scholarly journals Novel Cell Wall Hydrolase CwlC fromBacillus thuringiensisIs Essential for Mother Cell Lysis

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Xiaomin Chen ◽  
Tantan Gao ◽  
Qi Peng ◽  
Jie Zhang ◽  
Yunrong Chai ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Thuringiensis ◽  
Insecticidal Activity ◽  
Mother Cell ◽  
Uv Light ◽  
Point Mutations ◽  
Cell Lysis ◽  
Specific Gene ◽  
Content Type ◽  
Cell Wall Hydrolase

ABSTRACTIn this study, a sporulation-specific gene (tentatively namedcwlC) involved in mother cell lysis inBacillus thuringiensiswas characterized. The encoded CwlC protein consists of an N-terminalN-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified fromEscherichia coliwere able to directly bind to and digest theB. thuringiensiscell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is anN-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated thatcwlCis transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK). Deletion ofcwlCcompletely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase forB. thuringiensismother cell lysis during sporulation. EngineeredB. thuringiensisstrains targetingcwlC, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCEMother cell lysis has been well studied inBacillus subtilis, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis inBacillus thuringiensis. CwlC ofB. thuringiensisonly shows 9 and 21% sequence identity with knownB. subtilismother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ betweenB. subtilisandB. thuringiensis. ThecwlCgene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.

scholarly journals Interplay between Antibiotic Efficacy and Drug-Induced Lysis Underlies Enhanced Biofilm Formation at Subinhibitory Drug Concentrations

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Wen Yu ◽  
Kelsey M. Hallinen ◽  
Kevin B. Wood
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Cell Lysis ◽  
Living Cells ◽  
Cell Wall Synthesis ◽  
Drug Induced ◽  
Trade Off ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Antibiotic Efficacy

ABSTRACTSubinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression of many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation inEnterococcus faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, increases in extracellular DNA (eDNA) levels, and increases in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with nonantibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes in antibiotic-induced biofilm formation due to external perturbations, and we verified these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA levels, and biofilm formation inE. faecaliswhile also providing a predictive quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

scholarly journals Antimicrobial Activity of Exebacase (Lysin CF-301) against the Most Common Causes of Infective Endocarditis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Aubrey Watson ◽  
Jun Taek Oh ◽  
Karen Sauve ◽  
Patricia A. Bradford ◽  
Cara Cassino ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
Infective Endocarditis ◽  
Broth Microdilution ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Microdilution Method ◽  
Cell Wall Hydrolase ◽  
Broth Microdilution Method ◽  
Common Causes

ABSTRACT Exebacase, a recombinantly produced lysin (cell wall hydrolase), and comparator antibiotics were tested by the broth microdilution method against strain sets of Staphylococcus and Streptococcus spp., which are the most common causes of infective endocarditis in humans. Exebacase was active against all Staphylococcus spp. tested, including S. aureus and coagulase-negative staphylococci (MIC50/90, 0.5/1 μg/ml). Activity against Streptococcus spp. was variable, with S. pyogenes, S. agalactiae, and S. dysgalactiae (MIC50/90, 1/2 μg/ml) among the most susceptible.

scholarly journals Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin ResistanceIn Vivo

2011 ◽  
Vol 56 (1) ◽  
pp. 208-217 ◽  
Author(s):  
Keunsook K. Lee ◽  
Donna M. MacCallum ◽  
Mette D. Jacobsen ◽  
Louise A. Walker ◽  
Frank C. Odds ◽  
...  
Keyword(s):  
Weight Loss ◽  
Cell Wall ◽  
Candida Albicans ◽  
Point Mutations ◽  
Low Frequency ◽  
Normal Cells ◽  
Content Type ◽  
Echinocandin Resistance

ABSTRACTCandida albicanscells with increased cell wall chitin have reduced echinocandin susceptibilityin vitro. The aim of this study was to investigate whetherC. albicanscells with elevated chitin levels have reduced echinocandin susceptibilityin vivo. BALB/c mice were infected withC. albicanscells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreasedC. albicansdensity in kidney lesions. In contrast, mice infected with high-chitinC. albicanscells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when testedin vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content ofC. albicanscells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation inFKS1resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin againstC. albicanswas reducedin vivodue to either elevation of chitin levels in the cell wall or acquisition ofFKS1point mutations.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis

2019 ◽  
Vol 103 (10) ◽  
pp. 4103-4112
Author(s):  
Jing Lv ◽  
Xin Zhang ◽  
Tantan Gao ◽  
Tingting Cui ◽  
Qi Peng ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Mother Cell ◽  
Cell Lysis

scholarly journals Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis

2016 ◽  
Vol 60 (11) ◽  
pp. 6510-6517 ◽  
Author(s):  
Tomoyuki Homma ◽  
Austin Nuxoll ◽  
Autumn Brown Gandt ◽  
Patrick Ebner ◽  
Ina Engels ◽  
...  
Keyword(s):  
Cell Wall ◽  
Teichoic Acid ◽  
Cell Lysis ◽  
Acid Synthesis ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Synergistic Inhibition ◽  
Dual Targeting ◽  
Cell Densities ◽  
Treatment Of Infection

ABSTRACTTeixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediateStaphylococcus aureus(VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens.

scholarly journals The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase/Peptidase Domain

2006 ◽  
Vol 72 (7) ◽  
pp. 5108-5112 ◽  
Author(s):  
David M. Donovan ◽  
Juli Foster-Frey ◽  
Shengli Dong ◽  
Geneviève M. Rousseau ◽  
Sylvain Moineau ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Agalactiae ◽  
Point Mutations ◽  
Cell Lysis ◽  
Binding Domain ◽  
Cell Wall Binding ◽  
Lysis Activity ◽  
Cell Wall Binding Domain

ABSTRACT The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.

scholarly journals Aspergillus fumigatus Cyp51A and Cyp51B Proteins Are Compensatory in Function and Localize Differentially in Response to Antifungals and Cell Wall Inhibitors

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Mark T. Roundtree ◽  
Praveen R. Juvvadi ◽  
E. Keats Shwab ◽  
D. Christopher Cole ◽  
William J. Steinbach
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Therapeutic Option ◽  
Point Mutations ◽  
Normal Growth ◽  
Wall Stress ◽  
Stress Conditions ◽  
Compensatory Response ◽  
Content Type ◽  
Red Led

ABSTRACT Triazole antifungals are the primary therapeutic option against invasive aspergillosis. However, resistance to azoles has increased dramatically over the last decade. Azole resistance is known to primarily occur due to point mutations in the azole target protein Cyp51A, one of two paralogous 14-α sterol demethylases found in Aspergillus fumigatus. Despite the importance of Cyp51A, little is known about the function of its paralog, Cyp51B, and the behavior of these proteins within the cell or their functional interrelationship. In this study, we addressed two important aspects of the Cyp51 proteins: (i) we characterized their localization patterns under normal growth versus stress conditions, and (ii) we determined how the proteins compensate for each other’s absence and respond to azole treatment. Both the Cyp51A and Cyp51B proteins were found to localize in distinct endoplasmic reticulum (ER) domains, including the perinuclear ER and the peripheral ER. Occasionally, the Cyp51 proteins concentrated in the peripheral ER network of tubules along the hyphal septa and at the hyphal tips. Exposure to voriconazole, caspofungin, and Congo red led to significant increases in fluorescence intensity in these alternative localization sites, indicative of Cyp51 protein translocation in response to cell wall stress. Furthermore, deletion of either Cyp51 paralog increased susceptibility to voriconazole, though a greater effect was observed following deletion of cyp51A, indicating a compensatory response to stress conditions.

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