scholarly journals An M29 Aminopeptidase from Listeria Monocytogenes Contributes to In Vitro Bacterial Growth but not to Intracellular Infection

2020 ◽  
Vol 8 (1) ◽  
pp. 110 ◽  
Author(s):  
Xian Zhang ◽  
Chiyu Guan ◽  
Yi Hang ◽  
Fengdan Liu ◽  
Jing Sun ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Optimal Growth ◽  
Defined Medium ◽  
Intracellular Infection ◽  
Physiological Processes ◽  
Wide Range ◽  
Catalytic Motif ◽  
Listeria Infection

Aminopeptidases that catalyze the removal of N-terminal residues from polypeptides or proteins are crucial for physiological processes. Here, we explore the biological functions of an M29 family aminopeptidase II from Listeria monocytogenes (LmAmpII). We show that LmAmpII contains a conserved catalytic motif (EEHYHD) that is essential for its enzymatic activity and LmAmpII has a substrate preference for arginine and leucine. Studies on biological roles indicate that LmAmpII is required for in vitro growth in a chemically defined medium for optimal growth of L. monocytogenes but is not required for bacterial intracellular infection in epithelial cells and macrophages, as well as cell-to-cell spreading in fibroblasts. Moreover, LmAmpII is found as dispensable for bacterial pathogenicity in mice. Taken together, we conclude that LmAmpII, an M29 family aminopeptidase, can efficiently hydrolyze a wide range of substrates and is required for in vitro bacterial growth, which lays a foundation for in-depth investigations of aminopeptidases as potential targets to defend Listeria infection.

scholarly journals Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

1994 ◽  
Vol 36 (4) ◽  
pp. 301-310 ◽  
Author(s):  
Maria Cristina de Cunto Brandileone ◽  
Rosemeire Cobo Zanella ◽  
Vera Simonsen Dias Vieira ◽  
Claudio Tavares Sacciii ◽  
Lucimar Gonçalves Milagres ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Culture Supernatant ◽  
Culture Media ◽  
Membrane Vesicles ◽  
Normal Growth ◽  
Growth Conditions ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Iron Deficient

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

scholarly journals Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Chelsea E. Witte ◽  
Aaron T. Whiteley ◽  
Thomas P. Burke ◽  
John-Demian Sauer ◽  
Daniel A. Portnoy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Genetic Screen ◽  
Content Type ◽  
Forward Genetic Screen ◽  
Innate Immune Signaling ◽  
Food Borne Illness ◽  
Multidrug Resistance Transporters

ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.

scholarly journals S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase–dependent hydrogen sulfide synthesis

2019 ◽  
Vol 294 (28) ◽  
pp. 11011-11022 ◽  
Author(s):  
Pramod K. Yadav ◽  
Victor Vitvitsky ◽  
Hanseong Kim ◽  
Andrew White ◽  
Uhn-Soo Cho ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Cleavage Reaction ◽  
Physiological Processes ◽  
Structural Framework ◽  
Wide Range ◽  
Mercaptopyruvate Sulfurtransferase ◽  
Substrate Inhibitor

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80–90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

scholarly journals ANTIMICROBIAL ACTIVITY OF LYSOZYME TOWARDS LISTERIA MONOCYTOGENES AT VARIOUS MEDIUM CONDITIONS

2019 ◽  
Vol 19 (1S) ◽  
pp. 161-162
Author(s):  
M N Berlov ◽  
S V Legkovoy ◽  
E S Umnyakova ◽  
V N Kokryakov
Keyword(s):  
Antimicrobial Activity ◽  
Listeria Monocytogenes ◽  
Ionic Strength ◽  
Bacterial Growth ◽  
Human Leukocyte ◽  
Antimicrobial Action ◽  
Positive Bacterium ◽  
Enzymatic Action ◽  
Low Ionic Strength

The in vitro antimicrobial action of human leukocyte lysozyme from on gram-positive bacterium Listeria monocytogenes under various medium conditions was studied. It was shown that in a low ionic strength buffer (without NaCl), lower doses of lysozyme are required to reveal the microbicidal effect than in the case of 0.075 or 0.15 M NaCl. The bacterial growth phase does not significantly affect the antimicrobial activity of lysozyme. The results obtained are consistent with the two-stage mechanism of the antimicrobial action of lysozyme, which includes enzymatic and non-enzymatic action.

scholarly journals A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting

Materials ◽  
2021 ◽  
Vol 14 (4) ◽  
pp. 858
Author(s):  
Magdalena B. Łabowska ◽  
Karolina Cierluk ◽  
Agnieszka M. Jankowska ◽  
Julita Kulbacka ◽  
Jerzy Detyna ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
3D Printing ◽  
Cell Cultures ◽  
Polymeric Materials ◽  
Optimal Growth ◽  
Vital Functions ◽  
Wide Range ◽  
In Vitro Cell Cultures ◽  
Living Tissues

Sustaining the vital functions of cells outside the organism requires strictly defined parameters. In order to ensure their optimal growth and development, it is necessary to provide a range of nutrients and regulators. Hydrogels are excellent materials for 3D in vitro cell cultures. Their ability to retain large amounts of liquid, as well as their biocompatibility, soft structures, and mechanical properties similar to these of living tissues, provide appropriate microenvironments that mimic extracellular matrix functions. The wide range of natural and synthetic polymeric materials, as well as the simplicity of their physico-chemical modification, allow the mechanical properties to be adjusted for different requirements. Sodium alginate-based hydrogel is a frequently used material for cell culture. The lack of cell-interactive properties makes this polysaccharide the most often applied in combination with other materials, including gelatin. The combination of both materials increases their biological activity and improves their material properties, making this combination a frequently used material in 3D printing technology. The use of hydrogels as inks in 3D printing allows the accurate manufacturing of scaffolds with complex shapes and geometries. The aim of this paper is to provide an overview of the materials used for 3D cell cultures, which are mainly alginate–gelatin hydrogels, including their properties and potential applications.

scholarly journals Characterization of Chromosomal Regions Conserved in Yersinia pseudotuberculosis and Lost by Yersinia pestis

2008 ◽  
Vol 76 (10) ◽  
pp. 4592-4599 ◽  
Author(s):  
Flavie Pouillot ◽  
Corinne Fayolle ◽  
Elisabeth Carniel
Keyword(s):  
Yersinia Pestis ◽  
Yersinia Pseudotuberculosis ◽  
Rna Stability ◽  
Optimal Growth ◽  
Defined Medium ◽  
Open Reading Frames ◽  
Pcr Screening

ABSTRACT The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37°C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37°C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.

A comparison of selected methods for measuring the virulence properties ofListeriaspp.

2006 ◽  
Vol 52 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Sally Chiu ◽  
Paul B Vanderlinde ◽  
Gary A Dykes
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Virulence Genes ◽  
Listeriolysin O ◽  
Wide Range ◽  
Virulence Protein ◽  
Associated Proteins ◽  
Polymerase Chain ◽  
Virulence Properties

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.Key words: Listeria monocytogenes, virulence, pathogenicity.

scholarly journals Morin inhibits Listeria monocytogenes virulence in vivo and in vitro by targeting listeriolysin O and inflammation

2020 ◽  
Author(s):  
Gen Li ◽  
Guizhen Wang ◽  
Meng Li ◽  
Li Li ◽  
Hongtao Liu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Host Immune System ◽  
Listeriolysin O ◽  
Inflammatory Reactions ◽  
Bacterial Proliferation ◽  
Aquaculture Industry ◽  
Infected Cells ◽  
Listeria Infection

Abstract Background: Listeria monocytogenes ( L. monocytogenes ) is a global opportunistic intracellular pathogen that can cause many diseases, including meningitis and abortion in humans and animals; thus, L. monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes ; LLO can promote cytosolic bacterial proliferation and aid in evading the attacks of the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions. Results: Here, on the basis of our previous studies, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization and found that morin could effectively alleviate the inflammation induced by Listeria in vivo and in vitro and has an obvious protective effect on infected cells and mice. Conclusions: Thus, we further prove that morin is a valuable drug precursor to be developed for the treatment of Listeria infection.

scholarly journals Efecto in vitro de extractos ricos en saponinas de Pithecellobium saman y Sapindus saponaria sobre el crecimiento de dos bacterias celulolíticas ruminales

2005 ◽  
Vol 6 (1) ◽  
pp. 20
Author(s):  
Alba Lucía Santos ◽  
Hugo Jiménez ◽  
Adalgiza Cano
Keyword(s):  
Bacterial Growth ◽  
Bacterial Species ◽  
Defined Medium ◽  
Fibrobacter Succinogenes ◽  
Gram Negative Bacteria ◽  
Tropical Fruits ◽  
Ruminococcus Flavefaciens ◽  
Quillaja Saponaria ◽  
Sapindus Saponaria

<p>Se evaluó el efecto de extractos ricos en saponinas (ERS) provenientes de frutos de árboles nativos sobre el crecimiento de dos especies de bacterias celulolíticas del rumen. <em>Ruminococcus flavefaciens </em>y <em>Fibrobacter succinogenes </em>se cultivaron en medios de cultivo que contenían 0, 3 y 5 mg<strong>·</strong>ml−1 de ERS de los frutos de <em>Sapindus saponaria, Pithecellobium saman </em>y saponinas purificadas de <em>Quillaja saponaria. </em>Se determinó la curva de crecimiento de las bacterias por cuantificación espectrofotométrica a 620 nm. La utilización de 3 y 5 mg<strong>·</strong>ml−1 de ERS de <em>S. saponaria </em>y 5 mg<strong>·</strong>ml−1 de ERS de <em>P. saman </em>redujeron (P &lt; 0,05) el crecimiento de <em>R. flavefaciens</em>; mientras que la adición de 3 mg<strong>·</strong>ml−1 de ERS de <em>P. saman </em>incrementó el crecimiento de la bacteria después de la hora 11 en comparación con el tratamiento control. Por otro lado, el crecimiento de <em>F. succinogenes </em>fue inhibido (P &lt; 0,05) completamente por la utilización de 3 y 5 mg<strong>·</strong>ml−1 de ERS de <em>S. saponaria </em>y 5 mg<strong>·</strong>ml−1 de <em>P. saman</em>. La adición de ERS de <em>Q. saponaria </em>sólo afectó el crecimiento de <em>F. succinogenes </em>cuando se adicionaron 5 mg<strong>·</strong>ml−1. Los resultados muestran que la adición de ERS provenientes de frutos de árboles tropi­cales afectan el crecimiento de dos de las más importantes bacterias celulolíticas del rumen, en especial, de la bacteria Gram-negativa <em>F. succinogenes</em>.</p><p> </p><p><strong>The effect of saponine extracts from <em>Pithecellobium saman </em>and <em>Sapindus saponaria </em>on <em>in vitro </em>ruminal cellulolytic bacterial growth</strong></p><p>An experiment was carried out to evaluate the effects of saponin-rich extracts (SRE) from three tropical fruits on the growth of two rumen bacterial species. <em>Ruminococcus flavefaciens </em>and <em>Fibrobacter succinogenes </em>were cultured in a defined medium that contained 0, 3.0 and 5.0 mg<strong>·</strong>ml−1 SRE from <em>Sapindus saponaria </em>and <em>Pithecellobium saman </em>fruit and also purified saponin from <em>Quillaja saponaria</em>. Bacterial growth was spectrophotometrically measured at 620nm. Adding 3 and 5 mg<strong>·</strong>ml−1 of <em>S. saponaria </em>SRE concentrations and 5 mg<strong>·</strong>ml−1 of <em>P. saman </em>SRE reduced (P&lt;0.05) <em>R. flavefaciens </em>growth whilst <em>F. succinogenes </em>growth, was completely inhibited (P&lt;0.05) by 3 and 5 mg<strong>·</strong>ml−1 <em>S. saponaria </em>SRE and 5 mg<strong>·</strong>ml−1 <em>P. saman </em>SRE. Reduced <em>F. succinogenes </em>growth was only observed with 5 mg<strong>·</strong>ml−1 purified <em>Q. saponaria </em>saponin. Results from this experiment showed that SRE from three tropical fruits affected the growth of two of the most important cellulolytic rumen bacteria, especially Gram negative bacteria <em>F. succinogenes</em>.</p>

scholarly journals Morin inhibits Listeria monocytogenes virulence in vivo and in vitro by targeting listeriolysin O and inflammation

2020 ◽  
Author(s):  
Gen Li ◽  
Guizhen Wang ◽  
Meng Li ◽  
Li Li ◽  
Hongtao Liu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Host Immune System ◽  
Listeriolysin O ◽  
Inflammatory Reactions ◽  
Bacterial Proliferation ◽  
Aquaculture Industry ◽  
Infected Cells ◽  
Listeria Infection

Abstract Background: Listeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many diseases, including meningitis and abortion in humans and animals; thus, L. monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes; LLO can promote cytosolic bacterial proliferation and aid in evading the attacks of the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions.Results: Here, on the basis of our previous studies, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization and found that morin could effectively alleviate the inflammation induced by Listeria in vivo and in vitro and has an obvious protective effect on infected cells and mice.Conclusions: Thus, we further prove that morin is a valuable drug precursor to be developed for the treatment of Listeria infection.

Sign in / Sign up

Export Citation Format

Share Document