic cycle (Baker, et al, 2011). Lysogenic infection in E.coli is exemplified by bacteriophage lambda where the viral DNA replication takes place without destroying the bacterial cell. Here, the virion infects the host cell and instead of triggering more virus reproduction, it recombines with the host genome causing no damage to the cell which is known as prophage. These viruses can remain dormant within the bacterial cell for years and only cause infection under certain circumstances. T4-bacteriophage is a virulent virus that causes lytic infection to E.coli. The first step in this cycle is adsorption where the virus recognises the cell receptors on the surface of the host cell and attaches itself to the host using its tail fibres. Once attached to the host cell, the virus must penetrate through the cell membrane of E.coli. The base plate of the virus comes in contact with the cell wall of the host cell causing conformational change in structure of the virus. This allows the sheath to contract and the core is pushed through the wall into the cell membrane where the viral DNA is injected into the host cell (Todar, 2008). DNA viruses have the same genome configuration as the host cell; hence the replication process used in host cell can be used for viral replication. T4- bacteriophage transcribes nucleases that break down the bacterial DNA which is used to produce more viral genome. The newly made viral nucleic acid and structural proteins are assembled together to form virulent viruses. The lysozymes produced by the bacteriophages as a late viral protein lyses the cell wall of the E.coli causing it to burst open hence releasing virulent virus which have to potential to infect other cells. The lifecycle of a T4-bacteriophage takes about 25-35 minutes to finish (Mayer, 2010).

Figure 1: The left picture shows the electron microscopic image of T4- bacteriophage and the right shows the model of the T4-bacteriophage.
The head of the virus contains a capsid formed by icosahedral structure that holds the phage’s double stranded DNA. The collar connects the head and the tail structures. The tail consists of core, sheath, base plate and tail fibres which are involved in recognising host cells and then attaching phage to specific receptors on the host surfaces (Todar, 2008).
The virus-infected bacterial cell generally shows some changes in their phenotype such as altered shape, detachment from the substrate, cell lysis, membrane fusion, membrane permeability, inclusion bodies and apoptosis. These changes are known as the cytopathic effects of a virus (Cann, 2005). In this experiment, the lysis of E.coli is examined using a spectrophotometer which determines the absorbance of the cultures to indicate the growth of the bacterial cell within the flasks. Also, it is important for clinical and research virologists to know the number of infectious virus particles in a sample which is known as the titre. The plaque assay gives the most accurate results when determining the titre of the phage. T4- bacteriophages can be grown on bacterial lawn. Infected E.coli cells are lysed so, they will form visible plaques on the agar plate which are counted to calculate the titre of the virus.
The experiment aims to firstly infect the bacterial cells with T4-bacteriophage in order to indirectly observe the cytopathic effects of virus infection by monitoring changes in bacterial cell growth, compared to an uninfected (control) culture. An absorbance reading is taken using a spectrophotometry as the cells in the culture grow and divide making the culture opaque and thus increasing the absorbance of the culture. Also, the experiment plans to assess the progress of T4-phage production during the infection of E.coli by taking samples of extracellular virus at regular time intervals post infection. Furthermore, the virus from each samples were quantified using plaque assays to demonstrate the progress of virus amplification that occurred during the process of infection in the bacterial cells.
Materials and methods
E.coli was used as the host cell of T4-bacteriophages in the following experiments.
Measuring the changing cell density of cultures using a spectrophotometer: Two cultures were made; the control culture (C) consists of mixture of LB broth and E.coli whereas the T4- infected culture (T) was made by mixing LB broth, E.coli and 100μl of bacteriophage. The immediate absorbance readings at 0 seconds were measured in the spectrophotometer at wavelength 600nm and LB broth solution was used as a blank. The flasks containing the two cultures were placed in the orbital shaker covered with foil lids throughout the readings of absorbance. During the 1 hour incubation, 10 µl of the T4-infected sample was placed in a C-chip Haemocytometer and the bacterial cells were counted under the microscope which was used to calculate the multiplicity of infection (MOI). After 1 hour incubation, the absorbance readings were taken at 15 minutes interval until the cultures had been monitored for at least 2 hours.
Harvesting virus sample: At 40 min post infection, 1 ml of each of the cultures were taken out and placed into sterile microfuge tube. The tubes were centrifuged at 6,500 rpm for 5 min and 750 µl of supernatant was removed from the T4-infected tube. It was then incubated over night and another sample was collected after 23.30 hours post infection.
Preparation for plaque assay: A serial dilution of 10-7, 10-8 and 10-9 were made from the overnight T4-infected sample and dilution of 10-4, 10-5 and 10-6 were made from the 40 min p.i. T4-infected sample by using sterile M9 medium. 1 ml of E.coli culture was transferred to each of the two bottles containing 0.1 ml of the each diluted sample. The bottles were placed in water bath at 37°C for 15 minutes for bacterial infections to begin. One small bottle of molten soft agar was taken from 42°C bath and poured into labelled agar plates and evenly mixed with the bottle containing E.coli and diluted sample. This process was followed for all 6 agar plates. Then, the plates were incubated at 37°C overnight after they were set completely.
Plaque counts: After the incubation period, the agar plates were examined and the number of plaques formed on each plate were counted and recorded.
Results
The absorbance readings taken from the spectrophotometer at 600 nm of both the control and the T4-infected cultures is shown in table 1. At 0 min pi, both the cultures had similar readings (0.05 and 0.054 for C and T respectively) as they were just made. After one hour incubation period (60 min pi), there was increase in the absorbance for both of the cultures. However control had higher absorbance reading than T4-infected (0.106 and 0.064 respectively). Overall, the absorbance readings of both the cultures increases with the time except for 90 min pi in T4-infected where there is a decrease in absorbance reading by 0.028. The readings recorded for control is higher than T4-infected at each time period and the highest reading is at 120 min pi in the control culture (0.192).

Absorbance (600 nm)

Time (min pi)

Control

T4-infected

0.057

0.054

0.106

0.064

0.116

0.075

0.147

0.047

105

0.151

0.058

120

0.192

0.153

Table 1: absorbance reading of the cell cultures at 600nm

Figure 2: Absorbance reading of control and T4-infected cultures against time
Calculating the Multiplicity of infection (MOI)- the number of virions (pfu) per cell
T4-titre = 2.74 x 1010 pfu/ml
Cell count in T4-infected using C-chip Haemocytometer = 2808 cells per grid.
Number of cells per mL = 2808 x 10,000 = 2.808×107 cells/mL
Number of cells per flask = 2.808×107 x 23.1 (total volume) = 6.48 x 108 cells/flask
MOI = (0.1x titre)/cells per flask = 2.74×109/ 6.48×108 = 4.22 pfu/cell.

Time (min pi)

Dilution factor

Average plaque count (from both plates)

Dilution factor

Average plaque count (from both plates)

T4 titre (pfu/ml)

Log 10 titre

10-6

266

–

2.66 x 109

9.425

10-5

162

10-5

328

2.45 x108

8.389

–

10-6

184

1.84 x 109

9.265

–

10-6

284.5

2.845x 109

9.454

10-5

80.5

–

8.05 x 107

7.906

100

10-5

169.5

10-5

127.5

1.46 x 108

8.164

120

10-5

122

–

1.22 x 108

8.806

Table 2: class data of the plaque counts of T4-infected E.coli
The growth curve of the T4-infected E.coli is shown in figure 3. A growth curve of a virus normally shows the eclipse period, latent period, rise period and the burst size. At 0 min pi, there is high number of extracellular cells (2.66 x 109 pfu/ml) as the virus has not been taken up by the E.coli cells. As the time increase to 20 min pi, the curve levels fell down due to penetration of viruses into the cell (as shown in figure 3). This phase is known as eclipse phase where the input virus begins to uncoat so, no infectious virus is detected. Latent period covers the period from the time of disappearance of infecting virus (eclipse phase) to re-appearance of it in E.coli. The rise phase is when there is a gradual increase in T4-phage titre as viral replication occurs and new cells are formed. Then the virus T4-tire levels off towards the end as cell lysis take place releasing the newly formed virus particles. However, the rise phase (shown is figure 3) doesn’t not rise gradually. The T4-titre rises until 60 min pi (2.845x 109) and instead of levelling off, there is a huge drop in the virus titre (8.05 x 107) at 80 min pi.
In addition, the T4-virus sampled after overnight incubation (23.30 hours post infection), the dilution factors 10-7, 10-8 and 10-9 resulted in plaque counts of 12, 50 and 59 respectively. However, the plaques were only observed in one plate of each dilution and in the other plates no plaque were observed (Table 3 in appendix).
Discussion
Albrecht, et al (1996) states Infection caused by cytocidal viruses is normally associated with alterations in cell morphology, cell physiology and sequential biosynthetic events. The changes in cell morphology can sometimes be detectable which is known as cytopathic effects and they can be rounding of infected cells, formation of syncytia, and appearance of nuclear/cytoplasmic inclusion bodies. T4- bacteriophage usually causes death of host cell after replication causing cytopathic effects. A spectrophotometer is used to detect these cytopathic effects of T4-phage in E.coli. The spectrophotometer can only detect opacity due to the presence of bacterial cell and cannot detect virus as they are very small. So, as there is growth in cells in the culture, they become more opaque increasing the absorbance reading of the culture. The T4-infected culture didn’t have as much growth as the control as its absorbance readings are lower in all of the time period (as shown in figure 2).
T4-phages can only replicate within the host cell therefore it must be grown in a bacterial cell. As shown in figure 3, at time 0 min pi, the amount of virus titre is the highest (2.66 x 109 pfu/ml). This is because the process has just begun as the T4-phage injects its ds DNA into the host cell after cell contact which is known as adsorption. Then, the virus titre drops down to 2.45 x108 pfu/ml at 20 min pi as it is attached to the host cell by receptor binding in order to penetrate into the host cell. At 40 min pi, the virus titre level begins to rise as the virus start to replicate within the host cell. The virus titre is suppose to increase till a certain time period and will gradually level off as the virus cell replication increases and after the host cell is filled with viral components, cell lysis occur releasing the newly formed virus phages to infect the surrounding bacteria. However, as shown in figure 3 this does not happen. The virus titre rises till post infection 60 min but there is a huge drop in virus titre after (8.05 x 107pfu/ml).
There are errors in some of the results of the experiment like the decrease in absorbance reading of T4-infected at 90 min pi and the huge drop in virus titre at 60 min pi. Also, an overnight sample was taken from T4-infected culture at 23.30 hours post infection and plaque assay was carried out with dilution factors of 10-7, 10-8 and 10-9. There is no plaque formation in one of the two plates in each dilution (shown in table 3 in appendix). These errors in results can be due to various factors such as contamination in the samples due to poor sterilise technique, insufficient mixing and pipetting errors when making serial dilutions.
Also, in plaque assay, the morphology of plaque depends on various environmental factors like the phage, the host and the growth conditions (Maloy, 2002). The size of the plaque is proportional to the efficiency of adsorption, the length of latent phase and the burst size. Also, the phages are affected by various physical and chemical factors like temperature, acidity, ions, etc. The other error can be made when counting the plaques in plaque assay. The counting is subjective to the counter as different counter would get different results for the same plate. The colonies were concentrated so it would be hard to differentiate between single colonies as they grow very close together. For more accurate counting of the plaque, the plates with 10-200 plaques would be chosen as there were three dilution factors of plates to choose from and anything with >200 plaque counts were not counted. Also, the experiments could be repeated more than once and for longer period of time when monitoring T4-infected E.coli so, average could be taken out which would give more accurate and reliable results.
In conclusion, T4-bacteriophages are virulent infecting E.coli cells by lytic pathway and this can be measured by examining the cytopathic effects using spectrophotometer. Also, plaque assay of T4-infected E.coli helps to determine the virus titre – concentration of virus in a sample. T4- bacteriophages are a model organism to study and are involved in advancement of modern genetics and molecular biology. Also, they could be used to treat bacterial diseases as the theory states that phages can selectively kill the host cell without damaging the human cells. Some potential applications that are being considered include adding phage suspension to grafts in order to control skin infections and intravenous fluids for blood infections (Talaro, et al, 2007).
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Appendix