scholarly journals The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence

2016 ◽  
Vol 12 (11) ◽  
pp. e1006001 ◽  
Author(s):  
Daniel A. Pensinger ◽  
Kyle M. Boldon ◽  
Grischa Y. Chen ◽  
William J. B. Vincent ◽  
Kyle Sherman ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes

Green Synthesis of Silver Nanoparticles Using Artocarpus hirsutus Seed Extract and its Antibacterial Activity

2020 ◽  
Vol 21 (10) ◽  
pp. 980-989
Author(s):  
Sampath Shobana ◽  
Sunderam Veena ◽  
S.S.M. Sameer ◽  
K. Swarnalakshmi ◽  
L.A. Vishal
Keyword(s):  
Silver Nanoparticles ◽  
Cell Wall ◽  
Antibacterial Activity ◽  
Listeria Monocytogenes ◽  
Nanoparticle Synthesis ◽  
Enterobacter Aerogenes ◽  
Seed Extract ◽  
Diffusion Method ◽  
Multi Drug Resistance ◽  
Gastrointestinal Bacteria

Aims: To evaluate the antibacterial activity of Artocarpus hirsutus mediated seed extract for nanoparticle synthesis. Background: Gastrointestinal bacteria are known for causing deadly infections in humans. They also possess multi-drug resistance and interfere with clinical treatments. Applied nanotechnology has been known to combat such infectious agents with little interference from their special attributes. Here we synthesize silver nanoparticles from Artocarpus hirsutus seed extract against two gastro-intestinal bacterial species: Enterobacter aerogenes and Listeria monocytogenes. Objective: To collect, dry, and process seeds of Artocarpus hirsutus for nanoparticle synthesis. To evaluate the morphological interaction of silver nanoparticles with bacteria. Methods: Artocarpus hirsutus seeds were collected and processed and further silver nanoparticles were synthesized by the co-precipitation method. The synthesized nanoparticles were characterized using XRD, UV, FTIR, and SEM. These nanoparticles were employed to study the antibacterial activity of nanoparticles against Enterobacter aerogenes and Listeria monocytogenes using well diffusion method. Further, morphological interaction of silver nanoparticles on bacteria was studied using SEM. Result: Silver nanoparticles were synthesized using Artocarpus hirsutus seed extract and characterization studies confirmed that silver nanoparticles were spherical in shape with 25-40 nm size. Antibacterial study exhibited better activity against Enterobacter aerogenes with a maximum zone of inhibition than on Listeria monocytogenes. SEM micrographs indicated that Enterobacter aerogenes bacteria were more susceptible to silver nanoparticles due to the absence of cell wall. Also, the size and charge of silver nanoparticles enable easy penetration of the bacterial cell wall. Conclusion: In this study, silver nanoparticles were synthesized using the seed extract of Artocarpus hirsutus for the first time exploiting the fact that Moraceae species have high phytonutrient content which aided in nanoparticle synthesis. This nanoparticle can be employed for large scale synthesis which when coupled with the pharmaceutical industry can be used to overcome the problems associated with conventional antibiotics to treat gastrointestinal bacteria.

scholarly journals A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

2021 ◽  
Vol 9 (6) ◽  
pp. 1323
Author(s):  
Etai Boichis ◽  
Nadejda Sigal ◽  
Ilya Borovok ◽  
Anat A. Herskovits
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Protein Pair ◽  
Growth Conditions ◽  
Wall Structure ◽  
Antibiotic Resistant ◽  
Extracellular Milieu ◽  
Virion Release ◽  
Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

2008 ◽  
Vol 74 (22) ◽  
pp. 6848-6858 ◽  
Author(s):  
F. Abram ◽  
E. Starr ◽  
K. A. G. Karatzas ◽  
K. Matlawska-Wasowska ◽  
A. Boyd ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Intact Cell ◽  
Microscopic Analysis ◽  
Phenotypic Characterization ◽  
Carbon Utilization ◽  
Two Dimensional Gel Electrophoresis ◽  
Sigma B ◽  
The Impact

ABSTRACT Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

scholarly journals Cell shape and antibiotic resistance is maintained by the activity of multiple FtsW and RodA enzymes inListeria monocytogenes

2019 ◽  
Author(s):  
Jeanine Rismondo ◽  
Sven Halbedel ◽  
Angelika Gründling
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Immune System ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Protein Complexes ◽  
Inducible Promoter ◽  
Severe Form ◽  
Human Pathogen ◽  
Division Site

AbstractRod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that theBacillus subtilisRodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogenListeria monocytogenesencodes up to six FtsW/RodA homologs, however their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly,L. monocytogenesencodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter.L. monocytogenesalso possesses three RodA homologs, RodA1, RodA2 and RodA3 and their combined absence is lethal. Cells of arodA1/rodA3double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression ofrodA3andftsW2is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, arodA3mutant was more susceptible to the β-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights thatL. monocytogenesencodes a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division and antibiotic resistance.ImportanceThe human pathogenListeria monocytogenesis usually treated with high doses of β-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically onL. monocytogenesand the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form ofListeriainfection, which can be fatal in up to 30% of cases. The development of new strategies to treatListeriainfections is therefore necessary. Here we show that the expression of some of the FtsW and RodA enzymes ofL. monocytogenesis induced by the presence of β-lactam antibiotics and their combined absence makes bacteria more susceptible to this class of antibiotics. The development of antimicrobials that inhibit the activity or production of FtsW/RodA enzymes might therefore help to improve the treatment ofListeriainfections and thereby lead to a reduction in mortality.

scholarly journals EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

2020 ◽  
Author(s):  
Jeanine Rismondo ◽  
Lisa M. Schulz ◽  
Maria Yacoub ◽  
Ashima Wadhawan ◽  
Michael Hoppert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cell Wall Integrity ◽  
Growth Defect ◽  
Cell Wall Biosynthesis ◽  
High Concentrations

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity. IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

scholarly journals Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

2010 ◽  
Vol 432 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Emily R. Slepkov ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Phospholipase C ◽  
Bacterial Pathogen ◽  
Amino Acid Residues ◽  
C Terminus ◽  
N Terminus ◽  
Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

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