Periodic Reporting for period 1 - LYSM (Unravelling the potential of LysM-domain proteins to awaken silent secondary metabolites genes in predatory bacteria through predator-prey interactions.)
Okres sprawozdawczy: 2023-09-01 do 2025-08-31
LYSM project focuses on a group of uncharacterized proteins from the soil predator Myxococcus xanthus that are involved in predation of other microorganisms, and their potential involvement in the signalling cascade activating the normally silenced gene clusters for secondary metabolites production that are only expressed during this interaction. The research performed on the LysM proteins has proved that they are important for efficient predation on different prey organisms. These proteins (predicted to act as peptidoglycan hydrolases with different target sites on these macromolecules) have shown to act extracellularly improving predation of M. xanthus rather than directly acting on the prey killing process. Considering that deletion mutants of the lysM genes displays altered expression of some secondary metabolites genes, evidence so far suggests that LysM proteins may be linked to the prey-sensing process that allows M. xanthus to detect and discern between different microorganisms to select and produce the proper array of lytic factors to kill their prey. The data we have gathered so far has allowed us to build a model where the LysM proteins would degrade the peptidoglycan remains from the prey to generate peptidoglycan fragments that can be used as MAMPs (Microbial Associated Molecular Patterns) to be sensed by the predator.
Deletion mutants of the different lysM genes showed that each gene has a significant role during predation in M. xanthus. Bioinformatic and structural analyses using Alphafold modelling show that these proteins are likely to act as peptidoglycan hydrolases, cutting in specific positions on the peptide bonds of peptidoglycans. Predation assays using the forementioned mutants show that each LysM protein exhibits specificity for different prey. Furthermore, a fourth lysM gene, not included in the original proposal (MXAN_7387), and predicted to act as a glycoside hydrolase has also been analyzed, revealing a more generalist role for this protein, acting as a complementary LysM protein aiding in predation against all prey species tested. According to the predicted enzymatic activity for each LysM protein, together with MXAN_7387, the action of the LysM proteins would generate different peptidoglycan fragments in prey species with different peptidoglycan composition.
The lysM genes are expressed at low levels during predation suggesting that, either they encode potent lytic enzymes, or that these proteins foster predation by triggering a wider and more complex response in the predator. In order to test this, purified LysM proteins were used against prey cultures, and the results have shown that they had no potent lytic activity, as previously suspected, when added extracellularly. However, when added in presence of the predator, LysM proteins were able to improve predation in M. xanthus and to restore mutant phenotypes.
Assays using transcriptional fusions of secondary metabolite genes, which are normally silent in absence of prey, indicate that they are induced in presence of different prey as previous transcriptomic data suggested. Expression of these genes was shown to be altered in single mutants for lysM genes, as well as in a quadruple mutant for all four lysM genes (the original 3 subject genes plus the complementary MXAN_7387 gene). However, for some reporters such as those of the myxovirescin gene cluster, mutation of lysM genes results in a basal overexpression that is unsensitive to the presence of prey, suggesting a general dysregulation of predation genes under these conditions. This evidence would support a model where LysM proteins would degrade prey peptidoglycans or peptidoglycan remains to generate specific fragments that would serve as MAMPs that would signal M. xanthus of the presence and type of prey.
We are currently establishing a colorimetric method based on Remazol brilliant blue to easily assess the peptidoglycan hydrolase activity of the LysM proteins for a screening of the protein variants. Similarly, we are working on the identification of a good candidate for a silent gene cluster to act as a reporter to use for screening of their inducer activity after point mutations, since the reporters tested so far are expressed at levels that can only be measured by enzymatic activity assays and not by colorimetric assays.
The lysM genes are expressed at low levels during predation suggesting that, either they encode potent lytic enzymes, or that these proteins foster predation by triggering a wider and more complex response in the predator. In order to test this, purified LysM proteins were used against prey cultures, and the results have shown that they had no potent lytic activity, as previously suspected, when added extracellularly. However, when added in presence of the predator, LysM proteins were able to improve predation in M. xanthus and to restore mutant phenotypes.
Assays using transcriptional fusions of secondary metabolite genes, which are normally silent in absence of prey, indicate that they are induced in presence of different prey as previous transcriptomic data suggested. Expression of these genes was shown to be altered in single mutants for lysM genes, as well as in a quadruple mutant for all four lysM genes (the original 3 subject genes plus the complementary MXAN_7387 gene). However, for some reporters such as those of the myxovirescin gene cluster, mutation of lysM genes results in a basal overexpression that is unsensitive to the presence of prey, suggesting a general dysregulation of predation genes under these conditions. This evidence would support a model where LysM proteins would degrade prey peptidoglycans or peptidoglycan remains to generate specific fragments that would serve as MAMPs that would signal M. xanthus of the presence and type of prey.
We are currently establishing a colorimetric method based on Remazol brilliant blue to easily assess the peptidoglycan hydrolase activity of the LysM proteins for a screening of the protein variants. Similarly, we are working on the identification of a good candidate for a silent gene cluster to act as a reporter to use for screening of their inducer activity after point mutations, since the reporters tested so far are expressed at levels that can only be measured by enzymatic activity assays and not by colorimetric assays.
The outcome of this project has had a satisfactory impact on the scientific society. The identification of the four LysM proteins as predation-relevant hydrolytic enzymes targeting the peptidoglycan has been quite impactful, especially among scientists in the bacterial predation field. Despite many genes coding for hydrolytic enzymes that are induced during predation in M. xanthus (ranging from 32 to 46 depending on the prey), only two enzymes have been identified so far with a clear role during predation (LlpM and MepA). Thereby, characterization of the four LysM proteins from M. xanthus has meant a significant addition to the current knowledge about the tools used by bacterial predators to kill and consume their prey.
Additionally, we have, for the first time, preliminary evidence that peptidoglycan fragments could be acting as MAMPs in bacterial predation to detect and discern between different prey species. In eukaryotes, peptidoglycan fragments have been long known to be sensed by the immune system to detect pathogenic bacteria, acting as PAMPs (Pathogen Associated Molecular Patterns). When Koropatnick et al. (2004) discovered that the bioluminescent the squid Euprymna scolopes was also sensing peptidoglycan fragments to detect its non-pathogenic symbiotic bacteria Vibrio fischeri in its light organ, they coined the term MAMPs to include also patterns from non-pathogenic bacteria. The potential for MAMPs to be used during bacterial predation to detect prey, activating the predatory response and producing secondary metabolites and hydrolytic enzymes, has had a great reception in the scientific community when our results have been discussed during the different dissemination activities of this project. Scientific communication activities have also been successful, raising awareness of the antibiotic crisis among the general population and spreading the knowledge that not all microorganisms are harmful, and that in fact many of them are beneficial for our health, environment and society. Since some of the communication activities were aimed at high school students, we managed to increase interest in the biological sciences and motivate the young population to make a responsible use of antibiotics, setting the path for a new generation of scientists that have in mind the problems humanity is currently facing.
Additionally, we have, for the first time, preliminary evidence that peptidoglycan fragments could be acting as MAMPs in bacterial predation to detect and discern between different prey species. In eukaryotes, peptidoglycan fragments have been long known to be sensed by the immune system to detect pathogenic bacteria, acting as PAMPs (Pathogen Associated Molecular Patterns). When Koropatnick et al. (2004) discovered that the bioluminescent the squid Euprymna scolopes was also sensing peptidoglycan fragments to detect its non-pathogenic symbiotic bacteria Vibrio fischeri in its light organ, they coined the term MAMPs to include also patterns from non-pathogenic bacteria. The potential for MAMPs to be used during bacterial predation to detect prey, activating the predatory response and producing secondary metabolites and hydrolytic enzymes, has had a great reception in the scientific community when our results have been discussed during the different dissemination activities of this project. Scientific communication activities have also been successful, raising awareness of the antibiotic crisis among the general population and spreading the knowledge that not all microorganisms are harmful, and that in fact many of them are beneficial for our health, environment and society. Since some of the communication activities were aimed at high school students, we managed to increase interest in the biological sciences and motivate the young population to make a responsible use of antibiotics, setting the path for a new generation of scientists that have in mind the problems humanity is currently facing.