Identification of amino acid residues critical for distinguishing mono- and di-Carboxylate substrates in JEN 1

The knowledge of the mechanisms underlying the transport of carboxylic acids is crucial towards an efficient biological production of carboxylates which have been used for many years in industry namely for the production of biodegradable polymers and as substitute for petroleum-derived chemicals. In Saccharomyces cerevisiae, Jen1p is major monocarboxylate H+ symporter specific primarily for lactate, pyruvate and for acetate (Casal et al, 1999). A phylogenetic tree of ScJen1p homologues (Casal et al, 2008) showed the existence of two main clusters: a Jen1 group of proteins (monocarboxylate transporters) and a Jen2-like proteins (dicarboxylate transporters). In this work, we rationally design, combine and analyse novel mutations in two conserved regions located in TMS5 and TMS11 of Jen1p, which we predicted to affect more dramatically Jen1p specificity. The domain in TMS5 was identified by structure/function studies based on phylogenetic molecular comparisons among Jen1p homologues with different specificities and is critical for distinguishing mono- and di-carboxylate permeases. The conserved aminoacids in TMS11 domain pointed to the importance of this domain that was demonstrated to be involved in substrate binding. We thus identify several residues critical for Jen1p activity, among which some also function as critical specificity determinants for the distinction of mono- from di-carboxylates which constitutes a first step towards the elucidation and genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily (TC#2.A.1.12.2) and a tool for the in silico prediction of the function of Jen1p homologues in other fungi of industrial importance.Fundação para a Ciência e a Tecnologia (FCT) SFRH/BPD/22976/2005 (ISS) and SFRH/BD/61530/2009 (JSP

Structural-functional studies of plasma membrane carboxylate transporters in yeast

Tese de doutoramento Programa Doutoral em Biologia Molecular e Ambiental (especialidade em Biotecnologia Molecular)Carboxylic acids comprise a heterogeneous group of compounds that are important in overall cell functionality. They can be both carbon sources and waste products from cell metabolism. This thesis aimed at deepening the current knowledge on microbial monocarboxylate permeases through structural-functional approaches. A first approach was to investigate molecular determinants in Jen1p, a Saccharomyces cerevisiae lactate/proton symporter and its homologues. We have rationally designed and analysed several mutations in TMS-II, TMS-V and TMS-XI, predicted to be involved in substrate specificity and function. From the residues analysed we verified that F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from dicarboxylates, while R188 (TMS-II) and N501 (TMS-XI) were essential residues for function. Using a model based on Jen1p overall structural similarity with the GlpT permease, we showed that all polar residues critical for function in TMS-VII and TMS-XI are aligned in an imaginary axis that lies parallel to the protein pore. Furthermore, substrate docking studies revealed a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function (R188, H383, N501 and Q498). Aiming at obtaining a lactic acid producer strain of S. cerevisiae the l-LDH gene from Lactobacillus casei was expressed in S. cerevisiae W303-1A and in the isogenic mutants jen1Δ, ady2Δ and jen1Δ ady2Δ. The monocarboxylate permeases Jen1 and Ady2 were shown to be modulators of lactic acid production. In this work, an additional role in lactate uptake was demonstrated for Ady2. The Ady2 Escherichia coli homologue SatP (YaaH) from the AceTr family of acetate transporters was also characterized in the scope of this work. This transporter is highly specific for acetic acid (a monocarboxylate) and for succinic acid (a dicarboxylate), with affinity constants at pH 6.0 of 1.24 ± 0.13 mM for acetic acid and 1.18 ± 0.10 mM for succinic acid. In glucose-grown cells the ΔyaaH mutant is compromised for the uptake of both labelled acetic and succinic acids. SatP, together with ActP, described previously as an acetate transporter, affect the use of acetic acid as sole carbon and energy source. We have also demonstrated the critical role of SatP amino acid residues L131 and A164 on the enhanced ability to transport lactate. Additionally, AcpA from the filamentous fungus Aspergillus nidulans, also from the AceTr family of transporters, was shown to mediate the uptake of propionate, as well as of other short-chain monocarboxylic acids (benzoate, formate and butyrate). We have shown that the expression of this permease is activated upon conidiospore germination, reaching its maximum at the time of germ tube emergence, and is present at basal levels in germlings and young mycelium. Furthermore, we showed that although ammonia increases moderately AcpA-mediated acetate uptake, AcpA is not involved in ammonia export.Os ácidos carboxílicos são um grupo heterogéneo de compostos de grande relevância bioquímica uma vez que podem ser usados como única fonte de carbono e energia por vários seres vivos ou constituir sub-produtos do metabolismo celular. O trabalho descrito na presente tese pretendeu aprofundar o conhecimento sobre a estrutura e a funcionalidade de proteínas membranares transportadoras de ácidos monocarboxílicos em células de microrganismos. Numa primeira abordagem realizaram-se estudos moleculares sobre a permease de lactato/piruvato de Saccharomyces cerevisiae Jen1 e seus homólogos. Foram analisadas mutações em resíduos de aminoácidos potencialmente envolvidos na definição da especificidade para o substrato (ácidos mono- ou dicarboxílicos), localizados nos domínios transmembranares II, V e XI. Este estudo revelou que os aminoácidos F270 (TMS-V) e Q498 (TMS-XI) são cruciais para a especificidade deste transportador e determinam a distinção entre ácidos mono- e dicarboxílicos. Verificou-se igualmente que os resíduos N501 (TMS-XI) e R188 (TMS-II) são essenciais para estrutura da proteína Jen1, e determinam a sua funcionalidade. Foi construído um modelo da estrutura 3D desta proteína usando as similaridades estruturais com a permease GlpT. Neste modelo os aminoácidos críticos para a função encontravam-se alinhados num eixo paralelo ao poro da proteína validando os resultados obtidos in vivo com os respetivos mutantes. A modelação da proteína com lactato revelou uma possível rota de translocação do ácido, que inclui resíduos polares identificados experimentalmente como funcionalmente importantes (R188, H383, N501 and Q498). Abordou-se ainda o papel biotecnológico das permeases de ácidos carboxílicos de S. cerevisiae (Jen1 e Ady2) na bioprodução de ácido láctico. Para tal, a enzima L-lactato desidrogenase (L-LDH) de Lactobacillus casei foi expressa em células da levedura S. cerevisiae W303-1A, incluindo as estirpes mutantes jen1Δ, ady2Δ e jen1Δ ady2Δ. Estes estudos comprovaram o papel destas permeases no exporte de ácido láctico. Os resultados obtidos permitiram ainda identificar o papel da permease Ady2 no transporte de lactato. No âmbito desta tese estudou-se ainda o homólogo da proteína Ady2 na bactéria Escherichia coli, codificado pelo gene yaaH, ambos membros da família de transportadores AceTr. Os resultados demonstraram que o transportador YaaH (SatP) é específico para succinato (um ácido dicarboxílico) e para acetato (um ácido monocarboxílico) com as seguintes constantes de afinidade a pH 6.0: 1.24 ± 0.13 mM para o ácido acético e 1.18 ± 0.10 mM para o ácido sucínico. O mutante ΔyaaH apresenta menor capacidade de transporte de ácido acético e sucínico em células crescidas em glucose. Para além disso, o transportador SatP, juntamente com o transportador anteriormente descrito ActP, afetaram a utilização de ácido acético como única fonte de carbono e energia. Em SatP os aminoácidos L131 e A164 revelaram-se fundamentais para a capacidade de transportar ácido láctico. Os estudos foram ainda estendidos ao transportador AcpA do fungo filamentoso Aspergillus nidulans, também um membro da família AceTr. O seu perfil de especificidade foi analisado revelando que esta permease para além de mediar o transporte de acetato, medeia o transporte de propionato bem como de outros ácidos monocarboxílicos de cadeia curta (benzoato, formato e butirato). Observou-se que esta permease é expressa na germinação dos conidiósporos, atingindo um máximo de expressão aquando do aparecimento do tubo germinativo, mantendo-se a um nível basal no aparecimento do micélio. Para além disso, demonstrou-se que apesar de a amónia aumentar moderadamente o transporte de acetato mediado por AcpA, esta proteína não está envolvida no exporte de amóni

Identification of amino acid residues critical for the substrate translocation in lactate permease JEN1p of saccharomyces cerevisiae

Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate:H+ symporter specific primarily for lactate, pyruvate and for acetate (TC # 2.A.1.12.2) (Casal et al., 1999). A phylogenetic tree of ScJen1p homologues (Casal et al., 2008) showed the existence of two main clusters: a Jen1 group (monocarboxylate transporters) and a Jen2-like (dicarboxylate transporters). Structure-function relationships in Jen1p have been approached by using a rational mutational analysis of conserved amino acid residues (Soares-Silva et al., 2007). Analysis of the conserved sequence 379NXX[S/T]HX[S/T]QDXXXT391, located in transmembrane segment seven (TMS-VII), showed that residues N379, H383 or D387 are necessary for function and specificity, while Q386 is important for the kinetics of Jen1p-mediated transport. In this work, we rationally designed and analyzed novel mutations in conserved regions located in TMS-II, TMS-V and TMS-XI of Jen1p, which we predicted to affect Jen1p specificity (distinction between mono and dicarboxylates) and function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is critical for function. Using a model based on Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI are aligned along the protein pore and substrate docking studies reveal a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function. Overall, our results constitute a first step towards the genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily and a tool for the in silico prediction of the function of Jen1p homologues in other fungi (Soares-Silva et al., 2011).I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

A substrate translocation trajectory in the monocarboxylate/h+ symporter jen1

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387, located in transmembrane segment VII (TMS-VII), is part of the substrate translocation pathway. In this work, we rationally design and analyse novel mutations concerning residues in TMS-V and TMS-XI. Our analysis identifies several residues critical for Jen1p function. Among these, F270 (TMS-V) and Q498 (TMS-XI) function as specificity determinants for the distinction of mono- from di-carboxylates, whereas N501 is irreplaceable for function. Using a novel theoretical model created on the basis of Jen1p similarity with GltP permease, we demonstrate that all polar residues in TMS-VII and TMS-XI, shown previously and herein to be critical for function and/or specificity (N379, H383, D387, Q498, N501), are perfectly aligned in a row along an imaginary axis that lies parallel to a protein pore. The model also predicts that the flexible side-chain of an additional polar residue, R188 in TMSII, faces the pore and subsequent mutational analysis showed that this aminoacid, similar to most polar residues of the pore, is irreplaceable for function. Finally, our model shows that the location of F270 and Q498 could justify their role in substrate specificity. Independent substrate docking approaches reveal a ‘trajectory-like’ displacement of the substrate within the Jen1p pore. In this inward-facing trajectory the flexible side-chain of R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

Identification of amino acid residues critical for distinguishing mono- and di-carboxylate permeases in the lactate/pyruvate:H+symporter subfamily

Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies and, more recently, lactate as been used for production of biodegradable polymers and as substitute for petroleum-derived chemicals. Understanding in detail the mechanisms underlying the transport of carboxylic acids is crucial towards an efficient biological production of these compounds. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate H+ symporter specific primarily for lactate, pyruvate and for acetate, encoded by the JEN1 gene (TC 2.A.1.12.2) (Casal et al, 1999). A phylogenetic tree of ScJen1p homologues (Casal et al, 2008) showed the existence of two main clusters: a Jen1 group of proteins (monocarboxylate transporters) and a Jen2-like proteins (dicarboxylate transporters). By homology threading of the Jen1p with the LacY permease we were able to obtain a 3D dimensional model, that together with site directed mutagenesis strategies, pointed to the existence of common structure between these two permeases. Furthermore, we have also shown that a highly conserved motif in 7th transmembrane segment (TMS7) is part of the substrate translocation pathway (Soares-Silva et al, 2007). Conserved mutations in this motif affect the kinetics of Jen1p, as well as, its specificity towards physiological substrates. In this work, we rationally design, combine and analyse novel mutations in two other conserved regions located in TMS5 and TMS11 of Jen1p, which we predicted to affect more dramatically Jen1p specificity. The domain in TMS5 was identified by structure/function studies based on phylogenetic molecular comparisons among Jen1p homologues with different specificities and is critical for distinguishing mono- and di-carboxylate permeases. The conserved aminoacids in TMS11 domain pointed to the importance of this domain that was demonstrated to be involved in substrate binding. We thus identify several residues critical for Jen1p activity, among which some also function as critical specificity determinants for the distinction of mono- from di-carboxylates. Overall, our results constitute a first step towards the elucidation and genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily (TC#2.A.1.12.2) and a tool for the in silico prediction of the function of Jen1p homologues in other fungi of industrial importance.Fundação para a Ciência e a Tecnologia (FCT) fellowships SFRH/BPD/22976/2005 (ISS) and SFRH/BD/61530/2009 (JSP

Discovery and initial characterization of members of the new YaaH family of microbial acetate transporters

The emergence of probiotics and prebiotics has revived the importance of short-chain fatty acids (SCFAs) associated to colonic and systemic health improvement. Although biosynthesis and degradation of SCFAs and other short-chain carboxylic acids, such as lactate, pyruvate or citrate are well understood, the transport of these acids is still a matter of discussion. The presence of SCFAs transporters in cellular membranes is ubiquitous, displaying great level of homology among Bacteria, Archaea and Eukaryotes, indicating the ancient nature of these transporters and their high level of conservation. The mechanism of substrate uptake of these transporters including specificity, kinetics and bioenergetic studies is a field poorly explored. This work constitutes a first approach to establish the mode of action of the SCFA transporters specifically those belonging to the YaaH family. The YaaH family, (TC# 2.A.96, http://www.tcdb.org/tcdb) is presumed to be a family of acetate transporters. Its members possess 6 putative transmembrane span domains and are spread by the 3 domains of life: Bacteria, Eukaryotes and Archaea. This work aims at studying the YaaH protein from E. coli as well as it homologues from the yeast Saccharomyces cerevisiae (ScAdy2) and the fungi Aspergillus nidulans (AcpA). We have constructed a disrupted E. coli strain in the yaaH gene showing that the yaaH mutant cells are compromised for the uptake of the labelled acetic acid in comparison with the isogenic wt strain. This is the first experimental data that demonstrates the physiological role of the yaaH gene in the transport of acetate in bacteria. Using the yeast and fungi strains we were able to measure the kinetic parameters associated with these transporters and assign a specificity profile to this family. These transporters are specific primarily to acetate and are inhibited by other short chain acids such as benzoic, formic, propionic and butyric acids.FCT PhD grant SFRH/BD/61530/2009 (JSP

Following Darwin’s footsteps: Evaluating the impact of an activity designed for elementary school students to link historically important evolution key concepts on their understanding of natural selection

While several researchers have suggested that evolution should be explored from the initial years of schooling, little information is available on effective resources to enhance elementary school students’ level of understanding of evolution by natural selection (LUENS). For the present study, we designed, implemented, and evaluated an educational activity planned for fourth graders (9 to 10 years old) to explore concepts and conceptual fields that were historically important for the discovery of natural selection. Observation field notes and students’ productions were used to analyze how the students explored the proposed activity. Additionally, an evaluation framework consisting of a test, the evaluation criteria, and the scoring process was applied in two fourth-grade classes (N = 44) to estimate elementary school students’ LUENS before and after engaging in the activity. Our results show that our activity allowed students to link the key concepts, resulting in a significant increase of their understanding of natural selection. They also reveal that additional activities and minor fine-tuning of the present activity are required to further support students’ learning about the concept of differential reproduction.info:eu-repo/semantics/publishedVersio

2-hydroxylation of Acinetobacter baumannii lipid A contributes to virulence

Acinetobacter baumannii causes a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasing isolation of multidrug resistant strains. There is a major gap in knowledge on the infection biology of A. baumannii, and only few virulence factors have been characterized including the lipopolysaccharide. The lipid A expressed by A. baumannii is hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of the lipid A have been already characterized. Here we report the characterization of A. baumannii LpxO, which encodes the enzyme responsible for the 2-hydroxylation of the lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyses the 2-hydroxylation of the laurate transferred by A. baumannii LpxL. LpxO-dependent lipid A 2-hydroxylation protects A. baumannii from polymyxin B, colistin, and human β-defensin 3. LpxO contributes to survival of A. baumannii in human whole blood, and is required for pathogen survival in the waxmoth Galleria mellonella LpxO also protects Acinetobacter from G. mellonella antimicrobial peptides and limits the expression of them. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of the lipid A limits the activation of MAPK JNK to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine IL-10 via the activation of the transcriptional factor CREB. IL-10, in turn, limits the production of inflammatory cytokines following A. baumannii infection. Altogether, our studies suggest that LpxO is a candidate to develop anti A. baumannii drugs.</p

A Klebsiella pneumoniae antibiotic resistance mechanism that subdues host defences and promotes virulence

Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Recent studies highlight the emergence of multidrug-resistant K.\ua0pneumoniae strains which show resistance to colistin, a last-line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB-associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K.\ua0pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ-governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFβ-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria