Functional Identification of Proteus mirabilis eptC gene encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains

The polar and lateral flagella from Plesiomonas shigelloides are glycosylated with legionaminic acid

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now

Estudio de la biosíntesis del núcleo de lipopolisacarido (LPS) en "Proteus mirabilis"

[eng] Urinary tract infection (UTIs) is an extremely common disease. Proteus mirabilis is a common cause of UTI in individuals with functional or structural abnormalities or with long-term catheterization, it forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are flagella, fimbriae, outer membrane proteins, hemolysins, amino acid deaminase, protease, capsule and lipopolysaccharide (LPS). Study of LPS core is particularly relevant for several reasons: it is a conserved region, although it is increasingly clear that there is some variability at the genus or groups of similar genera, its chemical structure modulates the endotoxic activity of lipid A, alteration of the LPS core, which generates less virulent bacteria, encourages the search of substances that interfere with the biosynthesis of this region, and conserved regions of the core LPS could be useful as antigens in preventing diseases caused by pathogens that contain these conserved regions. The specific aims of this project have been to identify and functionally characterize genes involved in core LPS biosynthesis in P. mirabilis, to elucidate the mechanism of incorporation of galactosamine (GalN) to the core LPS, to identify genes coding for phosphoethanolamine (PEtN) modifications, and to characterize and to study the biological effects of the gene encoding the PEtN transferase involved in the modification of the second heptose residue (L,D-HepII). We found that P. mirabilis has most of the genes for the biosynthesis of LPS core grouped in the waa cluster in the chromosome. Despite this, additional genes required for core LPS biosynthesis are found outside the waa cluster. The pentasaccharide of the inner core, shared by all Enterobacteriaceae, is biosynthesized in P. mirabilis, by the sequential activity of a bifunctional transferase (WaaA) and three heptosyltransferases (WaaC, WaaF, and WaaQ). These enzymes are transcribed from genes located inside the waa cluster, and are conserved in P. mirabilis strains analyzed; for more, they show a high identity and similarity level to homologues proteins of Escherichia coli, Klebsiella penumoniae and Serratia marcescens. The waaL gene, coding for the O-antigen polymerase ligase, is found adjacent to the classic waa cluster. Downstream this gene, four genes encoding enzymes belonging to the 4 (walM, walN, and WalR), and 9 (walO) glycosyltransferase family were found. Even if members of these families were related to LPS core biosynthesis in several Gram-negative bacteria, in P. mirabilis they do not appear to be involved in the biosynthesis of the reported core LPS structures. The presence of the disaccharide hexosamine (HexN)-1,4-galacturonic acid (GalA) is a feature of P. mirabilis LPS outer core. Depending on the nature of the HexN outer core residue, two different homologues for N-acetyl-hexosamine transferases are present in the waa cluster: wabH or wabP. Altought the incorporation of glucosamine into LPS core requires an acetylglucosaminyltransferase (WabH) and a deacetilase (WabN), the incorporation of GalN requires three enzymes: an acetylgalactosaminyltransferase (WabP), a deacetilase (WabN) and an epimerase (gne). An amplification test with specific primers for this two different homologues can be used to predict the HexN nature in P. mirabilis LPS cores. The strain-specific genes wamB and wamC code for a galactosyltransferase and a heptosyltransferase respectively in strain R110 of P. mirabilis. The enzyme encoded by gene wamD is a N-acetylglucosaminyltransferase, and it is found in strain 51/57 of P. mirabilis. WamA, coded by wamA gene in the waa cluster of strains R110, 50/57, TG83 and HI4320, is a heptosyltransferase responsible for the incorporation of a quarter residue of heptose (Hep), in DD configuration, to the GalA II of the outer core. In P. mirabilis strain 51/57, a gene coding a protein of the Mig-14 family was identified inside the waa cluster, this localization appears to be an exception in the Enterobacteriaceae family. Inspection of the whole genome of P. mirabilis HI4320 did not allow the identification of a mig-14 similar gene. There are three putative PEtN transferases in the genome of P. mirabilis: PMI3040, PMI3576, and PMI3104. The gene identified as eptC (PMI3104) transfers the moiety of PEtN to the O-6 position of L,D-Hep II (HepII6PEtN). The absence of the positive charge due to PEtN residue doesn't affect the bacterial growth kinetics in lab conditions in rich or defined media, but causes a moderate destabilization of the outer-membrane. Despite the lack of the PEtN residue on the Hep II in P. mirabilis LPS core, has no statistically effects during urinary tract infection assays in mouse model, the absence of this modification causes an increase sensitivity to complement in non-immune human sera.[spa] P. mirabilis no es una causa frecuente de infecciones urinarias en el huésped normal, más bien infecta el tracto urinario con alteraciones funcionales o anatómicas, o instrumentación crónica como el cateterismo. P. mirabilis está a menudo asociado con cálculos urinarios e incrustaciones de los catéteres y es, particularmente importante, en pacientes con cateterización prolongada. Las infecciones del tracto urinario asociadas a cateterización son mundialmente reconocidas como la causa más común de infección asociada a tratamientos en ambiente hospitalario. El LPS es un factor de virulencia importante en bacterias Gram negativas patógenas. También conocido como endotoxina, es una molécula glicolipídica que constituye la estructura mayoritaria de la cara externa de la membrana externa (OM). En Proteus mirabilis la mayoría de los genes responsables de la biosíntesis de núcleo de LPS están localizados en el cromosoma, en el agrupamiento génico waa. A pesar de esto, algunos genes adicionales, necesarios para la biosíntesis del núcleo de LPS, se encuentran ubicados fuera del agrupamiento génico waa. El pentasacárido del núcleo interno, común a todas las Enterobacteriáceae, se biosintetiza en P. mirabilis, por la actividad secuencial de una transferasa bifunciona (WaaA) y tres heptosiltransferasas (WaaC, WaaF, y WaaQ). La presencia del disacárido HexN‐1,4‐GalA es característica del núcleo externo de LPS en P. mirabilis. Dependiendo de la naturaleza del residuo de HexN, se encuentran, en el agrupamiento génico waa, dos HexNAc transferasas diferentes: wabH o wabP. El gen eptC (PMI3104) codifica para la enzima que transfiere el residuo de fosfoetanolamina a la posición O-6 de la L,D-Hep II (HepII6PEtN), en el núcleo de LPS de P. mirabilis. La ausencia de la carga positiva del residuo de fosfoetanolamina no afecta a la cinética de crecimiento de las bacterias en condiciones standard de laboratorio sea en medios ricos o definidos. La ausencia del residuo fosfoetanolamina provoca una desestabilización moderada de la membrana externa que se traduce en una disminución de la MIC para SDS

Lipopolysaccharide (LPS) core biosynthesis in "Proteus mirabilis" / Estudio de la biosíntesis del núcleo de lipopolisacarido (LPS) en "Proteus mirabilis"

Urinary tract infection (UTIs) is an extremely common disease. Proteus mirabilis is a common cause of UTI in individuals with functional or structural abnormalities or with long-term catheterization, it forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are flagella, fimbriae, outer membrane proteins, hemolysins, amino acid deaminase, protease, capsule and lipopolysaccharide (LPS). Study of LPS core is particularly relevant for several reasons: it is a conserved region, although it is increasingly clear that there is some variability at the genus or groups of similar genera, its chemical structure modulates the endotoxic activity of lipid A, alteration of the LPS core, which generates less virulent bacteria, encourages the search of substances that interfere with the biosynthesis of this region, and conserved regions of the core LPS could be useful as antigens in preventing diseases caused by pathogens that contain these conserved regions. The specific aims of this project have been to identify and functionally characterize genes involved in core LPS biosynthesis in P. mirabilis, to elucidate the mechanism of incorporation of galactosamine (GalN) to the core LPS, to identify genes coding for phosphoethanolamine (PEtN) modifications, and to characterize and to study the biological effects of the gene encoding the PEtN transferase involved in the modification of the second heptose residue (L,D-HepII). We found that P. mirabilis has most of the genes for the biosynthesis of LPS core grouped in the waa cluster in the chromosome. Despite this, additional genes required for core LPS biosynthesis are found outside the waa cluster. The pentasaccharide of the inner core, shared by all Enterobacteriaceae, is biosynthesized in P. mirabilis, by the sequential activity of a bifunctional transferase (WaaA) and three heptosyltransferases (WaaC, WaaF, and WaaQ). These enzymes are transcribed from genes located inside the waa cluster, and are conserved in P. mirabilis strains analyzed; for more, they show a high identity and similarity level to homologues proteins of Escherichia coli, Klebsiella penumoniae and Serratia marcescens. The waaL gene, coding for the O-antigen polymerase ligase, is found adjacent to the classic waa cluster. Downstream this gene, four genes encoding enzymes belonging to the 4 (walM, walN, and WalR), and 9 (walO) glycosyltransferase family were found. Even if members of these families were related to LPS core biosynthesis in several Gram-negative bacteria, in P. mirabilis they do not appear to be involved in the biosynthesis of the reported core LPS structures. The presence of the disaccharide hexosamine (HexN)-1,4-galacturonic acid (GalA) is a feature of P. mirabilis LPS outer core. Depending on the nature of the HexN outer core residue, two different homologues for N-acetyl-hexosamine transferases are present in the waa cluster: wabH or wabP. Altought the incorporation of glucosamine into LPS core requires an acetylglucosaminyltransferase (WabH) and a deacetilase (WabN), the incorporation of GalN requires three enzymes: an acetylgalactosaminyltransferase (WabP), a deacetilase (WabN) and an epimerase (gne). An amplification test with specific primers for this two different homologues can be used to predict the HexN nature in P. mirabilis LPS cores. The strain-specific genes wamB and wamC code for a galactosyltransferase and a heptosyltransferase respectively in strain R110 of P. mirabilis. The enzyme encoded by gene wamD is a N-acetylglucosaminyltransferase, and it is found in strain 51/57 of P. mirabilis. WamA, coded by wamA gene in the waa cluster of strains R110, 50/57, TG83 and HI4320, is a heptosyltransferase responsible for the incorporation of a quarter residue of heptose (Hep), in DD configuration, to the GalA II of the outer core. In P. mirabilis strain 51/57, a gene coding a protein of the Mig-14 family was identified inside the waa cluster, this localization appears to be an exception in the Enterobacteriaceae family. Inspection of the whole genome of P. mirabilis HI4320 did not allow the identification of a mig-14 similar gene. There are three putative PEtN transferases in the genome of P. mirabilis: PMI3040, PMI3576, and PMI3104. The gene identified as eptC (PMI3104) transfers the moiety of PEtN to the O-6 position of L,D-Hep II (HepII6PEtN). The absence of the positive charge due to PEtN residue doesn't affect the bacterial growth kinetics in lab conditions in rich or defined media, but causes a moderate destabilization of the outer-membrane. Despite the lack of the PEtN residue on the Hep II in P. mirabilis LPS core, has no statistically effects during urinary tract infection assays in mouse model, the absence of this modification causes an increase sensitivity to complement in non-immune human sera.P. mirabilis no es una causa frecuente de infecciones urinarias en el huésped normal, más bien infecta el tracto urinario con alteraciones funcionales o anatómicas, o instrumentación crónica como el cateterismo. P. mirabilis está a menudo asociado con cálculos urinarios e incrustaciones de los catéteres y es, particularmente importante, en pacientes con cateterización prolongada. Las infecciones del tracto urinario asociadas a cateterización son mundialmente reconocidas como la causa más común de infección asociada a tratamientos en ambiente hospitalario. El LPS es un factor de virulencia importante en bacterias Gram negativas patógenas. También conocido como endotoxina, es una molécula glicolipídica que constituye la estructura mayoritaria de la cara externa de la membrana externa (OM). En Proteus mirabilis la mayoría de los genes responsables de la biosíntesis de núcleo de LPS están localizados en el cromosoma, en el agrupamiento génico waa. A pesar de esto, algunos genes adicionales, necesarios para la biosíntesis del núcleo de LPS, se encuentran ubicados fuera del agrupamiento génico waa. El pentasacárido del núcleo interno, común a todas las Enterobacteriáceae, se biosintetiza en P. mirabilis, por la actividad secuencial de una transferasa bifunciona (WaaA) y tres heptosiltransferasas (WaaC, WaaF, y WaaQ). La presencia del disacárido HexN‐1,4‐GalA es característica del núcleo externo de LPS en P. mirabilis. Dependiendo de la naturaleza del residuo de HexN, se encuentran, en el agrupamiento génico waa, dos HexNAc transferasas diferentes: wabH o wabP. El gen eptC (PMI3104) codifica para la enzima que transfiere el residuo de fosfoetanolamina a la posición O-6 de la L,D-Hep II (HepII6PEtN), en el núcleo de LPS de P. mirabilis. La ausencia de la carga positiva del residuo de fosfoetanolamina no afecta a la cinética de crecimiento de las bacterias en condiciones standard de laboratorio sea en medios ricos o definidos. La ausencia del residuo fosfoetanolamina provoca una desestabilización moderada de la membrana externa que se traduce en una disminución de la MIC para SDS

Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains

La formazione degli insegnanti e la Chimica

La Divisione di Didattica della Società Chimica Italiana è impegnata nella formazione degli insegnanti in servizio con la Scuola Nazionale di Didattica della Chimica “Giuseppe Del Re” che si svolge annualmente e di norma a San Miniato (PI), e la Scuola Permanente per l’Aggiornamento degli Insegnanti di Scienze (SPAIS), che ha luogo, sempre annualmente, in Sicilia. Per la formazione iniziale, nell’ambito della Scuola di Didattica e Ricerca Educativa “Ulderico Segre” del 2017 sono state elaborate linee guida per il conseguimento dei 24CFU

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now

The Seeds of Gotham's Doom - Eleonora Aquilini

<div>Mr. Bloom is releasing a parasite resulting in deaths across Gotham. Can Batman use his knowledge of bacteriology and parasites to save Gotham? Find out on this week's episode of School of Batman!</div><div><br></div><div>Our guest this week is Eleonora Aquilini, who has a PhD in Environmental Microbiology from the University of Barcelona and is currently a post doc at the University of Montpellier. You can find Eleonora on Twitter: twitter.com/EleoAquilini.</div><div>__________________</div><div><br></div><div>Impact Moderato by Kevin MacLeod is licensed under a Creative Commons Attribution license (creativecommons.org/licenses/by/4.0/)</div><div>Source: incompetech.com/music/royalty-fre…isrc=USUAN1100618</div><div>Artist: incompetech.com/</div><div><br></div><div>Cool Vibes - Film Noire by Kevin MacLeod is licensed under a Creative Commons Attribution license (creativecommons.org/licenses/by/4.0/)</div><div>Source: incompetech.com/music/royalty-fre…isrc=USUAN1100863</div><div>Artist: incompetech.com/</div><div><br></div><div>Mechanolith by Kevin MacLeod is licensed under a Creative Commons Attribution license (creativecommons.org/licenses/by/4.0/)</div><div>Source: incompetech.com/music/royalty-fre…isrc=USUAN1100879</div><div>Artist: incompetech.com/</div