Final Report Summary - BACTERIAL STRESS (STUDIES ON THE BACTERIAL STRESS RESPONSE AND STRESS-INDUCED CROSS-RESISTANCE)
FP7-PEOPLE-2010-IIF Project (ID: 274660), entitled "STUDIES ON THE BACTERIAL STRESS RESPONSE AND STRESS-INDUCED CROSS-RESISTANCE".
Marie Curie Fellow: Antón Vila Sanjurjo, PhD (AVS)
Grupo PRONAMAR, Dept. Química Fundamental
Facultade de Ciencias
Universidade de A Coruña (UDC)
Campus Zapateira, s/n
15.071 - A Coruña - España (Spain).
tlf: (34) 981-167000 ext:2659
e-mail: antonvila.s@gmail.com
SPECIFIC AIM I: THE ROLE OF THE RIBOSOME IN STRESS-INDUCED CROSS-RESISTANCE: Most bacteria, including many human and animal pathogens, can acquire a high-resistance state as a consequence of exposure to stress. For example, when logarithmically growing E. coli cells exhaust an essential nutrient in the medium, they enter the so-called stationary phase of growth (1), in which the cells become highly resistant to a variety of environmental stresses (2). Since in their natural environments, bacteria seldom encounter conditions that allow exponential growth, understanding the mechanism/s behind stress-induced cross-resistance is of uttermost importance for the control of bacterial populations. In particular, the epidemiology and virulence of many human, animal, and plant pathogens appears to be related to stress-induced cross-resistance (3,4,5,6,7,8), and phenomena such as biofilm formation appear to be the direct consequence of exposure to environmental stress (9,10). Therefore, understanding these bacterial survival strategies has important medical and economical implications.
This specific aim was based on our previuos observation of the abnormal growth at low temperatures of E. coli cells carrying certain ksg-resistance (ksgr) mutations affecting 16S rRNA (11) (Vila-Sanjurjo, unpublished). We have focused in the characterization of this strange phenotype by performing growth measurements with the mutants both in liquid and solid media. These experiments showed that two types of ksgr mutations in 16S rRNA, i.e. mutants carrying base changes at position G926 and mutants with altered dimethyl As stem loop, display s tress- i nduced c ross s ensitivity or SICS. Specifically, the cells become ksg sensitive (ksgs) at low temperatures in solid medium. The SICS phenotype is not a mere consequence of the original ksg-resistance phenotype, as ksgr mutations at position 794 of 16S rRNA do not result in the SICS phenotype. Experiments in liquid medium showed that the SICS phenotype requires the passage of the cells through stationary phase. These results are also supported by functional experiments showing complete absence of translation inhibition by ksg in the cold, for cells undergoing cold shock during logarithmic phase. All these observations identify the ribosome as a critical element for the induction of cross-resistance during stationary phase and establish ksgr mutations as valid models for the analysis of this phenomenon. At the same time, a complete understanding of the growth features of the SICS phenotype is necessary to set up the initial conditions for genome-wide analysis by Ribosome Profiling (12) and proteomics analysis to be performed in collaboration with Dr. Lori Kohlstaedt (Director, Proteomics/Mass Spectrometry Laboratory at UC Berkeley) . Genome-wide experiments will provide an unprecedented view of the genetic changes involved in the bacterial stress response during stationary phase and how these changes are related to the SICS phenotype. Since the experimental set up for RP has been already established in the lab, we are now well poised to launch these experiments which will bring closure to this Specific Aim.
In addition to the growth and the RP experiments, we had proposed the construction of a series of reporter constructs to test the function of mutant ribosomes. These include constructs based on both beta-galactosidase activity and fluorescence. As a result, a substantial amount of research time has been devoted to the construction and characterization of these plasmids. Beta-galactosidase-based plasmids having all possible base substitutions at the first position of the initiation codon are currently being tested with the ksgr mutants. To do this in a high-throughput fashion, we have implemented 96-well plate technology, as originally proposed. Fluorescent constructs have also been made and successfully tested. To be useful for microscopy studies, however, a second fluorophore is being added to these constructs. These construct promise to become a powerful tool to follow translation via light microscopy in single cells. In particular, we expect to use these constructs to visualize how translation is related to the SICS phenotype.
CONCLUSIONS: The characterization of the SICS phenotype could offer an unprecedented view of the involvement of the bacterial ribosome in the stress response and the development of stress-induced cross-resistance. As a result, these studies will offer important insights in some the molecular events responsible for the achievement of a more resistant state by Gram negative bacteria during stress.
SPECIFIC AIM II: ANALYSIS OF THE RESPONSE TO IRON DEFICIENCY AND THE PROCESS OF IRON UPTAKE IN FISH PATHOGEN PHOTOBACTERIUM DAMSELAE SUBSP . PISCICIDA: Iron is an essential nutrient for most organisms. However, in spite of being the fourth most abundant element in earth’s crust, soluble iron(III) remains largely unavailable to microorganisms, with a concentration in the range of 10–18 M (reviewed in (13). The low concentration of soluble iron(III) implies that this compound cannot diffuse into the cell's cytoplasm where its concentration reaches about 1 μM. In order to scavenge for iron, many gram-negative bacteria, including some important human and animal pathogens, produce small iron(III)-chelating organic molecules called siderophores. Siderophores are actively transported into the cell by specific outer membrane receptor proteins belonging to the beta -barrel family. A ll of the outer membrane receptors possess a 22 antiparallel beta-stranded beta-barrel and an N-terminal globular domain (plug) that occludes the opening of the beta-barrel (14). It has been determined that iron scavenging is a major virulence factor in infectious processes carried out by Gram negative pathogens (15). As a result, compounds interfering with this process are expected to result in powerful antibacterials. Our main goal is to achieve the rational design of such antibacterials by first understanding the structural relationship between siderophores and their transporters.
Work in this specific Aim has been performed in two parallel fronts within PRONAMAR, the group led by Carlos Jiménez (Scientist in charge): the chemical synthesis branch (CSB), devoted to the synthesis of siderophores and the structural biology branch (SBB, led by AVS), whose goal is the production of siderophore receptors for their crystallization. During year I, SBB developed vectors for the expression of the siderophore transporter FvtA, from the fish pathogen Vibrio anguillarum, and performed the first attempts to purify the protein. In parallel, SBB attempted to build and characterize Fe(III)-vanchrobactin complexes (vanchrobactin is the siderophore normally transported by FvtA) for their eventual co-crystallization with FvtA. At the same time, PRONAMAR's CSB pursued the production of analogs of vanchrobactin with features of interest. Experimental failures in all these three fronts, together with the acquisition of a grant from the Spanish Government to fund PRONAMAR's research activities (Grant AGL2012-39274-C02-02 to Carlos Jiménez), prompted us to slightly shift the subject of our scientific focus during year II. Thus, we switched our attention from the fish pathogen Vibrio anguillarum to Photobacterium damselae subsp. piscicida. This bacterium is the causative agent of the marine fish disease pasteurellosis or photobacteriosis, one of the fish bacterial diseases with greater economic impact (16). The search for new treatments against this infectious disease has become a priority in marine aquaculture. Early studies made by our research group allowed the isolation and chemical characterization of the siderophore piscibactin from P. damselae subsp. piscicida, as well as the identification of a gene cluster encoding its synthesis and transport. We could also identify the putative siderophore outer membrane receptor, FrpA (17,18). An important advantage of this experimental system is that Fe(III)-piscibactin complexes are easily made, as shown by our group, in contrast to the vanchrobactin/FvtA case (18).
Much like our previous efforts with FvtA/vanchrobactin, the work load was split between PRONAMAR's CSB, devoted to the chemical synthesis of piscibactin, and SBB, whose focus was the overexpression of the protein FrpA in E. coli for its crystallization. In both cases, we have reached important milestones. First, together with our collaborators, our group has determined the structure of piscibactin (18). Second, the chemical synthesis of the siderophore is well on its way (Souto et al. unpublished). Regarding the expression of FrpA, we used the knowledge gained during the previous work with FvtA and designed several types of expression vectors to allow the production of the recombinant protein in E. coli . Similarly to FvtA's case, initial attempts to express FrpA in E. coli failed (Vila-Sanjurjo et al. unpublished). We eventually succeeded by using a vector in which the native targeting sequence from FrpA was replaced by the well-known pelB targeting sequence (19). Specifically, our vector encoded a chimeric protein in which the mature protein was preceded by a pelB-10His fusion peptide (20). After affinity purification, the identity of the eluted protein was confirmed by peptide mass fingerprinting using MALDI-TOF/TOF Mass Spectrometry. FrpA was detected in three cellular fractions, 1) the insoluble fraction, likely representing inclusion bodies; 2) an abundant membrane form, likely representing an intermediate form of the protein on its way to the outer membrane; and 3) a less abundant, outer membrane form, likely representing native FrpA. Thus, the isolation of FrpA in both its native and denatured forms appears quite possible. We are currently attempting to purify the protein from inclusion bodies for its refolding, as described by Saleem et al. (21). Additionally, we will attempt to increase the outer-membrane FrpA fraction by overexpressing genes known to be involved in the targeting of Beta-barrel proteins (22). The purified protein will be used for crystallization assays and as an antigen for the development of new vaccines against pasteurellosis. At the same time, these studies will serve as the basis for a pipeline aimed at the structural characterization of siderophore-transporter complexes involved in the pathogenicity of Gram-negative bacteria of human interest.
CONCLUSIONS: It is expected that understanding the structural details of the interaction between siderophore and their outer-membrane receptors will lead to the rational design of siderophore analogs with features of interest. Our group possesses the knowledge to act on all fronts of this scientific endeavor. The current Marie Curie project has constituted the first step towards the establishment of a pipeline dedicated to the analysis of siderophore-transporter complexes of pathogenic Gram-negative bacteria, with the idea of hijacking the mechanism of iron scavenging as a way to deliver antibacterials.
SPECIFIC AIM III: THE ROLE OF THE MITOCHONDRIAL RIBOSOME IN HUMAN DISEASE: SEARCHING FOR MUTATIONS IN MITOCHONDRIAL rRNA WITH HIGH DISRUPTIVE POTENTIAL: This project has been developed in collaboration with several researchers from the Mitochondrial Research Group (MRG) at the University of Newcastle (UK).
Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially-encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. The functional analysis of these mutations has been largely hampered by the lack of methods for direct manipulation of the mt-genome and the impossibility of recreating mitochondrial translation in vitro. We have developed a new analytical method, named HIA (Heterologous Inferential Analysis), for the study of the large collection of uncharacterized mitochondrial rRNA (mt-rRNA) mutations. HIA combines conservational information with functional and structural data obtained from heterologous ribosomal sources. As a result, HIA's predictive power is far superior to the traditional reliance on simple conservation indexes.
Our search through the literature for mt-12S rRNA variations possibly associated with disruption of mitochondrial function resulted in the identification of 161 different variations, corresponding to 152 different sites, almost 16% of the mt-12S rRNA molecule. HIA revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive potential on the function of the mitoribosome. Specifically, out of the 46 extremely rare variations analyzed, we found 1 whose disruptive potential is "unlikely disruptive", 18 which cannot be classified due to local structural differences between the mitochondrial and the heterologous ribosomes ("undetermined"), 14 which are regarded as “NEE” (not enough evidence), 5 which are "likely disruptive”, and 9 which are "expectedly disruptive". In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data has been used to predict the pathogenicity of mt-rRNA mutations. This is the case of the 255G>A (m.902G>A) and 533U>G (m.1180T>G) mutations (23,24), whose identification in homoplasmy necessarily implies the existence of a deficient translational apparatus in the mitochondria of the affected tissues. In other cases, HIA analysis allows the prioritisation of variants for additional investigation. Eventually, HIA-inspired investigation of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool. The results of this analysis has been recently reported (25).
We are currently performing HIA with variations mapping to mt-16S rRNA, the RNA component of the mitochondrial large ribosomal subunit (mt-LSU). A total of 181 mt-16S rRNA variations have been analyzed of which 82 are extremely rare and are being subjected to HIA.
Finally, as a byproduct of HIA we have performed comparative analysis of all the rRNA genes present in the large collection of GenBank mt-genomes (about 18,000 as of February 2013). We are currently performing a thorough statistical regression analysis of the correlation between rRNA conservation and the number of variant appearances in GenBank. The analysis shows a clear correlation between the two variables, in agreement with the well-known conservation of rRNA structure/function observed in other systems. We believe that these data will have important implications for the understanding of the structure and function of the mitoribosome and, as a result, will also contribute to our understanding of its involvement in human disease and ageing.
CONCLUSIONS: This research provides, for the first time, a method for scoring the disruptive and, in some cases, the pathogenic potential of a subset of mitochondrial DNA mutations, namely those mapping to the rRNA genes. This comes at a time when the involvement of mitochondrial DNA mutations in human disease is gaining an increasing recognition within the scientific and medical community.
As expected, the addition of Specific Aim III at the end of year I, has ensured that publishable results are available at the end of the funding period. Regarding the other two Specific Aims, a portion of the proposed work could not be completed during the funding period. This is partly due to experimental setbacks and to the restrictions imposed by the Spanish economic situation, which resulted in the lack of vital human and physical resources initially ascribed to the project. Despite these facts, we have made enormous progress towards the achievement of the proposed goals. Such progress consists of both positive experimental results and the development of important molecular tools. Notably, new funding has been secured to ensure that the core of the proposed goals will be completed within a reasonable amount of time via the continuation of AVS's contract at UDC.
ADDITIONAL WORK NOT DESCRIBED IN THE ORIGINAL PROPOSAL:
RIBOSOME PROFILING: We have started the analysis of dengue infection by means of Ribosome Profiling, as prompted by the ILIR grant achieved in collaboration with NAMRU-6, Perú. AVS's participation in this work was proposed and approved as part of the modifications made to the original proposal after year I. AVS has been a crucial part in establishing the experimental and bioinformatic protocols used in this project. So far, the results are highly promising and we expect to publish them in a high-profile journal soon.
DNA BARCODING OF SPELEOTHEM ASSOCIATED ORGANISMS: In collaboration with the Geological Institute Parga Pondal at UDC and the UDC associated company AllGenetics & Biology SL., we have started the characterization of the micro-flora associated with speleothemes in Galician granitic caves by DNA barcoding (26). The initial results show a highly complex bacterial community associated to these formations. We are currently analyzing the presence of other organisms within the micro-flora. Our goal is to establish whether or not speleothem formation requires the action of microbes. We believe that this research has the potential to uncover new biological processes of industrial interest.
OVERALL CONCLUSIONS AND SOCIO-ECONOMIC IMPACT OF THE PROJECT: The work developed under the 3 Specific Aims above has important economic and medical implications. On the one hand, Specific Aims I and II are related to the study of some of the weapons used by Gram negative bacteria during infectious and invasive processes. As a result, these studies could have important implications for the development of new strategies to control bacterial populations. On the other hand, Specific Aim III is devoted to the study of potentially harmful mutations in mitochondrial DNA, affecting the function of the mitoribosome. Thus, our studies are expected to result in powerful diagnostic tools for mitochondrial disease.
DISSEMINATION OF RESULTS: Specific Aim III has yielded the most significant achievement so far, namely the recent publication of a paper in the high-impact journal Human Molecular Genetics (Impact factor: 7.692 5-Yr impact factor: 7.541) entitled “The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential”. We expect at least three more manuscripts that will follow up on the published results.
Regarding Specific Aims I and II, the work is well advanced but not yet ready for publication. We expect to complete this work within the time frame created by the lab's new funding situation. In both cases, we expect to obtain high-profile publications.
In addition, AVS has participated in the international meeting “Targeting Mitochondria 2012” in Berlin, with a poster presentation. AVS has also been invited to give a presentation at the “Institut für Biologie und Biotechnologie der Pflanzen” of the University of Münster, Germany, entitled “The role of the mitochondrial ribosome in human disease: Analysis of mutations in 12S mitochondrial rRNA with a high disruptive potential”. This same topic has been recently featured in an interview by a local radio station.
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9. Schuwirth B.S. Borovinskaya M.A. Hau C.W. Zhang W., Vila-Sanjurjo A., Holton J.M. and Cate J.H. (2005) Structures of the bacterial ribosome at 3.5 A resolution. Science, 310, 827-834.
10. Schuwirth B.S. Day J.M. Hau C.W. Janssen G.R. Dahlberg A.E. Cate J.H. and Vila-Sanjurjo A. (2006) Structural analysis of kasugamycin inhibition of translation. Nat. Struct. Mol. Biol., 13, 879-886.
11. Vila-Sanjurjo A., Squires C.L. and Dahlberg A.E. (1999) Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of escherichia coli. J. Mol. Biol., 293, 1-8.
12. Ingolia N.T. Ghaemmaghami S., Newman J.R. and Weissman J.S. (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324, 218-223.
13. Seibel P., Di Nunno C., Kukat C., Schafer I., Del Bo R., Bordoni A., Comi G.P. Schon A., Capuano F., Latorre D. et al. (2008) Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids. Nucleic Acids Res., 36, 5872-5881.
14. Krewulak K.D. and Vogel H.J. (2008) Structural biology of bacterial iron uptake. Biochim. Biophys. Acta, 1778, 1781-1804.
15. Terhorst C., Moller W., Laursen R. and Wittmann-Liebold B. (1973) The primary structure of an acidic protein from 50-S ribosomes of escherichia coli which is involved in GTP hydrolysis dependent on elongation factors G and T. Eur. J. Biochem., 34, 138-152.
16. Romalde J.L. (2002) Photobacterium damselae subsp. piscicida: An integrated view of a bacterial fish pathogen. Int. Microbiol., 5, 3-9.
17. Osorio C.R. Juiz-Rio S. and Lemos M.L. (2006) A siderophore biosynthesis gene cluster from the fish pathogen photobacterium damselae subsp. piscicida is structurally and functionally related to the yersinia high-pathogenicity island. Microbiology, 152, 3327-3341.
18. Souto A., Montaos M.A. Rivas A.J. Balado M., Osorio C.R. Rodríguez J., Lemos M.L. and Jiménez C. (2012) Structure and biosynthetic assembly of piscibactin, a siderophore from photobacterium damselae subsp. piscicida, predicted from genome analysis. European Journal of Organic Chemistry, 2012, 5693-5700.
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25. Smith P.M. Elson J.L. Greaves L.C. Wortmann S.B. Rodenburg R.J. Lightowlers R.N. Chrzanowska-Lightowlers Z.M. Taylor R.W. and Vila-Sanjurjo A. (2013) The role of the mitochondrial ribosome in human disease: Searching for mutations in 12S mitochondrial rRNA with high disruptive potential. Hum. Mol. Genet., .
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Marie Curie Fellow: Antón Vila Sanjurjo, PhD (AVS)
Grupo PRONAMAR, Dept. Química Fundamental
Facultade de Ciencias
Universidade de A Coruña (UDC)
Campus Zapateira, s/n
15.071 - A Coruña - España (Spain).
tlf: (34) 981-167000 ext:2659
e-mail: antonvila.s@gmail.com
SPECIFIC AIM I: THE ROLE OF THE RIBOSOME IN STRESS-INDUCED CROSS-RESISTANCE: Most bacteria, including many human and animal pathogens, can acquire a high-resistance state as a consequence of exposure to stress. For example, when logarithmically growing E. coli cells exhaust an essential nutrient in the medium, they enter the so-called stationary phase of growth (1), in which the cells become highly resistant to a variety of environmental stresses (2). Since in their natural environments, bacteria seldom encounter conditions that allow exponential growth, understanding the mechanism/s behind stress-induced cross-resistance is of uttermost importance for the control of bacterial populations. In particular, the epidemiology and virulence of many human, animal, and plant pathogens appears to be related to stress-induced cross-resistance (3,4,5,6,7,8), and phenomena such as biofilm formation appear to be the direct consequence of exposure to environmental stress (9,10). Therefore, understanding these bacterial survival strategies has important medical and economical implications.
This specific aim was based on our previuos observation of the abnormal growth at low temperatures of E. coli cells carrying certain ksg-resistance (ksgr) mutations affecting 16S rRNA (11) (Vila-Sanjurjo, unpublished). We have focused in the characterization of this strange phenotype by performing growth measurements with the mutants both in liquid and solid media. These experiments showed that two types of ksgr mutations in 16S rRNA, i.e. mutants carrying base changes at position G926 and mutants with altered dimethyl As stem loop, display s tress- i nduced c ross s ensitivity or SICS. Specifically, the cells become ksg sensitive (ksgs) at low temperatures in solid medium. The SICS phenotype is not a mere consequence of the original ksg-resistance phenotype, as ksgr mutations at position 794 of 16S rRNA do not result in the SICS phenotype. Experiments in liquid medium showed that the SICS phenotype requires the passage of the cells through stationary phase. These results are also supported by functional experiments showing complete absence of translation inhibition by ksg in the cold, for cells undergoing cold shock during logarithmic phase. All these observations identify the ribosome as a critical element for the induction of cross-resistance during stationary phase and establish ksgr mutations as valid models for the analysis of this phenomenon. At the same time, a complete understanding of the growth features of the SICS phenotype is necessary to set up the initial conditions for genome-wide analysis by Ribosome Profiling (12) and proteomics analysis to be performed in collaboration with Dr. Lori Kohlstaedt (Director, Proteomics/Mass Spectrometry Laboratory at UC Berkeley) . Genome-wide experiments will provide an unprecedented view of the genetic changes involved in the bacterial stress response during stationary phase and how these changes are related to the SICS phenotype. Since the experimental set up for RP has been already established in the lab, we are now well poised to launch these experiments which will bring closure to this Specific Aim.
In addition to the growth and the RP experiments, we had proposed the construction of a series of reporter constructs to test the function of mutant ribosomes. These include constructs based on both beta-galactosidase activity and fluorescence. As a result, a substantial amount of research time has been devoted to the construction and characterization of these plasmids. Beta-galactosidase-based plasmids having all possible base substitutions at the first position of the initiation codon are currently being tested with the ksgr mutants. To do this in a high-throughput fashion, we have implemented 96-well plate technology, as originally proposed. Fluorescent constructs have also been made and successfully tested. To be useful for microscopy studies, however, a second fluorophore is being added to these constructs. These construct promise to become a powerful tool to follow translation via light microscopy in single cells. In particular, we expect to use these constructs to visualize how translation is related to the SICS phenotype.
CONCLUSIONS: The characterization of the SICS phenotype could offer an unprecedented view of the involvement of the bacterial ribosome in the stress response and the development of stress-induced cross-resistance. As a result, these studies will offer important insights in some the molecular events responsible for the achievement of a more resistant state by Gram negative bacteria during stress.
SPECIFIC AIM II: ANALYSIS OF THE RESPONSE TO IRON DEFICIENCY AND THE PROCESS OF IRON UPTAKE IN FISH PATHOGEN PHOTOBACTERIUM DAMSELAE SUBSP . PISCICIDA: Iron is an essential nutrient for most organisms. However, in spite of being the fourth most abundant element in earth’s crust, soluble iron(III) remains largely unavailable to microorganisms, with a concentration in the range of 10–18 M (reviewed in (13). The low concentration of soluble iron(III) implies that this compound cannot diffuse into the cell's cytoplasm where its concentration reaches about 1 μM. In order to scavenge for iron, many gram-negative bacteria, including some important human and animal pathogens, produce small iron(III)-chelating organic molecules called siderophores. Siderophores are actively transported into the cell by specific outer membrane receptor proteins belonging to the beta -barrel family. A ll of the outer membrane receptors possess a 22 antiparallel beta-stranded beta-barrel and an N-terminal globular domain (plug) that occludes the opening of the beta-barrel (14). It has been determined that iron scavenging is a major virulence factor in infectious processes carried out by Gram negative pathogens (15). As a result, compounds interfering with this process are expected to result in powerful antibacterials. Our main goal is to achieve the rational design of such antibacterials by first understanding the structural relationship between siderophores and their transporters.
Work in this specific Aim has been performed in two parallel fronts within PRONAMAR, the group led by Carlos Jiménez (Scientist in charge): the chemical synthesis branch (CSB), devoted to the synthesis of siderophores and the structural biology branch (SBB, led by AVS), whose goal is the production of siderophore receptors for their crystallization. During year I, SBB developed vectors for the expression of the siderophore transporter FvtA, from the fish pathogen Vibrio anguillarum, and performed the first attempts to purify the protein. In parallel, SBB attempted to build and characterize Fe(III)-vanchrobactin complexes (vanchrobactin is the siderophore normally transported by FvtA) for their eventual co-crystallization with FvtA. At the same time, PRONAMAR's CSB pursued the production of analogs of vanchrobactin with features of interest. Experimental failures in all these three fronts, together with the acquisition of a grant from the Spanish Government to fund PRONAMAR's research activities (Grant AGL2012-39274-C02-02 to Carlos Jiménez), prompted us to slightly shift the subject of our scientific focus during year II. Thus, we switched our attention from the fish pathogen Vibrio anguillarum to Photobacterium damselae subsp. piscicida. This bacterium is the causative agent of the marine fish disease pasteurellosis or photobacteriosis, one of the fish bacterial diseases with greater economic impact (16). The search for new treatments against this infectious disease has become a priority in marine aquaculture. Early studies made by our research group allowed the isolation and chemical characterization of the siderophore piscibactin from P. damselae subsp. piscicida, as well as the identification of a gene cluster encoding its synthesis and transport. We could also identify the putative siderophore outer membrane receptor, FrpA (17,18). An important advantage of this experimental system is that Fe(III)-piscibactin complexes are easily made, as shown by our group, in contrast to the vanchrobactin/FvtA case (18).
Much like our previous efforts with FvtA/vanchrobactin, the work load was split between PRONAMAR's CSB, devoted to the chemical synthesis of piscibactin, and SBB, whose focus was the overexpression of the protein FrpA in E. coli for its crystallization. In both cases, we have reached important milestones. First, together with our collaborators, our group has determined the structure of piscibactin (18). Second, the chemical synthesis of the siderophore is well on its way (Souto et al. unpublished). Regarding the expression of FrpA, we used the knowledge gained during the previous work with FvtA and designed several types of expression vectors to allow the production of the recombinant protein in E. coli . Similarly to FvtA's case, initial attempts to express FrpA in E. coli failed (Vila-Sanjurjo et al. unpublished). We eventually succeeded by using a vector in which the native targeting sequence from FrpA was replaced by the well-known pelB targeting sequence (19). Specifically, our vector encoded a chimeric protein in which the mature protein was preceded by a pelB-10His fusion peptide (20). After affinity purification, the identity of the eluted protein was confirmed by peptide mass fingerprinting using MALDI-TOF/TOF Mass Spectrometry. FrpA was detected in three cellular fractions, 1) the insoluble fraction, likely representing inclusion bodies; 2) an abundant membrane form, likely representing an intermediate form of the protein on its way to the outer membrane; and 3) a less abundant, outer membrane form, likely representing native FrpA. Thus, the isolation of FrpA in both its native and denatured forms appears quite possible. We are currently attempting to purify the protein from inclusion bodies for its refolding, as described by Saleem et al. (21). Additionally, we will attempt to increase the outer-membrane FrpA fraction by overexpressing genes known to be involved in the targeting of Beta-barrel proteins (22). The purified protein will be used for crystallization assays and as an antigen for the development of new vaccines against pasteurellosis. At the same time, these studies will serve as the basis for a pipeline aimed at the structural characterization of siderophore-transporter complexes involved in the pathogenicity of Gram-negative bacteria of human interest.
CONCLUSIONS: It is expected that understanding the structural details of the interaction between siderophore and their outer-membrane receptors will lead to the rational design of siderophore analogs with features of interest. Our group possesses the knowledge to act on all fronts of this scientific endeavor. The current Marie Curie project has constituted the first step towards the establishment of a pipeline dedicated to the analysis of siderophore-transporter complexes of pathogenic Gram-negative bacteria, with the idea of hijacking the mechanism of iron scavenging as a way to deliver antibacterials.
SPECIFIC AIM III: THE ROLE OF THE MITOCHONDRIAL RIBOSOME IN HUMAN DISEASE: SEARCHING FOR MUTATIONS IN MITOCHONDRIAL rRNA WITH HIGH DISRUPTIVE POTENTIAL: This project has been developed in collaboration with several researchers from the Mitochondrial Research Group (MRG) at the University of Newcastle (UK).
Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially-encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. The functional analysis of these mutations has been largely hampered by the lack of methods for direct manipulation of the mt-genome and the impossibility of recreating mitochondrial translation in vitro. We have developed a new analytical method, named HIA (Heterologous Inferential Analysis), for the study of the large collection of uncharacterized mitochondrial rRNA (mt-rRNA) mutations. HIA combines conservational information with functional and structural data obtained from heterologous ribosomal sources. As a result, HIA's predictive power is far superior to the traditional reliance on simple conservation indexes.
Our search through the literature for mt-12S rRNA variations possibly associated with disruption of mitochondrial function resulted in the identification of 161 different variations, corresponding to 152 different sites, almost 16% of the mt-12S rRNA molecule. HIA revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive potential on the function of the mitoribosome. Specifically, out of the 46 extremely rare variations analyzed, we found 1 whose disruptive potential is "unlikely disruptive", 18 which cannot be classified due to local structural differences between the mitochondrial and the heterologous ribosomes ("undetermined"), 14 which are regarded as “NEE” (not enough evidence), 5 which are "likely disruptive”, and 9 which are "expectedly disruptive". In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data has been used to predict the pathogenicity of mt-rRNA mutations. This is the case of the 255G>A (m.902G>A) and 533U>G (m.1180T>G) mutations (23,24), whose identification in homoplasmy necessarily implies the existence of a deficient translational apparatus in the mitochondria of the affected tissues. In other cases, HIA analysis allows the prioritisation of variants for additional investigation. Eventually, HIA-inspired investigation of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool. The results of this analysis has been recently reported (25).
We are currently performing HIA with variations mapping to mt-16S rRNA, the RNA component of the mitochondrial large ribosomal subunit (mt-LSU). A total of 181 mt-16S rRNA variations have been analyzed of which 82 are extremely rare and are being subjected to HIA.
Finally, as a byproduct of HIA we have performed comparative analysis of all the rRNA genes present in the large collection of GenBank mt-genomes (about 18,000 as of February 2013). We are currently performing a thorough statistical regression analysis of the correlation between rRNA conservation and the number of variant appearances in GenBank. The analysis shows a clear correlation between the two variables, in agreement with the well-known conservation of rRNA structure/function observed in other systems. We believe that these data will have important implications for the understanding of the structure and function of the mitoribosome and, as a result, will also contribute to our understanding of its involvement in human disease and ageing.
CONCLUSIONS: This research provides, for the first time, a method for scoring the disruptive and, in some cases, the pathogenic potential of a subset of mitochondrial DNA mutations, namely those mapping to the rRNA genes. This comes at a time when the involvement of mitochondrial DNA mutations in human disease is gaining an increasing recognition within the scientific and medical community.
As expected, the addition of Specific Aim III at the end of year I, has ensured that publishable results are available at the end of the funding period. Regarding the other two Specific Aims, a portion of the proposed work could not be completed during the funding period. This is partly due to experimental setbacks and to the restrictions imposed by the Spanish economic situation, which resulted in the lack of vital human and physical resources initially ascribed to the project. Despite these facts, we have made enormous progress towards the achievement of the proposed goals. Such progress consists of both positive experimental results and the development of important molecular tools. Notably, new funding has been secured to ensure that the core of the proposed goals will be completed within a reasonable amount of time via the continuation of AVS's contract at UDC.
ADDITIONAL WORK NOT DESCRIBED IN THE ORIGINAL PROPOSAL:
RIBOSOME PROFILING: We have started the analysis of dengue infection by means of Ribosome Profiling, as prompted by the ILIR grant achieved in collaboration with NAMRU-6, Perú. AVS's participation in this work was proposed and approved as part of the modifications made to the original proposal after year I. AVS has been a crucial part in establishing the experimental and bioinformatic protocols used in this project. So far, the results are highly promising and we expect to publish them in a high-profile journal soon.
DNA BARCODING OF SPELEOTHEM ASSOCIATED ORGANISMS: In collaboration with the Geological Institute Parga Pondal at UDC and the UDC associated company AllGenetics & Biology SL., we have started the characterization of the micro-flora associated with speleothemes in Galician granitic caves by DNA barcoding (26). The initial results show a highly complex bacterial community associated to these formations. We are currently analyzing the presence of other organisms within the micro-flora. Our goal is to establish whether or not speleothem formation requires the action of microbes. We believe that this research has the potential to uncover new biological processes of industrial interest.
OVERALL CONCLUSIONS AND SOCIO-ECONOMIC IMPACT OF THE PROJECT: The work developed under the 3 Specific Aims above has important economic and medical implications. On the one hand, Specific Aims I and II are related to the study of some of the weapons used by Gram negative bacteria during infectious and invasive processes. As a result, these studies could have important implications for the development of new strategies to control bacterial populations. On the other hand, Specific Aim III is devoted to the study of potentially harmful mutations in mitochondrial DNA, affecting the function of the mitoribosome. Thus, our studies are expected to result in powerful diagnostic tools for mitochondrial disease.
DISSEMINATION OF RESULTS: Specific Aim III has yielded the most significant achievement so far, namely the recent publication of a paper in the high-impact journal Human Molecular Genetics (Impact factor: 7.692 5-Yr impact factor: 7.541) entitled “The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential”. We expect at least three more manuscripts that will follow up on the published results.
Regarding Specific Aims I and II, the work is well advanced but not yet ready for publication. We expect to complete this work within the time frame created by the lab's new funding situation. In both cases, we expect to obtain high-profile publications.
In addition, AVS has participated in the international meeting “Targeting Mitochondria 2012” in Berlin, with a poster presentation. AVS has also been invited to give a presentation at the “Institut für Biologie und Biotechnologie der Pflanzen” of the University of Münster, Germany, entitled “The role of the mitochondrial ribosome in human disease: Analysis of mutations in 12S mitochondrial rRNA with a high disruptive potential”. This same topic has been recently featured in an interview by a local radio station.
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