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1.
Microb Drug Resist ; 27(1): 1-2, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33464170
2.
PLoS Negl Trop Dis ; 14(12): e0008878, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33264290

RESUMEN

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.

3.
Proc Natl Acad Sci U S A ; 117(49): 31398-31409, 2020 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-33229580

RESUMEN

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.

4.
Mol Microbiol ; 2020 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-32979868

RESUMEN

Lipoproteins are characterized by a fatty acid moiety at their amino-terminus through which they are anchored into membranes. They fulfill a variety of essential functions in bacterial cells, such as cell wall maintenance, virulence, efflux of toxic elements including antibiotics, and uptake of nutrients. The posttranslational modification process of lipoproteins involves the sequential action of integral membrane enzymes and phospholipids as acyl donors. In recent years, the structures of the lipoprotein modification enzymes have been solved by X-ray crystallography leading to a greater insight into their function and the molecular mechanism of the reactions. The catalytic domains of the enzymes are exposed to the periplasm or external milieu and are readily accessible to small molecules. Since the lipoprotein modification pathway is essential in proteobacteria, it is a potential target for the development of novel antibiotics. In this review, we discuss recent literature on the structural characterization of the enzymes, and the in vitro activity assays compatible with high-throughput screening for inhibitors, with perspectives on the development of new antimicrobial agents.

5.
J Biol Chem ; 295(49): 16785-16796, 2020 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-32978253

RESUMEN

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15-25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host-pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

6.
FEMS Microbiol Rev ; 2020 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-32897324

RESUMEN

Microbiota, and the plethora of signalling molecules that they generate, are a major driving force that underlies a striking range of inter-individual physioanatomic and behavioural consequences for the host organism. Among the bacterial effectors, one finds peptidoglycan, the major constituent of the bacterial cell surface. In the steady-state, fragments of peptidoglycan are constitutively liberated from bacterial members of the gut microbiota, cross the gut epithelial barrier and enter the host system. The fate of these peptidoglycan fragments, and the outcome for the host, depends on the molecular nature of the peptidoglycan, as well the cellular profile of the recipient tissue, mechanism of cell entry, the expression of specific processing and recognition mechanisms by the cell, and the local immune context. At the target level, physiological processes modulated by peptidoglycan are extremely diverse, ranging from immune activation to small molecule metabolism, autophagy and apoptosis. In this review, we bring together a fragmented body of literature on the kinetics and dynamics of peptidoglycan interactions with the mammalian host, explaining how peptidoglycan functions as a signaling molecule in the host under physiological conditions, how it disseminates within the host, and the cellular responses to peptidoglycan.

7.
Front Immunol ; 11: 2007, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32849665

RESUMEN

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response.

8.
PLoS Pathog ; 16(8): e1008639, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32790743

RESUMEN

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Leptospira/inmunología , Leptospirosis/inmunología , Lipopolisacáridos/metabolismo , Lipoproteínas/metabolismo , Antígenos O/metabolismo , Receptor Toll-Like 4/fisiología , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Citocinas/metabolismo , Femenino , Leptospirosis/metabolismo , Leptospirosis/microbiología , Leptospirosis/patología , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Lipoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/fisiología , Antígenos O/genética , Transducción de Señal , Receptor Toll-Like 2/fisiología
9.
PLoS Pathog ; 15(5): e1007811, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31107928

RESUMEN

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.


Asunto(s)
Memoria Inmunológica/inmunología , Leptospira interrogans/inmunología , Leptospirosis/prevención & control , Macrófagos Peritoneales/inmunología , Proteína Adaptadora de Señalización NOD2/agonistas , Bibliotecas de Moléculas Pequeñas/farmacología , Receptor Toll-Like 2/agonistas , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Memoria Inmunológica/efectos de los fármacos , Leptospirosis/inmunología , Leptospirosis/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal
10.
PLoS Pathog ; 13(12): e1006725, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29211798

RESUMEN

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Evasión Inmune , Leptospira interrogans/inmunología , Leptospira interrogans/patogenicidad , Lipoproteínas/metabolismo , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Peptidoglicano/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Femenino , Humanos , Evasión Inmune/genética , Inmunidad Innata , Leptospira/inmunología , Leptospira interrogans/genética , Leptospirosis/genética , Leptospirosis/inmunología , Leptospirosis/microbiología , Lipoproteínas/genética , Lipoproteínas/inmunología , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Peptidoglicano/química , Peptidoglicano/inmunología , Unión Proteica , Transducción de Señal , Especificidad de la Especie , Virulencia/genética , Virulencia/inmunología
11.
Nat Commun ; 8(1): 776, 2017 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-28974686

RESUMEN

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the "divisome") and lateral wall growth (the "elongasome"). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.


Asunto(s)
Proteínas Bacterianas/química , Pared Celular/metabolismo , Helicobacter pylori/genética , Proteínas de Unión a las Penicilinas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Helicobacter pylori/metabolismo , Modelos Moleculares , Proteínas de Unión a las Penicilinas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia
12.
Sci Rep ; 7(1): 9909, 2017 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-28855660

RESUMEN

Helicobacter pullorum is an avian bacterium that causes gastroenteritis, intestinal bowel and hepatobiliary diseases in humans. Although H. pullorum has been shown to activate the mammalian innate immunity with release of nitric oxide (NO), the proteins that afford protection against NO and reactive nitrogen species (RNS) remain unknown. Here several protein candidates of H. pullorum, namely a truncated (TrHb) and a single domain haemoglobin (SdHb), and three peroxiredoxin-like proteins (Prx1, Prx2 and Prx3) were investigated. We report that the two haemoglobin genes are induced by RNS, and that SdHb confers resistance to nitrosative stress both in vitro and in macrophages. For peroxiredoxins, the prx2 and prx3 expression is enhanced by peroxynitrite and hydrogen peroxide, respectively. Mutation of prx1 does not alter the resistance to these stresses, while the single ∆prx2 and double ∆prx1∆prx2 mutants have decreased viability. To corroborate the physiological data, the biochemical analysis of the five recombinant enzymes was done, namely by stopped-flow spectrophotometry. It is shown that H. pullorum SdHb reacts with NO much more quickly than TrHb, and that the three Prxs react promptly with peroxynitrite, Prx3 displaying the highest reactivity. Altogether, the results unveil SdHb and Prx3 as major protective systems of H. pullorum against nitrosative stress.


Asunto(s)
Infecciones por Helicobacter/microbiología , Helicobacter/patogenicidad , Estrés Nitrosativo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Helicobacter/genética , Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Humanos , Intestinos/microbiología , Intestinos/patología , Hígado/microbiología , Hígado/patología , Macrófagos/metabolismo , Macrófagos/microbiología , Viabilidad Microbiana/genética , Mutación , Óxido Nítrico/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Virulencia
13.
Antibiotics (Basel) ; 6(1)2017 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-28241458

RESUMEN

Lytic transglycosylases (Lts) are involved in recycling, cell division, and metabolism of the peptidoglycan. They have been understudied for their usefulness as potential antibacterial targets due to their high redundancy in Gram-negative bacteria. Bulgecin A is an O-sulphonated glycopeptide that targets primarily soluble lytic tranglycosylases (Slt). It has been shown that bulgecin A increases the efficacy of ß-lactams that target penicillin bindings proteins (PBPs). Here, we present the high-resolution crystal structure of LtgA from Neisseria meningitidis strain MC58, a membrane bound homolog of Escherichia coli Slt, in complex with bulgecin A. The LtgA-bulgecin A complex reveals the mechanism of inhibition by bulgecin A at near atomic resolution. We further demonstrate that bulgecin A is not only a potent inhibitor of LtgA, but most importantly, it restores the efficacy of ß-lactam antibiotics in strains of N. meningitidis and Neisseria gonorrhoeae that have reduced susceptibility to ß-lactams. This is particularly relevant for N. gonorrhoeae where no vaccines are available. This work illustrates how best to target dangerous pathogens using a multiple drug target approach, a new and alternative approach to fighting antibiotic resistance.

14.
J Am Chem Soc ; 139(15): 5330-5337, 2017 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-28333455

RESUMEN

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.


Asunto(s)
Amidohidrolasas/metabolismo , Bacillus anthracis/enzimología , Bacillus cereus/enzimología , Carbono/metabolismo , Prolina/metabolismo , Amidohidrolasas/química , Amidohidrolasas/aislamiento & purificación , Sitios de Unión , Carbono/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Hidroxilación , Modelos Moleculares , Prolina/química
15.
Microb Drug Resist ; 22(6): 477-86, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27447281

RESUMEN

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.


Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/patología , Helicobacter pylori/enzimología , Peptidoglicano/metabolismo , Amidohidrolasas/genética , Amoxicilina/farmacología , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , División Celular , Expresión Génica , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ácidos Murámicos/química , Ácidos Murámicos/metabolismo , Mutación , Peptidoglicano/química , Estómago/efectos de los fármacos , Estómago/microbiología , Estómago/patología , Virulencia
17.
PLoS Genet ; 11(7): e1005338, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26162030

RESUMEN

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.


Asunto(s)
Adaptación Fisiológica/genética , Estructuras de la Membrana Celular/inmunología , Moraxella catarrhalis/genética , Neisseria meningitidis/genética , Mucosa Respiratoria/microbiología , Evolución Biológica , Proteínas de Ciclo Celular/genética , Humanos , Moraxella catarrhalis/inmunología , Moraxella catarrhalis/fisiología , Nasofaringe/microbiología , Neisseria meningitidis/inmunología , Neisseria meningitidis/fisiología , Peptidoglicano/química , Peptidoglicano/inmunología , Mucosa Respiratoria/inmunología
18.
Science ; 349(6251): 989-93, 2015 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-26160380

RESUMEN

Changes to the symbiotic microbiota early in life, or the absence of it, can lead to exacerbated type 2 immunity and allergic inflammations. Although it is unclear how the microbiota regulates type 2 immunity, it is a strong inducer of proinflammatory T helper 17 (T(H)17) cells and regulatory T cells (T(regs)) in the intestine. Here, we report that microbiota-induced T(regs) express the nuclear hormone receptor RORγt and differentiate along a pathway that also leads to T(H)17 cells. In the absence of RORγt(+) T(regs), T(H)2-driven defense against helminths is more efficient, whereas T(H)2-associated pathology is exacerbated. Thus, the microbiota regulates type 2 responses through the induction of type 3 RORγt(+) T(regs) and T(H)17 cells and acts as a key factor in balancing immune responses at mucosal surfaces.


Asunto(s)
Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestinos/microbiología , Microbiota/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Colitis Ulcerosa/inmunología , Colon/inmunología , Colon/microbiología , Vida Libre de Gérmenes , Homeostasis , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Intestinos/inmunología , Ratones , Modelos Inmunológicos , Nematospiroides dubius , Organismos Libres de Patógenos Específicos , Infecciones por Strongylida/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th2/inmunología , Vitamina A/metabolismo
19.
PLoS Negl Trop Dis ; 8(12): e3359, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25474719

RESUMEN

Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts.


Asunto(s)
Enfermedades Renales/microbiología , Leptospira interrogans/aislamiento & purificación , Leptospirosis/microbiología , Imagen Óptica/métodos , Animales , Femenino , Enfermedades Renales/sangre , Leptospira interrogans/genética , Leptospirosis/sangre , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Sepsis/microbiología
20.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 10): 2631-9, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25286847

RESUMEN

Peptidoglycan O-acetylesterase (Ape1), which is required for host survival in Neisseria sp., belongs to the diverse SGNH hydrolase superfamily, which includes important viral and bacterial virulence factors. Here, multi-domain crystal structures of Ape1 with an SGNH catalytic domain and a newly identified putative peptidoglycan-detection module are reported. Enzyme catalysis was performed in Ape1 crystals and key catalytic intermediates along the SGNH esterase hydrolysis reaction pathway were visualized, revealing a substrate-induced productive conformation of the catalytic triad, a mechanistic detail that has not previously been observed. This substrate-induced productive conformation of the catalytic triad shifts the established dogma on these enzymes, generating valuable insight into the structure-based design of drugs targeting the SGNH esterase superfamily.


Asunto(s)
Esterasas/química , Esterasas/metabolismo , Neisseria meningitidis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Peptidoglicano/metabolismo , Conformación Proteica
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