Final Report Summary - SYM-BIOTICS (Dual exploitation of natural plant strategies in agriculture and public health: enhancing nitrogen-fixation and surmounting microbial infections)
The unexpected discovery of 800 plant peptides produced in Sinorhizobium meliloti -Medicago truncatula symbiosis prompted us to investigate their function in symbiosis and explore their novel activities beyond symbiosis. In symbiosis, the peptides provoke an irreversible, host-directed multistep differentiation process of bacteria culminating in the formation of polyploid, non-cultivable nitrogen fixing bacteroids. The genes coding for these peptides are activated by the intracellular presence of bacteria. Consecutive expression of early and late genes is coordinated with growth of the host cells by repeated genome duplications. We investigated foremost ploidy dependent changes in the epigenome and found correlation between gene expression and ploidy-dependent opening of the chromatin as well as histone modifications.
>700 of the 800 symbiotic peptide genes code for cysteine-rich NCR peptides. But why are so many genes? In the soil there are countless strain variants of the bacterium partner and there are very many ecotypes and accessions of M. truncatula. In a given bacterium-plant interaction not all NCR genes are expressed. We have shown that certain NCR peptides can enforce symbiotic selectivity providing an additional level of host determinants of symbiotic specificity. Cationic NCR peptides show interaction with bacterial membranes and in vitro at higher doses provoke membrane damage, loss of membrane potential, various surface changes and even complete cell disruption. In the plant, NCRs do not kill their bacterium partners whose survival is ensured by the bacterial bacA gene. NCRs also enter the cytosol of bacteria. In nitrogen-fixing bacteroids we identified ~150 NCRs indicating their high stability and persisting functions. NCRs have multiple bacterial targets. For example, NCR247 has an extremely high protein-binding capacity. It interacts with several ribosomal proteins and contributes to the altered proteome and physiology of the endosymbiont. Moreover, its binding to FtsZ abolishes septum formation and cell division. Collectively NCRs can affect specific biological pathways at multiple points. However, due to the expansion of the NCR family by gene duplications, probably many gene functions are redundant. Unexpectedly, the loss of a single gene, NCR169 was detrimental for the symbiosis, demonstrating that there are essential NCRs with unique functions.
The fate of bacteroids is irreversible not only in M. truncatula but also in other IRLC legumes while the morphology of the endosymbionts shows great variations. Identifying the NCR gene repertoire of 10 selected IRLC legumes, we found a correlation between the complexity of the NCR families and the morphotypes of bacteroids.
As several synthetic cationic NCR peptides prevented growth of S. meliloti and killed them at higher concentrations, it seemed likely that cationic NCRs might effectively eliminate other microbes as well. Due to the alarming rise of infectious diseases, spread of antibiotic resistant bacteria, and lack of effective treatments, the Sym-Biotics project focused on the NCRs’ antimicrobial potential. 40 different synthetic NCR peptides were tested on various Gram negative and Gram positive pathogen bacteria and fungi.
Peptides with pI>9.5 exhibited broad range of antimicrobial activity, effectively killing many of the tested bacteria and fungi. However, their bactericidal spectrum was not identical indicating that in addition to the positive charge; the amino acid composition and sequence also contribute to the antimicrobial activity. The minimal inhibitory concentration of the peptides against various growing Candida strains was comparable to that of the antifungal amphotericin B. Importantly, the antimicrobial NCRs were not toxic for human and animal cell lines. Moreover, their multi-target action reduces the likelihood of developing resistance against them. These properties make them promising drug candidates. As peptide synthesis is still costly, we have been testing smaller derivatives of the peptides to find those which maintain their antimicrobial activity and could be sources of new antibiotics.
>700 of the 800 symbiotic peptide genes code for cysteine-rich NCR peptides. But why are so many genes? In the soil there are countless strain variants of the bacterium partner and there are very many ecotypes and accessions of M. truncatula. In a given bacterium-plant interaction not all NCR genes are expressed. We have shown that certain NCR peptides can enforce symbiotic selectivity providing an additional level of host determinants of symbiotic specificity. Cationic NCR peptides show interaction with bacterial membranes and in vitro at higher doses provoke membrane damage, loss of membrane potential, various surface changes and even complete cell disruption. In the plant, NCRs do not kill their bacterium partners whose survival is ensured by the bacterial bacA gene. NCRs also enter the cytosol of bacteria. In nitrogen-fixing bacteroids we identified ~150 NCRs indicating their high stability and persisting functions. NCRs have multiple bacterial targets. For example, NCR247 has an extremely high protein-binding capacity. It interacts with several ribosomal proteins and contributes to the altered proteome and physiology of the endosymbiont. Moreover, its binding to FtsZ abolishes septum formation and cell division. Collectively NCRs can affect specific biological pathways at multiple points. However, due to the expansion of the NCR family by gene duplications, probably many gene functions are redundant. Unexpectedly, the loss of a single gene, NCR169 was detrimental for the symbiosis, demonstrating that there are essential NCRs with unique functions.
The fate of bacteroids is irreversible not only in M. truncatula but also in other IRLC legumes while the morphology of the endosymbionts shows great variations. Identifying the NCR gene repertoire of 10 selected IRLC legumes, we found a correlation between the complexity of the NCR families and the morphotypes of bacteroids.
As several synthetic cationic NCR peptides prevented growth of S. meliloti and killed them at higher concentrations, it seemed likely that cationic NCRs might effectively eliminate other microbes as well. Due to the alarming rise of infectious diseases, spread of antibiotic resistant bacteria, and lack of effective treatments, the Sym-Biotics project focused on the NCRs’ antimicrobial potential. 40 different synthetic NCR peptides were tested on various Gram negative and Gram positive pathogen bacteria and fungi.
Peptides with pI>9.5 exhibited broad range of antimicrobial activity, effectively killing many of the tested bacteria and fungi. However, their bactericidal spectrum was not identical indicating that in addition to the positive charge; the amino acid composition and sequence also contribute to the antimicrobial activity. The minimal inhibitory concentration of the peptides against various growing Candida strains was comparable to that of the antifungal amphotericin B. Importantly, the antimicrobial NCRs were not toxic for human and animal cell lines. Moreover, their multi-target action reduces the likelihood of developing resistance against them. These properties make them promising drug candidates. As peptide synthesis is still costly, we have been testing smaller derivatives of the peptides to find those which maintain their antimicrobial activity and could be sources of new antibiotics.