Investigating the role of PBP2sal under different conditions in S. Typhimurium

Updated: May 14


Abstract


Penicillin binding proteins (PBPs) are enzymes involved in the bacterial growth and biosynthesis of the peptidoglycan cell wall. PBP2 and PBP2sal are two of many enzymes included in the elongasome protein complex that synthesizes the peptidoglycan found in S. Typhimurium.



The aim of this investigation is to potentially discover particular conditions in which PBP2 switches with PBP2sal. Additionally, if a switch would occur within a macrophage where Salmonellamultiplies, the particular penicillin binding protein can be targeted to treat patients with Salmonellosis. The study was performed by taking two strains, a wildtype and a mutant with omitted PBP2sal and compare them under different conditions including; Drop dilution method on copper chloride plates, growth curve in the bio screen, disc diffusion of antibiotics and detergent sensitivity. The role of PBP2sal in terms of its switching with PBP2, and its importance in surviving in these conditions, was investigated.


The copper chloride sensitivity demonstrated no clear visual difference between the wildtype and the mutant, which indicates PBP2sal is not involved, and thus, does not have an impact on the susceptibility to CuCl2. Measuring the growth curve in the bio screen performed, showed similar growth curves for the wild type and the mutant with overall no visual phenotype. Antibiotic sensitivity comparison did also not show a clear difference. However, once subjected to detergent the wild type survived better than the mutant.


Introduction

Salmonella enterica, is a rod-shaped gram-negative bacterium that survives inside the environment of a macrophage. There are two types of Salmonellosis, typhoidal and non-typhoidal. The typhoidal has the ability to survive within our immune systems and is what causes the turn towards antibiotics as a treatment method. It grows within the macrophages, being the hallmark of the typhoidal disease leading to typhoid fever (7). Salmonella causing Typhoidal fever, increases the risk of fatality up to 20% without a treatment (8). The non-typhoidal is normally what causes diarrhea and is treated through re-establishing the fluid balance within the body by simply drinking more water (9).


We areusing S. Typhimurium as a model-organism to study human typhoidal fever, since it is a version of Salmonella that causes typhoidal fever in mice.Globally, there are 86 million cases of salmonellosis with an approximate 200 000 deaths due to typhoid fever (1). Thus, antibiotics is used widely to treat Typhoidal fever. However, the usage of antibiotics is becoming less effective as the spread of antibiotics increases by the day. By 2050, an estimate of 10 000 000 deaths will be attributed to antimicrobial resistance (AMR) solely due to the spread of antibiotic resistance (1&2).The cell wall strengthens and maintains the shape of the bacterium to keep it from osmotic lysis (3), which make it essential for the survival of a bacterium and thus a common target for antibiotics.

A bacterium grows and divides through the process of binary fission (6) forming two identical daughter cells (10). The two ball-shaped daughter cells then undergo the process of elongating due to the elongasome, which is a protein complex that elongates the bacteria from the more ball-shaped morphology to long rods (10).The elongasome synthesizes the bacteria’s peptidoglycan where many enzymes including PBP2 and PBP2sal are involved in and switch between one another depending on the conditions of the bacteria (11).

Thus, a gene modification and omission of a particular penicillin binding protein, such as PBP2sal, could help to identify the protein’s functions in the bacteria and to learn more of the peptidoglycan synthesis during S. Typhimuriums elongation. The understanding of the proteins involved in the elongation process of a bacterium helps to identify new prevention methods and treatments.


Materials & Methods

This work involves a mutant of S. Typhimurium where the gene for PBP2sal has been removed. PBP2sal is yet to be officially identified, but the knowledge of the protein and its function comes from unpublished work by the García-Del Portillo lab and personal communication with Sonía Castanheira who is leading the project. This lab has previously identified PBP3sal (4). To further characterize PBP2sal, it was omitted by gene mutation and replaced by a gene resulting in kanamycin resistance prior to the start of the experiments. The new mutated culture got the name Δpbp2sal::KanR. In this following way, we aimed to find a phenotype. All the following experiments were performed three times to be proven reliable.


Drop-dilution method on Copper Chloride plates. To investigate whether PBP2sal plays a role in copper chloride sensitivity that accumulates within a macrophage, the wild type and mutant strains ofSalmonella were grown overnight in 2ml LB (containing 10mg/ml NaCl) and supplemented with different copper chloride concentrated plates; 0mM, 2mM, 3mM and 4mM copper chloride plates.


Growth curve in the Bio screen method. Since S. Typhimurium is an intracellular pathogen, it survives and multiplies within macrophages. Thus, a comparison between the strains in environments that mimick the intraphagosomal environment of a macrophage was performed. The bacteria were grown overnight in 2ml LB (containing 10mg/ml NaCl), and 1:25 diluted with PBS before they were placed into separate LB (nutrient with NaCl) , TY (nutrient without NaCl) and MM (minimal media with pH 5.8) medias inside the bio screen within honeycomb plates for 24h.


Disc diffusion method (Antibiotics). The two Strains’ antibiotic sensitivity was compared by letting the bacteria grow overnight in 2ml LB. The disks, 6mm in diameter, were infused with 5μl of antibiotic. Mainly antibiotics that target the cell wall were studied, but also ones that target inter alia RNA polymerase, ribosomes and DNA gyrase.

Antibiotics used were:

  • 5μl of Tetracycline from a of 10 mg/ml stock

  • 5μl of Vancomycin from a of 20 mg/ml stock

  • 5μl of Ampicillin from a of 5 mg/ml stock

  • 5μl Rifampicin from a 10 mg/ml stock

  • 5μl of G-Penicillin from a 10 mg/ml stock

  • 5μl Polymyxin B from a 10 mg/ml stock

  • 5μl Novobiocin from a 10 mg/ml stock

The next day, the diameter of the inhibitory rings were measured and written down in millimeters (mm).


Detergent sensitivity test. Detergents are used every day for cleaning and avoiding infectious diseases. Thus, the survival of the two strains were compared to observe the change in the bacteria’s interaction with the detergent. The bacteria were grown overnight in 2ml LB. Subsequently each strain was mixed with MiliQ water or 0.5% DOC in MiliQ. After a 30-minute incubation, 100μl of the diluted bacteria were poured onto LB plates and the colonies were counted the next day after incubation at 37°C overnight. Grown colonies were counted the next day and a mean was calculated for accuracy.


Results


Copper chloride sensitivity. Fig.1 presents six diluted drop series compared between the wild type and the mutant in regard to their visible growth. Both bacterial strains interact similarly in the TY plate, as well as the plates with copper chloride. From the control to the 2mM concentration, no clear visual difference is seen. However, from 3mM to 4mM concentration, higher sensitivity to copper chloride is demonstrated.

Growth curve. The mutant presented similar growth in LB media as the wild type (fig.2A). Only once at log phase is when the wild type grows steeper than the mutant, but the difference is not evident and they both eventually even out on the same value of approx. 1.4 OD. In TY media (fig.2B), the wild type gains a steeper growth curve and evens out above the mutant, which indicates that it simply grows better but only to a minimal extent. However, in minimal media of pH 5.8 (fig.2C), the mutant seems to grow much steeper after 75 minutes. With the result so far, the difference is not clear enough to indicate a phenotype.

Disc diffusion (Antibiotic sensitivity). The mutant and the wild types sensitivity towards eight different antibiotics were tested by measuring the inhibitory zone caused by the discs. The cell wall targeting antibiotics, Penicillin G and Ampicillin presented (fig.3A) no significant difference in inhibitory zone between the mutant and the wild type. Vancomycin caused an inhibitory zone of 5mm in one of the three trials for the wild type. However, a high standard deviation was obtained for that value, indicating the result is uncertain and not reliable.


Ribosome targeting enzymes such as Chloramphenicol and Tetracyclin did also not show a clear phenotypical difference with only 0.3mm and 0.6mm difference between the inhibitory zones of the wild type and the mutant respectively. Rifampicin targeting RNA Polymerase and Polymyxin targeting outer membrane showed similar values between the two strains as well. The Novobiocin (fig.3B) targeting DNA gyrase caused a cloudy inhibitory zone that was not measurable. An example (fig.3C) is presented of inhibitory zones caused by the wild type with all eight discs diffused with antibiotic on the plate.

Detergent sensitivity. In this study, Deoxycholate was used as a detergent to compare the % survival of the wild type and the mutant. A mean % survival was obtained and plot into a bar graph (fig.4). The wild type has a higher % survival than the mutant. The plates also presented a clear phenotype with more colonies present on the wild type plate compared to the mutant.

Discussion

With approximately 200,000 deaths annually worldwide, Salmonella is beginning to become a larger global problem with the simultaneous spread of antibiotic resistance (1&5). For a bacterium to grow, it needs to multiply and elongate to maintain its shape and stability. In order to elongate, elongasome protein complexes are needed which includes many enzymes such as PBP2/PBP2sal that help to build more peptidoglycan (11). The aim of this investigation is to find conditions in which PBP2sal plays a role in the elongation and in turn the survival of the bacteria as well as when it switches position with PBP2. Thus, this area of research could potentially find a new prevention method for Salmonellosis which only targets PBP2sal.


Copper chloride sensitivity. This study aimed to investigate whether PBP2sal plays a role in the survival of the S. Typhimurium in a copper environment. Since copper accumulates within a macrophage (12&13), this study could potentially broaden the understanding of the interaction between S. Typhimurium inside a macrophage. The results (fig.1) presented no clear phenotype. From the control plates to 4mM copper chloride, the increase in sensitivity to copper chloride was equal between the wild type and the mutant. This indicates that PBP2sal does not play a role in copper chloride sensitivity.


Growth curve in the bio screen. It was established in which the wild type and the mutant were put into different medias and their growth curves were measured over a day. Since PBP2/PBP2sal are involved in the biosynthesis of peptidoglycan, they need nutrients for the process to be completed. Thus, enclosing them in a system with only LB/TY/MM will show where they grow better and as well as in which condition each switch. Three different mean growth curves (fig.2) were obtained from three trials. The LB and TY did not show a visible difference, indicating that PBP2sal does not play a role in growth within such environments. However, MM began presenting a difference with the wild type growing more at the end of the 24h bio screen incubation. This could suggest that with further measuring of the growth curve, the mutant might actually have more growth, bypassing the wild type after a day time. As the theory suggests, in certain conditions PBP2/PBP2sal switch and so far, no clear difference has been observed in MM, implying PBP2sal does not play a role in the growth inside a macrophage. But proves to have potential for further investigating, since a difference was observed around the end of the experiment in MM.


Antibiotic sensitivity. Sensitivity towards antibiotics is important since antibiotic resistance is spreading rapidly. A table was made to collect all the values +/- standard deviation. The wild type and the mutant did not show a clear difference in sensitivity towards the eight antibiotics studied. Novobiocin’s cloudy inhibitory zone was intriguing however (fig.3C) it was not possible to measure the diameter accurately. A possible assumption could be that the gene that replaced PBP2sal in the strain, called NeoR, might have affected the result and possibly caused an inhibitory zone specific to the Kanamycin resistance. Thus, it could be possible that particularly Novobiocin would interact better or worse depending on what resistance would replace PBP2sal. It was expected that the antibiotics such as Penicillin G, Ampicillin and Vancomycin would present a switch between PBP2/PBP2sal since these antibiotics target the cell wall and so it is believed that a switch could occur to resist the antibiotic. If a switch between PBP2/PBP2sal would have been established, Penicillin G would then not be able to bind and inhibit PBP2sal, since it has a different shape of binding sight. However, the values did not show a clear difference as seen (fig.3A).


Detergent sensitivity. Detergent is used to keep bacteria and dirt away, making it reseasonable to investigate. This study presented a clear phenotype in the two strains (fig.4) in the bar graph, where the wild type survived remarkably better than the mutant. Since the detergent disrupts the outer membrane, there is a higher chance of the bacteria undergoing osmotic lysis. The results indicate PBP2sal plays a role in maintaining the stability in the cell wall from getting broken down once the outer membrane gets destabilized.


Concluding remarks

Conclusively, the results so far have proven that PBP2sal plays an important role in the survival of the bacteria in detergent. However, not essential in antibiotic sensitivity and copper sensitivity. Except for MM growth curve (fig.2C) where there has been seen a potential for a phenotype and better survival for the mutant than the wild type indicating a better growth without PBP2sal. Thereby further investigation with MM as well as detergent sensitivity could lead to potentially a new prevention method to avoid further spread of antibiotic resistance.


Acknowledgement

Many thank you-s to say, so few words adequate enough; I thank Karolinska Institute Biolclinicum, department of Molecular tumor and cellular biology (MTC) for granting me the opportunity to work and do research in a field I have immense passion for. Very grateful for all the help and cooperation our supervisor, Kim Vestö, provided during the past six weeks. Special thanks to Nicole Brindell, my lab partner for being a motivating and fun companion. I also want to acknowledge Gerald McInerney as well as Nicole Lazlo, Axel Schröder and Kira Taivassalo for arranging excellent and memorable social and educational events. It brought the group closer together and made me more interested and enthusiastic in regard to science and medicine.


References

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