Bacillus anthracis or as it is more commonly known as anthrax is a bacteria that can be quite harmful to mammals such as humans. In fact it has been used as a biological weapon in the Amerithrax case. Part of what makes B. anthracis so dangerous is that it exists mostly as spores, which then germinate once within their host organism. Spores are a way for cells to exist where they can quickly create new cells, and that are protected from harm. B. anthracis can infect humans through 3 different methods, through a cut on your skin, by ingestion of undercooked meat of an infected animal or by being inhaled. Once the spores have infiltrated another organism they are able to germinate which essentially means that they are able to begin to grow and divide quickly. Once infected by B. anthracis the consequences can be deadly. For this reason many scientists have been interested in studying the factors that impact its virulence, or its ability to cause damage to its host. One such study recently was conducted by Neha Dhasmana, looked at how the length of the bacterial chains is controlled. Previous studies have shown that the ability of B. anthracis to form long chains of multiple cells increases virulence. This study is investigating the role that the prkC gene plays in determining chain length and therefore the virulence.
In order to study this role of the prkC gene on chain length they used a mutant version of the B. anthracis that did not have the prkC gene, known as BAS ΔprkC and the wild type that did have the prkC gene which acted as the control, called BAS WT. They also used a third type which they called BAS ΔprkC::prkC, which means that they took the BAS ΔprkC strain that did not have the prkC gene and inserted it into the genome, essentially giving it back that gene. They then cultured the three different strains and used phase contrast microscopy and scanning electron microscopy to view the cells after 9 hours of incubation.
Figure 1) A) Cultures of the BAS WT and BAS ΔprkC strains B) phase contrast microscopy and C) scanning electron microscopy of the BAS WT, BAS ΔprkC and BAS ΔprkC::prkC strains showing the chain lengths.
From parts B and C of figure 1 you can clearly see that the strains that have functioning prkC genes, the BAS WT and the BAS ΔprkC::prkC, are forming longer chains than the strain that does not have the prkC gene. They also examined the chain lengths quantitatively using ImageJ software to show the difference in the chain length between the BAS ΔprkC and the BAS WT. They examined the chain length every 2 hours starting with the 2nd hour until 30 hours after the beginning of the growth period. They found that the BAS WT strain has significantly longer chain lengths compared to the BAS ΔprkC strain for every time point they measured except for the last 2.
Once they saw these results they were interested in examining why the removal of the prkC gene resulted in shorter chain lengths. Looking at previous work that has been done they found that the disruption of sap, which is a protein that along with EA1 forms on the cell walls during the phase when the population is undergoing exponential growth, causes chain lengths to get longer. This is because Sap and EA1 work together to disrupt a protein known as BslO, which helps the daughter cells to divide from each other during cell division. Without the presence of Sap inhibiting BslO it is able to properly break down the cell wall. To determine the expression level of these different proteins within the cells they performed quantitative western blotting at different amounts of time that the cells had been growing for. They found that in the BAS ΔprkC strain, Sap was upregulated for the majority of the time points they collected data for. These results are in line with previous studies that found that the inhibition of Sap resulted in longer chain lengths. They also found that BslO was upregulated in the BAS ΔprkC strain. They argue that the increase in Sap causes the restriction of BslO to the septal region where it can cause the separation of the daughter cells and that increased amounts of BslO enhance this effect which is why shorter chain lengths are observed.
They also noticed that the growth curves of the 3 strains were different (Figure 2A). Growth curve refers to the way that bacteria move through different phases where the overall population size is undergoing different changes. When comparing the 2 growth curves they found that the BAS ΔprkC strain had a lower replication rate in the logarithmic phase, which can not be explained by the differences in chain lengths (Figure 2A). Looking at figure 2A you can also see that the BAS WT has a higher value of OD 600 nm for all of the time points. OD 600 nm is a way to measure the concentration of cells, or essentially the number of cells based on the amount of light reflected, where a higher value indicates more cells. From this you can see that the population with the prkC gene is able to produce more cells. They decided to use transmission electron microscopy to determine what was causing the differences in growth curves. They found that the BAS ΔprkC strain had decreased cell wall and septal thickness (Figure 2 B and C). These findings are important because they show that the prkC gene also impacts the individual cell morphology, not just their ability to form chains.
Figure 2) A) showing the growth curves of the BAS WT and BAS ΔprkC strains, B) transmission electron micrographs showing the difference in cell was thickness between the 2 strains, C) Bar graph showing the difference in cell wall thickness between the strains with **** indicating that the difference is highly significant.
These findings are significant because they demonstrate that the deletion of the prkC gene causes the shortening of chain lengths in Bacillus anthracis, and that this is because of changes in the amount of the Sap and BslO proteins that are important in cell division, and that there are differences in the cell morphology based on the deletion of the prkC gene. This is important because the length of chains that B. anthracis is able to form plays an important role in its virulence, which means that understanding the way that its chaining is regulated can give us insight into how this deadly pathogen can be changed into less lethal forms. While this study did show that the prkC gene does play a role in chain length, an interesting next step would be to determine if the decreased thickness of the cell wall and septum, found when this gene is removed, plays a role in the mechanism of decreasing virulence. Furthermore, it would be interesting to see results of virulence of the BAS ΔprkC and BAS WT strains in a mouse model rather than just relying on previous studies to claim that the prkC gene impacts virulence.
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