scholarly journals Microenvironment ofMycobacterium smegmatisCulture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Ana Cristina Doria dos Santos ◽  
Victor Hugo de Souza Marinho ◽  
Pedro Henrique de Aviz Silva ◽  
Barbarella de Matos Macchi ◽  
Mara Silvia Pinheiro Arruda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Complete Medium ◽  
Alternative Source ◽  
Cholesterol Supplementation ◽  
Mycobacterial Cell Wall ◽  
Bacteria Culture ◽  
Phosphatidylinositol Mannosides

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as anin vitroexperimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenicMycobacterium smegmatisin MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDaandLAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages withM. smegmatis,after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.

scholarly journals Biosynthesis of β-(1→5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

2019 ◽  
Author(s):  
Yuria Chihara ◽  
Yutaka Tanaka ◽  
Minoru Izumi ◽  
Daisuke Hagiwara ◽  
Akira Watanabe ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Aspergillus Fumigatus ◽  
Pathogenic Fungus ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Fungal Cell ◽  
Animal Pathogens

ABSTRACTThe pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.IMPORTANCEβ-(1→5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

scholarly journals CpsA, a LytR-CpsA-Psr Family Protein in Mycobacterium marinum, Is Required for Cell Wall Integrity and Virulence

2015 ◽  
Vol 83 (7) ◽  
pp. 2844-2854 ◽  
Author(s):  
Qinglan Wang ◽  
Lin Zhu ◽  
Victoria Jones ◽  
Chuan Wang ◽  
Yifei Hua ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Mycobacterium Marinum ◽  
Cell Wall Integrity ◽  
Insertion Mutant ◽  
Uncharacterized Protein ◽  
Bacterial Colony ◽  
Content Type ◽  
Mycobacterial Cell Wall

LytR-CpsA-Psr family proteins play an important role in bacterial cell wall integrity. Although the pathogenic relevance of LytR-CpsA-Psr family proteins has been studied in a few bacterial pathogens, their function in mycobacteria remains uncharacterized. In this work, a transposon insertion mutant (cpsA::Tn) ofMycobacterium marinumwas studied. We found that inactivation of CpsA altered bacterial colony morphology, sliding motility, cell surface hydrophobicity, and cell wall permeability. Besides, thecpsAmutant exhibited a decreased arabinogalactan content, indicating that CpsA plays a role in cell wall assembly. Moreover, the mutant shows impaired growth within macrophage cell lines and is severely attenuated in zebrafish larvae and adult zebrafish. Taken together, our results indicated that CpsA, a previously uncharacterized protein, is important for mycobacterial cell wall integrity and is required for mycobacterial virulence.

scholarly journals The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

2002 ◽  
Vol 68 (4) ◽  
pp. 2018-2025 ◽  
Author(s):  
Hitoshi Shimoi ◽  
Kazutoshi Sakamoto ◽  
Masaki Okuda ◽  
Ratchanee Atthi ◽  
Kazuhiro Iwashita ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Repetitive Sequences ◽  
Alcoholic Beverage ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
A Cell ◽  
Characteristic Sequences ◽  
Foam Forming ◽  
Almost All

ABSTRACT Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.

scholarly journals Probiotic potential and safety characterization of Enterococcus hirae G24 isolated from indigenous raw goat milk

2020 ◽  
pp. 218-226
Author(s):  
Kamni Rajput ◽  
Ramesh Chandra Dubey
Keyword(s):  
Cell Surface ◽  
Pathogenic Bacteria ◽  
Cell Surface Hydrophobicity ◽  
Raw Milk ◽  
Surface Hydrophobicity ◽  
Rrna Gene ◽  
Enterococcus Hirae ◽  
Bacterial Cultures ◽  
Probiotic Potential

In this paper, an investigation on lactic acid bacterial isolates from ethnic goat raw milk samples were examined for their probiotic potential and safety parameters. For this purpose, isolated bacterial cultures were screened based on certain parameters viz., sugar fermentation, tolerance to temperature, salt, low pH, bile salts, and phenol resistance. After that, these bacterial cultures were more estimated in vitro for auto-aggregation, cell surface hydrophobicity, response to simulated stomach duodenum channel, antibiotic resistance, and antimicrobial activity. Besides, probiotic traits show the absence of gelatinase and hemolytic activity supports its safety. The isolate G24 showed good viability at different pH, bile concentration, phenol resistance and response to simulated stomach duodenum passage but it did not show gelatinase and hemolytic activities. Isolate G24 was susceptible to amikacin, carbenicillin, kanamycin, ciprofloxacin, co-trimazine, nitrofurantoin, streptomycin, and tetracycline. Isolate G24 also exhibited antimicrobial action against five common pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogens, and Salmonella typhimurium. It displayed the maximum auto-aggregation, cell surface hydrophobicity to different hydrocarbons. Following molecular characterization the isolate G24 was identified as Enterococcus hirae with 16S rRNA gene sequencing and phylogeny. E. hirae G24 bears the excellent properties of probiotics.

The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2641-2651 ◽  
Author(s):  
Amparo Galán ◽  
Manuel Casanova ◽  
Amelia Murgui ◽  
Donna M. MacCallum ◽  
Frank C. Odds ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Glutamic Acid ◽  
Germ Tube ◽  
Cell Surface Hydrophobicity ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Amino Acid Sequences ◽  
Calcofluor White

Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated by RIM101. A Δker1/Δker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δker1/Δker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with β-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δker1/Δker1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.

scholarly journals In vitro Post-Antifungal Effect of Posaconazole and Its Impact on Adhesion-Related Traits and Hemolysin Production of Oral Candida dubliniensis Isolates

2019 ◽  
Vol 28 (6) ◽  
pp. 552-558
Author(s):  
Arjuna Nishantha Bandara Ellepola ◽  
Ranil Samantha Dassanayake ◽  
Ziauddin Khan
Keyword(s):  
Oral Candidiasis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Candida Dubliniensis ◽  
In Vitro Assays ◽  
Drug Induced ◽  
Antifungal Effect ◽  
Buccal Epithelial Cells ◽  
Oral Candida

Objective: Candidal adherence to denture acrylic surfaces (DAS) and oral buccal epithelial cells (BEC), formation of candidal germ tubes (GT), candidal cell surface hydrophobicity (CSH), and hemolysin production are important pathogenic traits of Candida. The antifungal drug-induced post-antifungal effect (PAFE) also impacts the virulence of Candida. Candida dubliniensis isolates are associated with the causation of oral candidiasis which could be managed with posaconazole. Thus far there is no evidence on posaconazole-induced PAFE and its impact on adhesion-related attributes and production of hemolysin by C. dubliniensis isolates. Hence, the PAFE, adhesion to DAS and BEC, formation of GT, CSH, and hemolysin production of 20 oral C. dubliniensis isolates after brief exposure to posaconazole was ascertained. Materials and Methods: The PAFE, adherence to DAS and BEC, formation of GT, candidal CSH, and hemolysin production were investigated by hitherto described in vitro assays. Results: The mean PAFE (h) induced by posaconazole on C. dubliniensis isolates was 1.66. Exposure to posaconazole suppressed the ability of C. dubliniensis to adhere to DAS, BEC, formation of candidal GT, candidal CSH and to produce hemolysin by a reduction of 44, 33, 34, 36, and 15% (p < 0.005 to p < 0.001), respectively. Conclusion: Exposure of C. dubliniensis isolates to posaconazole for a brief period induced an antimycotic impact by subduing its growth in addition to suppressing pathogenic adherence-associated attributes, as well as production of hemolysin.

scholarly journals Characterization of Monolaurin Resistance in Enterococcus faecalis

2007 ◽  
Vol 73 (17) ◽  
pp. 5507-5515 ◽  
Author(s):  
Muriel Dufour ◽  
Janet M. Manson ◽  
Philip J. Bremer ◽  
Jean-Pierre Dufour ◽  
Gregory M. Cook ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Transmission Electron ◽  
Serious Infections ◽  
Autolytic Activity

ABSTRACT There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

Lactobacilli Isolated from Chicken Intestines: Potential Use as Probiotics

1999 ◽  
Vol 62 (3) ◽  
pp. 252-256 ◽  
Author(s):  
C. GUSILS ◽  
A. PÉREZ CHAIA ◽  
S. GONZÁLEZ ◽  
G. OLIVER
Keyword(s):  
Cell Surface ◽  
Mixed Culture ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Probiotic Properties ◽  
Culture Studies ◽  
Salmonella Gallinarum ◽  
Feed Formulation ◽  
Lactobacillus Animalis

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

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