Investigating The Impact Of PBP3SAL On Growth And Survival In S. Typhimurium

Updated: Jan 24

Written by Nicole Brindell*1,2 under the supervision of Kim Vestö *2

1. Karolinska Institute Summer Research School With Biomedical Orientation 2019 2. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; Mikael Rehn Group


Abstract


Typhoid fever is a serious disease caused by typhoidal Salmonella which is estimated to affect up to 27 million people annually with 460 000 resulting in death. Typhoidal Salmonella are intracellular pathogens capable of surviving and multiplying inside immune cells called macrophages where they systematically reproduce and cause infection. Thus, cell-division inside macrophages is crucial for the pathogenesis of Salmonella. The cell-division requires the synthesis of new cell wall by Penicillin Binding Protein 3, PBP3, along with a paralog named PBP3SAL. Currently, little is known about the importance of PBP3SAL and for this reason, in this study, we investigate the impact of PBP3sal on growth and survival in Salmonella Enterica serovar Typhimurium. This was done by comparing the survival and growth of Salmonella Enterica serovar Typhimurium with a mutant missing the gene for PBP3SAL in vitro through different environments modelling aspects of the pathogenesis. The investigated environments were acidic, containing copper chloride or hydrogen peroxide. Here we report that the presence of PBP3SAL could be beneficial for Salmonella in an environment containing hydrogen peroxide, of no importance in an acidic (pH 5.8) environment, as well as disadvantageous in an environment containing copper ions. We also present reason to further investigate the impact of PBP3SAL in the pathogenesis of typhoidal Salmonella to establish its eligibility as a target for new antibiotic medicine against typhoid fever.


Introduction


Salmonella is a diverse genus of rod-shaped bacteria which spreads through contaminated food and water. The estimated annual global burden of Salmonella related sicknesses is thought to be up to 182 million cases each year with 1.3 million deaths (1). There are two types of Salmonella causing different diseases, typhoidal and non-typhoidal. Typhoidal Salmonella causes diseases such as typhoid fever while non-typhoidal Salmonella causes diarrhoeal diseases (2). Each year, typhoidal Salmonella itself is estimated to cause a 27 million morbidity with 460 000 deaths (1). This study focuses on Salmonella enterica serovar Typhimurium, a model-organism for typhoid fever in mice. Hence, the pathogenesis description will be for typhoidal Salmonella.


Typhoidal Salmonella are intracellular pathogens, meaning that they despite immune responses manage to survive and reproduce inside the host's cells. For the pathogenesis of Salmonella, it means that the bacteria survives inside phagosomal immune cells called macrophages, successfully inhibiting its antimicrobial responses (3). In order for the pathogenesis to continue, the bacteria have to divide and multiply inside the macrophages (4-5). When dividing, the bacteria need new cell wall which is produced by PBP3, a cell division-specific cell wall synthase which is a component of the divisome complex required for the separation of daughter cells (6-7). Recently a paralog to PBP3 has been found named PBP3SAL which is absent in non-pathogenic bacteria and thought to be a specialized cell division protein with activity restricted to the acidic phagosomal environment (8). It is believed that the dynamic between PBP3 and PBP3SAL switches in the divisome depending on the environment such as acidic ones where PBP3SAL is thought to replace PBP3 to a further extent (8). However, the full mechanism of PBP3SAL is currently unknown.


This study focuses on investigating the impact of PBP3SAL on growth and survival in Salmonella enterica serovar Typhimurium in order to deepen the understanding of PBP3sal and its importance for the in vitro aspects of pathogenesis of Salmonella. This was done by comparing S. Typhimurium with a mutant lacking the gene for PBP3SAL in regards to growth and survival in environments containing copper chloride, hydrogen peroxide or acidic pH. If PBP3SAL is shown to be crucial for the pathogenesis of Salmonella, it could be a possible target for new antibiotic medicine against typhoid fever.

Materials & Methods


Bacteria

In this study, we have observed Salmonella enterica serovar Typhimurium and a mutant called Δpbp3sal :: kanR, where the gene for PBP3SAL has been erased. The mutant was already available in the laboratory when this study began.


Drop-dilution method on copper chloride & hydrogen peroxide plates

Since several studies suggest that macrophages produce reactive oxygen species like H2O2 (9-10) and copper ions (11-12) to restrict the growth of bacteria, the importance of PBP3SAL for the Salmonella when encountering these stresses was observed by a drop-dilution method to assay for sensitivity towards these parameters.

Both strains of bacteria were grown overnight in 2ml Luria Broth (LB) shaking at 220rpm and 37°C. The next day, the cultures were diluted 1:100 in 2ml TY (LB without NaCl) medium and grown for 2h, in 37°C and 220rpm. Then, 106CFU/ml bacterial cultures in PBS were prepared for all involved strains. Afterwards, a 6 time 1⁄2 dilution series was performed with the strains. Then, 5μl droplets from the strains were pipetted in a row onto the test plates and incubated overnight at 37°C. The plates were TY agar and contained CuCl2 molarities of 0mM, 2mM, 3mM, 4mM, and 5mM as well as hydrogen peroxide molarities of 0mM, 0.3mM, 0.4mM, 0.5mM and 0.6mM.


Copper chloride microscopy

Considering that PBP3 synthesizes new cell wall and hence the shape of Salmonella (6-7), in addition to that macrophages use copper ions to kill bacteria (11-12), it was of interest to investigate the impact of CuCl2 on the morphology of Salmonella through microscopy.

All strains of bacteria were grown overnight in 2ml LB shaking at 220rpm and 37°C. The following day, the cultures were diluted 1:100 in 2ml TY medium (control) and 2ml of TY in the presence of 1mM CuCl2 for two hours at 37°C, shaking at 200rpm. Then, the samples were centrifuged and washed twice with PBS. Later, the solutions were dyed with Nile red dye, which binds to the lipids of the bacteria, and incubated for 15 minutes at room temperature. Then, 10μl of the solutions were applied to glass slides and later covered with 5μl mounting media. Next, the glass slides were imaged through microscopy in order to investigate the effect of copper chloride on both strains of bacteria.


Growth curves in bioscreen

Since PBP3SAL is believed to be a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment, we investigated growth in parameters mimicking the intraphagosomal environment such as low pH 5.8 in minimal media, as well as the presence or lack of salt in LB and TY media. Both strains of bacteria were incubated overnight in 2ml LB with 37°C shaking at 220rpm. The following day, the overnight cultures were mixed to an optical density of 0.01 in media (i.e. LB, TY, MM with pH 5.8) and loaded into honeycomb plates in triplicates with negative controls, then to be placed in the bioscreen. For the following 24 hours, the reader measured the optical density every 15 minutes, shaked the plates for 5 seconds before every measurement and kept a temperature of 37°C.


Results


PBP3SAL increases sensitivity to CuCl2

Figure 1 presents the results from the dilution series on the copper chloride plates. To begin with, in the TY control plates (Fig 1), no difference between the growth of wild type and Δpbp3sal :: kanR could be seen. As in the control, no difference in growth between the two bacterial strains in the 3mM plates is seen (Fig 1). However, the growth of both bacteria is clearly restricted with increasing concentrations of CuCl2. This can be seen in the 4mM plate, where the last two droplets of the wild type strain are barely visible (Fig. 1).


Additionally, in the 4mM plate the two last droplets of each strain are clearly affected by the copper chloride (Fig. 1). At the same time, the Δpbp3sal :: kanR seems to grow more compared to the wild type. This pattern is also seen in the 5mM plate (Fig. 1) where there is close to no growth in the wild type strain while the Δpbp3sal :: kanR still has considerable growth in the three first droplets. In conclusion, it seems like the Δpbp3sal :: kanR is less sensitive to the higher concentrations of CuCl2 than the wild type since it has visibly higher growth.


The presence of PBP3SAL improves growth in H2O2 media

The following description will be for the H2O2 plates presented in Figure 2. In the TY control plates, no difference between the growth of wild type and Δpbp3sal :: kanR could be seen (Fig. 2). In the 0.4mM plate (Fig. 2), we can already see that the two strains differ by growth. In the last two droplets, there is evidently more growth in the wild type in comparison to the Δpbp3sal :: kanR. As well as in the 0.4mM plate, the 0.5mM plate results (Fig. 2) imply that the wild type is less sensitive to hydrogen peroxide than Δpbp3sal :: kanR since it presents more growth. Moreover, this is also seen in the 0.6mM plate (Fig. 2) where the Δpbp3sal :: kanR shows less growth than the wild type.

To summarize, the growth of both the wild type and Δpbp3sal :: kanR is drastically reduced with higher concentrations of H2O2. At the same time, the wild type seems to be less sensitive to the hydrogen peroxide in comparison with the Δpbp3sal :: kanR which lacks the gene for PBP3SAL.


Abnormal morphology of wild type Salmonella in 1mM CuCl2 The following description will be for Figure 3, presenting the results from the copper chloride microscopy. To begin with, the wild type control strain (Fig. 3) seems to grow normally with a regular shape of rod-shaped bacteria. For the Δpbp3sal :: kanR control strain (Fig. 3), the bacteria appear to be slightly elongated (Fig. 3,#1-2) in comparison to the wild type. However, the wild type 1mM CuCl2 bacteria demonstrate an odd morphology with being more elongated in comparison to the control (Fig. 3). Though drastically elongated bacteria were seen in the wild type 1mM CuCl2, the Δpbp3sal :: kanR 1mM CuCl2 bacteria demonstrate an absence of long filaments.


Similar growth curves of the bacteria in LB, TY and MM media

The results from the bioscreen growth curves will be described in the following description. In all the graphs presented in figure 4, both bacteria have been grown in different media during a time span of 24h. These media include LB, TY and minimal media (pH 5.8). The minimal media (Fig. 4C) was particularly chosen because it mimics the intraphagosomal environment inside the Salmonella containing vacuole of macrophages.

In all graphs (Fig. 4), there is no clear difference between the OD600nm growth of wild type and Δpbp3sal :: kanR measured by the bioscreen. In the graph presenting the growth of both strains in LB media (Fig. 4A) there seems to be some difference in the growth logarithmic phase, during the time span of 200-450 minutes, however, that is not considered enough to be a difference and later the difference between the wild type and Δpbp3sal :: kanR evens out. Compared to the LB media growth (Fig. 4A), the growth in TY media (Fig. 4B) is slower, as well as no difference in growth between the two tested types of strains. Although some difference can be seen between the wild type and Δpbp3sal :: kanR in the graph presenting the growth in minimal media (Fig. 4C), it is not enough to be considered a difference.


Discussion


Each year it is estimated that up to 27 million people are affected and 460 000 people die due to typhoid fever (1), a result of rod-shaped bacteria called Salmonella enterica categorized under serovar Typhi and Paratyphi. Typhoidal Salmonella are intracellular pathogens, which for the pathogenesis of Salmonella means that it survives inside immune cells called macrophages (3). Also, it is crucial that the bacteria divides and multiplies inside the macrophages in order for the pathogenesis to continue (4-5). For the bacterial cell division, it is important that the bacteria builds new cell wall for the division with the divisome, a protein complex which consists of several proteins. One of the proteins being PBP3 which synthesizes a cell wall between daughter cells required for the separation (6-7). In this study, we focus on deepening the understanding of PBP3SAL, a recently found paralog to PBP3 (8), and its importance for the pathogenesis of Salmonella by comparing wild type Salmonella enterica serovar Typhimurium with a mutant, Δpbp3sal :: kanR, where the gene for PBP3SAL has been removed.


Copper chloride microscopy and sensitivity

With the increasing molarities of CuCl2, a clear difference between the two types of bacteria is seen (Fig. 1). They imply that Δpbp3sal :: kanR is less sensitive to CuCl2 than the wild type. This is also supported by our microscopy results (Fig. 3), where the wild type appears to have more elongated bacteria in comparison to the Δpbp3sal :: kanR, suggesting a malfunction in the cell division and henceforth a higher sensitivity to copper ions. Since Δpbp3sal :: kanR lacks the gene for PBP3SAL in comparison to the wild type, this suggests that the difference is a result of the presence of PBP3SAL. For the reason that the wild type has less growth in comparison to Δpbp3sal :: kanR, as well as an abnormal morphology, it is believed that PBP3SAL has a negative impact on the growth of bacteria when Cu2+ is present. In addition, the different phenotypes are not due to a general growth difference because no difference is seen in the TY control plate (Fig.1) and control strains (Fig. 3). We expected to see that the mutant would survive to a lesser extent than the wild type since it lacks PBP3SAL, claimed to be needed to replace the PBP3 in the divisome in order to survive in macrophages (8). Therefore, it is surprising that PBP3SAL could be more sensitive to CuCl2 than PBP3 which indicates that the mechanism of PBP3SAL is more complex than envisioned.


Hydrogen peroxide sensitivity

In the results (Fig. 2), both the two types of bacteria are restricted by the increasing concentrations of H2O2, with the wild type presenting less sensitivity than Δpbp3sal :: kanR. These results cannot be explained with a general growth difference between the bacteria since no difference in growth is seen in the TY control plate (Fig. 2). Due to the difference in the genotype, meaning that the wild type has a gene for PBP3SAL in its genome while Δpbp3sal :: kanR lacks it, the results suggest that PBP3SAL effects the bacterial growth in H2O2 TY media. Since the wild type has more growth than Δpbp3sal :: kanR, this indicates that PBP3SAL reduces the bacteria’s sensitivity to H2O2. Considering that macrophages use H2O2 among other reactive oxygen species to kill bacteria (9-10), the presence of PBP3SAL in Salmonella could be an adaptation for the bacteria needed in order to survive inside the intraphagosomal environment. This would support Castanheira et. al. (8) in how PBP3SAL evolved in the pathogenic Salmonella enterica serovar Typhimurium as a crucial specialized peptidoglycan synthase inside macrophages.

Copper chloride and hydrogen peroxide sensitivity

According to Castanheira et. al. (8) the dynamic between PBP3 and PBP3SAL present in the divisome changes depending on the environment such as the intraphagosomal environment of macrophages. In acidic environments, such as the Salmonella-containing vacuole inside macrophages, it is shown that PBP3SAL becomes more prominent than PBP3 which indicates that PBP3sal is needed to replace PBP3 to a further extent for the survival of the bacteria. Therefore, we hypothesized that PBP3SAL would be more adapted to the intraphagosomal environment with copper ions, hydrogen peroxide and acidity than PBP3.


Furthermore, it is believed that Cu2+ restricts the bacteria through the creation of reactive oxygen species. Thus, when the copper chloride results indicated that PBP3SAL was more sensitive to CuCl2 than PBP3 (Fig. 1), we assumed a similar pattern would be seen in the hydrogen peroxide plates species than the wild type. Therefore, the hydrogen peroxide results were surprising since they contradicted our hypothesis when the wild type was shown to be less sensitive to hydrogen peroxide than (Fig. 2). This implies that the effect copper ions have on our Salmonella, is not related to the creation of reactive oxygen species.


Growth curves in different media

While PBP3sal is shown to have an effect on how Salmonella grows in CuCl2 and H2O2 (Fig. 1 and 2), PBP3SAL seems to have no effect on how Salmonella grows in our tested medias (LB, TY and MM 5.8) (Fig. 4). The wild type and Δpbp3sal :: kanR do not have considerably different OD600nm growths presented in figure 4, meaning that the presence of PBP3SAL does not have an impact on how well our bacteria divide and grow in the tested medias. However, we have reason to believe that the values presented in figure 4C for the growth in minimal media are faulty since in Castanheira et. al. (8), they display evidence that PBP3SAL proliferates under acidic conditions (pH≤5.8). This would indicate that the bacteria containing PBP3SAL (i.e. wild type) would present more growth than Δpbp3sal :: kanR lacking PBP3SAL, which is the opposite of our presented results, as well as indicating a faulty method used. Therefore, we believe that the method was flawed with a systematic error, that the media did not have the pH of 5.8 as believed which may have resulted in our contradicting results to the published study.


Concluding remarks

In this study, we have found that the presence of PBP3SAL in an environment containing hydrogen peroxide could be beneficial for Salmonella enterica serovar Typhimurium and disadvantageous in a copper chloride environment. In conclusion, our results could imply that PBP3SAL is a crucial specialized peptidoglycan synthase which is specially adapted to hydrogen peroxide environments inside macrophages. Finally, it may be concluded that if this pattern is confirmed in further studies, PBP3SAL could be an eligible new target for antibiotic medicine against typhoid fever.

Acknowledgements

First and foremost, I am grateful for my supervisor Kim Vestö for his sincere guidance and support during my five weeks at the Department of Microbiology, Tumor and Cell Biology at the Karolinska Institute. His memorable commitment to making my experience truly giving and educational has been inspiring. Secondly, I would like to thank Yas Asghari who has been an entertaining and supporting lab-partner making our five weeks together unforgettable. I would also like to acknowledge Gerald McInerney, Axel Schröder, Nicole Laszlo and Kira Taivassalo for hosting the Karolinska Institute Summer Research School With Biomedical Orientation 2019.


References

Cover Image Credit: https://imgflip.com/gif/5zdyu


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