scholarly journals Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic

2020 ◽  
Vol 11 ◽  
Author(s):  
Jees Sebastian ◽  
Rashmi Ravindran Nair ◽  
Sharmada Swaminath ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Surface ◽  
Outer Layer ◽  
Negative Charge ◽  
De Novo ◽  
Cell Surface Hydrophobicity ◽  
Surface Flow ◽  
Continuous Exposure ◽  
Mechanical Removal

Majority of the cells in the bacterial populations exposed to lethal concentrations of antibiotics for prolonged duration succumbs to the antibiotics’ sterilizing activity. The remaining cells survive by diverse mechanisms that include reduced permeability of the antibiotics. However, in the cells surviving in the continued presence of lethal concentrations of antibiotics, it is not known whether any cell surface alterations occur that in turn may reduce permeability of the antibiotics. Here we report the presence of a highly negatively charged, hydrophilic, thickened capsular outer layer (TCOL) on a small proportion of the rifampicin surviving population (RSP) of Mycobacterium tuberculosis (Mtb) cells upon prolonged continuous exposure to bactericidal concentrations of rifampicin in vitro. The TCOL reduced the intracellular entry of 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), a fluorochrome-conjugated rifampicin permeability probe of negligible bacteriocidal activity but comparable properties. Gentle mechanical removal of the TCOL enabled significant increase in the 5-FAM-rifampicin permeability. Zeta potential measurements of the cells’ surface charge and hexadecane assay for cell surface hydrophobicity showed that the TCOL imparted high negative charge and polar nature to the cells’ surface. Flow cytometry using the MLP and RSP cells, stained with calcofluor white, which specifically binds glucose/mannose units in β (1 → 4) or β (1 → 3) linkages, revealed the presence of lower content of polysaccharides containing such residues in the TCOL. GC-MS analyses of the TCOL and the normal capsular outer layer (NCOL) of MLP cells showed elevated levels of α-D-glucopyranoside, mannose, arabinose, galactose, and their derivatives in the TCOL, indicating the presence of high content of polysaccharides with these residues. We hypothesize that the significantly high thickness and the elevated negative charge of the TCOL might have functioned as a physical barrier restricting the permeability of the relatively non-polar rifampicin. This might have reduced intracellular rifampicin concentration enabling the cells’ survival in the continued presence of high doses of rifampicin. In the context of our earlier report on the de novo emergence of rifampicin-resistant genetic mutants of Mtb from the population surviving under lethal doses of the antibiotic, the present findings attain clinical significance if a subpopulation of the tubercle bacilli in tuberculosis patients possesses TCOL.

scholarly journals Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Prolonged Exposure ◽  
De Novo ◽  
Efflux Pump ◽  
Persister Cells ◽  
Efflux Pump Inhibitor ◽  
Antibiotic Resistant ◽  
Mechanical Removal ◽  
Derivatives Of

ABSTRACTPersisters constitute a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. Multiple mechanisms have been suggested for bacterial persistence against antibiotics. With mycobacteria being no exception to this behaviour, we had reported the de novo emergence of genetically antibiotic-resistant Mycobacterium tuberculosis from persister cells upon prolonged exposure to microbicidal concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we present evidence for reduced permeability to rifampicin as a mechanism for persistence of Mycobacterium tuberculosis in vitro. We observed that rifampicin persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-rifampicin. GC-MS analysis of TOL showed the presence of ∼7-fold, ∼5-fold and ∼2- fold higher levels of α-D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked mannose, respectively, among ∼2-fold higher levels of derivatives of several other types of sugars such as arabinose and galactose. Taken together, the present study reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro.

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.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

scholarly journals Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

2018 ◽  
Vol 115 (39) ◽  
pp. 9779-9784 ◽  
Author(s):  
Sangeeta Tiwari ◽  
Andries J. van Tonder ◽  
Catherine Vilchèze ◽  
Vitor Mendes ◽  
Sherine E. Thomas ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
De Novo ◽  
Target Space ◽  
Biosynthesis Pathway ◽  
Arginine Biosynthesis ◽  
Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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 Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

1994 ◽  
Vol 5 (7) ◽  
pp. 819-828 ◽  
Author(s):  
Y Wang ◽  
G M Fuller
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
De Novo ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Synthesis Inhibitors ◽  
Different Cell Types ◽  
Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

scholarly journals Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

2007 ◽  
Vol 6 (7) ◽  
pp. 1137-1149 ◽  
Author(s):  
Talibah Metcalf ◽  
Hanke van der Wel ◽  
Ricardo Escalante ◽  
Leandro Sastre ◽  
Christopher M. West
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Outer Layer ◽  
Fluorescent Protein ◽  
De Novo ◽  
Middle Layer ◽  
In Vivo Studies ◽  
Permeability Barrier ◽  
Spore Coat

ABSTRACT Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.

scholarly journals Development of Selenized Lactic Acid Bacteria and their Selenium Bioaccummulation Capacity

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 91
Author(s):  
Gabriela Krausova ◽  
Antonin Kana ◽  
Ivana Hyrslova ◽  
Iva Mrvikova ◽  
Miloslava Kavkova
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Cell Surface ◽  
Sodium Selenite ◽  
Antioxidant Properties ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
In Vivo Studies ◽  
Dpph Method

Selenized lactic acid bacteria (LAB) represent potentially safe and effective sources of selenium (Se), essential for human health, as lactic acid fermentation improves Se bioavailability and reduces its toxicity. LAB are generally recognized as safe (GRAS) and widely used in fermented dairy products. To facilitate selenized LAB implementation as a functional food, we developed and characterized new Se-enriched strains based on the food industry commercial strains Streptococcus thermophilus CCDM 144 and Enterococcus faecium CCDM 922A as representatives of two LAB genera. We evaluated Se bioaccumulation capacity, Se biotransformation and growth ability in the presence of different sodium selenite concentrations (0–50 mg/L), and antioxidant properties (2, 2-diphenyl-1-picrylhydrazyl (DPPH) method) and cell surface hydrophobicity between Se-enriched and parental strains in vitro. Sodium selenite addition did not negatively influence growth of either strain; thus, 50 mg/L was chosen as the optimal concentration based on strain accumulation capacity. Selenization improved the antioxidant properties of both strains and significantly increased their cell surface hydrophobicity (p < 0.05). To our knowledge, this represents the first report of Se-enriched strain hydrophobicity as well as the first on Se speciation in families Enterococcaceae and Streptococcaceae. Moreover, both tested strains demonstrated good potential for Se-enrichment, providing a foundation for further in vitro and in vivo studies to confirm the suitability of these Se-enriched strains for industrial applications.

Mechanisms for de novo biogenesis of an apical membrane compartment in groups of simple epithelial cells surrounded by extracellular matrix

1997 ◽  
Vol 110 (22) ◽  
pp. 2781-2794 ◽  
Author(s):  
G.K. Ojakian ◽  
W.J. Nelson ◽  
K.A. Beck
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Golgi Complex ◽  
De Novo ◽  
Apical Membrane ◽  
Free Cell ◽  
Cell Contacts ◽  
Lateral Membrane ◽  
Cell Cell

In open monolayers of epithelial cells grown in vitro, the apical membrane domain forms on the free cell surface that faces the culture medium. However, in vivo, the apical lumenal compartment arises within groups of cells that do not have a free cell surface. We designed in vitro culture conditions, using small colonies of MDCK cells overlaid with collagen, in which formation of the apical membrane must occur de novo by remodeling existing membrane domains that are contacted by other cells or extracellular matrix. Within 12 hours of collagen overlay, the apical membrane glycoprotein gp135 is removed from the free cell surface, while lateral membrane proteins (e.g. Na+,K+-ATPase) remain at sites of cell-cell contacts. Subsequently, lumenal structures, containing gp135 and the apically secreted protein gp81, formed within these cell-cell contacts. Na+,K+-ATPase, adherens junction (E-cadherin, alpha- and beta-catenins) and tight junction (ZO-1) proteins were localized on the lateral membrane adjacent to, but excluded from the gp135-positive lumenal compartment. Therefore, each lumen represents a newly formed apical compartment on the lateral membrane. The Golgi complex (alpha-mannosidase II and Golgi beta-spectrin), centrosomes (gamma-tubulin) and microtubules reorient to a cytoplasmic position adjacent to the newly-forming apical lumenal compartments. Significantly, addition of colchicine, nocodazole or brefeldin A inhibits apical lumen formation. These results demonstrate that simple epithelial cells form an apical lumenal compartment de novo through initial intermixing, and then sorting of apical and basal-lateral membrane proteins at sites of cell-cell contacts. In addition, apical lumen formation requires an intact microtubule network, microtubule-dependent reorientation of the Golgi complex and secretory apparatus, and fully functional protein delivery from the Golgi complex to the forming apical cell surface.

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