Periodic Reporting for period 4 - Growth regulation (The wide-spread bacterial toxin delivery systems and their role in multicellularity)
Okres sprawozdawczy: 2023-07-01 do 2023-12-31
In a multicellular entity, population heterogeneity on the level of individual cells is essential for division of labor. Population heterogeneity allows isogenic cells to respond differently to the same environmental cues, allowing different cells to perform different tasks. But how population heterogeneity arises in bacterial populations and how this affects multi-cellularity is not completely clear. We previously found that indications that delivery of anti-bacterial toxins among kin cells resulted in population heterogeneity in terms of growth in bacterial populations. Non-growing cells are not affected by antibiotic treatment and can cause a relapse of bacterial infections. Understanding how non-growing cells arise in bacterial populations is essential for designing interventions against recurrent infections. In addition, bacterial multi-cellular entities known as biofilms are inherently tolerant to most antibiotic treatment, causing problems on medical implants as well as in biotechnological applications. Understanding the molecular mechanisms that initiate bacterial multi-cellularity are of essence if we wish to prevent biofilm formation.
The aim of this study is to understand if and how bacterial toxin delivery systems contribute to generation of population heterogeneity and how this impacts bacterial evolution and/or multicellularity.
The project is divided into three parts where the first (1) part is focused on understanding the molecular mechanisms behind kin-delivery mediated population heterogeneity. In the second (2) part, we will investigate the biological consequences (i.e. effects on multi-cellularity and evolution) of such toxin mediated population heterogeneity in bacteria, and in the third (3) we will ask if eukaryotic homologs of these toxin delivery systems share functions (mechanistic or biologic) with the bacterial toxin delivery systems.
Overall, the results from this project have increased our understanding of multicellular behavior in bacteria as well as in eukaryotes. In the future, this knowledge can allow us to modulate bacterial behavior such as biofilm formation and antibiotic tolerance, or eukaryotic behavior like aberrant replication (cancer). Thus, the results from this project can be used for novel treatment options for bacterial and eukaryotic diseases.
The aim of this study is to understand if and how bacterial toxin delivery systems contribute to generation of population heterogeneity and how this impacts bacterial evolution and/or multicellularity.
The project is divided into three parts where the first (1) part is focused on understanding the molecular mechanisms behind kin-delivery mediated population heterogeneity. In the second (2) part, we will investigate the biological consequences (i.e. effects on multi-cellularity and evolution) of such toxin mediated population heterogeneity in bacteria, and in the third (3) we will ask if eukaryotic homologs of these toxin delivery systems share functions (mechanistic or biologic) with the bacterial toxin delivery systems.
Overall, the results from this project have increased our understanding of multicellular behavior in bacteria as well as in eukaryotes. In the future, this knowledge can allow us to modulate bacterial behavior such as biofilm formation and antibiotic tolerance, or eukaryotic behavior like aberrant replication (cancer). Thus, the results from this project can be used for novel treatment options for bacterial and eukaryotic diseases.
Our projects have gained substantial advances in the field. Our key findings is the molecular understanding of how toxin-delivery generates population heterogeneity in bacterial populations. Using numerous molecular biological tools, we find that some cells in a population sense the toxin because they lack sufficient immunity. The cells that sense the toxin, become transiently intoxicated, and change their gene-expression (Eriksson et al. in revision, poster presentation at the microbial stress responses Gordon conference, Massachusetts 2023 and EMBO Infection biology meeting, Paris 2023). Comparison between toxins with different toxic activities, has allowed us to see similarities and differences in these responses, of which some were quite unexpected. We have found that delivery of different toxins, with different toxic acitivities ranging from nucleases to ionophores, affects gene-expression of biofilm genes and the bacteria's ability to form biofilm in several bacterial species (Eriksson et al. in prep and Zaborskyte et al. in prep. Poster presentation at the microbial stress responses Gordon conference, Massachusetts 2023 and EMBO Infection biology meeting, Paris 2023). This is surprising, considering that different stress responses should be initiated by the different toxins. Our understanding of the molecular mechanism of how these toxins affect the cells, provide an explanation to why different toxins give the same response.
We also found that in addition to their conventional role in interbacterial competition, delivered toxins can regulate bacterial growth when expressed intracellularly, i.e. in the absence of delivery. Mainly, we find that toxin expression regulates growth rate of S.typhimurium in macrophages even in the presence of immunity (Stårsta et al. PLOS genetics 2020, oral presentation at the Salmonella Gordon conference, Massachusetts 2019) and that internal expression of toxins results in growth regulation during stress in E.coli (Stårsta et al .in prep. Oral presentation by Koskiniemi at the EMBO TA-workshop in Windsor). Thus, our results suggests that toxin delivery systems affect bacterial biology also in conditions when delivery is restricted. This is to our knowledge, the first evidence that delivered toxins are expressed internally like type-II-TA-modules.
In the wild, bacteria typically contain more than one toxin delivery system. In order to better understand how toxin delivery could be used in these wild isolates, we investigated what toxin delivery systems different bacteria harbor. We find that specific toxin delivery systems or toxins per se, are selected for in entertoxigenic E.coli but not necessarily in other E.coli (publication 2, Kjellin et al. Gut Microbes 2024. Poster presentation EMBO Infection biology meeting, Paris 2023). Thus, it is possible that the arsenal of toxin delivery systems and the specific sets of toxins that these can deliver, are important for deciding what niche a bacterium is able to colonize. More work is required to get the full picture regarding the environmental niches where different toxin(s) /delivery systems are important, and the bioinformatic pipelines that we developed during this project will be very useful in answering this.
By isolating and sequencing different isolates of closely related social amoeba, we have identified multiple Rhs-toxin homologs in these amoebas. The toxins originate from multiple horizontal gene transfer events between bacteria and amoeba, as they are localized to different parts of the genome and share little sequence homology in the toxin part. Molecular characterization of these toxins suggests that they are not toxic to bacteria, suggesting that they are not used for antibacterial protection as has been seen for some type 6 effectors previously. Structural predictions and preliminary data suggest that these toxins could act as ionophores, which could be involved in changing the metabolism of amoeba during multicellular development (Kjellin et al. in prep. Oral presentation at the Dictyostelium meeting, Stirling 2022).
We also found that in addition to their conventional role in interbacterial competition, delivered toxins can regulate bacterial growth when expressed intracellularly, i.e. in the absence of delivery. Mainly, we find that toxin expression regulates growth rate of S.typhimurium in macrophages even in the presence of immunity (Stårsta et al. PLOS genetics 2020, oral presentation at the Salmonella Gordon conference, Massachusetts 2019) and that internal expression of toxins results in growth regulation during stress in E.coli (Stårsta et al .in prep. Oral presentation by Koskiniemi at the EMBO TA-workshop in Windsor). Thus, our results suggests that toxin delivery systems affect bacterial biology also in conditions when delivery is restricted. This is to our knowledge, the first evidence that delivered toxins are expressed internally like type-II-TA-modules.
In the wild, bacteria typically contain more than one toxin delivery system. In order to better understand how toxin delivery could be used in these wild isolates, we investigated what toxin delivery systems different bacteria harbor. We find that specific toxin delivery systems or toxins per se, are selected for in entertoxigenic E.coli but not necessarily in other E.coli (publication 2, Kjellin et al. Gut Microbes 2024. Poster presentation EMBO Infection biology meeting, Paris 2023). Thus, it is possible that the arsenal of toxin delivery systems and the specific sets of toxins that these can deliver, are important for deciding what niche a bacterium is able to colonize. More work is required to get the full picture regarding the environmental niches where different toxin(s) /delivery systems are important, and the bioinformatic pipelines that we developed during this project will be very useful in answering this.
By isolating and sequencing different isolates of closely related social amoeba, we have identified multiple Rhs-toxin homologs in these amoebas. The toxins originate from multiple horizontal gene transfer events between bacteria and amoeba, as they are localized to different parts of the genome and share little sequence homology in the toxin part. Molecular characterization of these toxins suggests that they are not toxic to bacteria, suggesting that they are not used for antibacterial protection as has been seen for some type 6 effectors previously. Structural predictions and preliminary data suggest that these toxins could act as ionophores, which could be involved in changing the metabolism of amoeba during multicellular development (Kjellin et al. in prep. Oral presentation at the Dictyostelium meeting, Stirling 2022).
Understanding how bacteria communicate with one another and how they regulate growth in response to external stimuli is of central importance when designing new interventions against bacterial infections. We hypothesize that toxin delivery systems are conserved cell-cell communication systems, used to regulate growth and multicellular behavior. From our project, we have learned that bacterial toxin delivery among kin is an important mechanism for generation of population heterogeneity in a bacterial population. This type of heterogeneity can be useful as a bet-hedging strategy, but also to allow cells with the same genotype to respond differently to the same signals. Understanding of the molecular mechanisms of how different toxins function has given us information that can be used to design more efficient antimicrobial treatment methods in the future, but could also have consequences on treatment of other diseases including immunotherapy and cancer.