Analysis of light/dark synchronization of cell-wall-lessChlamydomonas reinhardtii(Chlorophyta) cells by flow cytometry

Abstract Abstract 623 The IgVH/IgVL repertoire of the leukemic clones in B-cell chronic lymphocytic leukemia (B-CLL) patients is biased as compared to the repertoire of various B-cell subsets in healthy donors. Approximately, 30% of B-CLL patients can be classified in homology subsets based on the B-cell receptor (BCR) heavy chain complementary determining region 3 (CDR3) amino acid sequence of their leukemic clone. Interestingly, within these homology subsets, recurrent somatic hypermutations have been identified and heavy chains are paired to characteristic light chains. The occurrence of B-CLL groups expressing highly similar BCRs strongly suggests a shared specificity and involvement of recurrent antigens in the development of B-CLL. We have identified a novel B-CLL homology subset of four patients with a leukemic clone expressing highly similar mutated IgVH coupled to nearly identical IgVL rearrangements. Interestingly, all four patients had subset-biased somatic replacement mutations at two positions in IgVH. We produced the BCR of three patients belonging to this homology subset as recombinant soluble IgM (Subset-IgM) as well as BCRs of twenty-six cases expressing other rearrangements (Control-IgM). The Subset-IgM did not show any signs of autoreactivity in tissue arrays and were not found polyreactive by ELISAs for various well-known autoantigens. Therefore, we tested for reactivity with common pathogens by flow cytometry. The Subset IgM did not stain any of 20 bacterial strains. Interestingly, the yeast Candida Albicans stained brightly with Subset-IgM, as well as two other Candida strains and zymosan (cell wall of Saccharomyces Cerevisiae) (Figure 1A). None of the twenty-six Control-IgM showed this specificity. The Subset-IgM also stained yeast-infected hair-follicles and cervical smears by immunohistochemistry (Figure 1B). By ELISA, the antigen was found to be mannan, a main constituent of the yeast cell wall. Preincubation of Subset-IgM with mannan abolished the reactivity to Zymosan. Furthermore, B-CLL cells of subset patients bound zymosan ex-vivo. Of note, when the subset characteristic light chain was replaced by light chains of control B-CLL, reactivity to mannan was abrogated. In addition, when cultured in vitro, mannan induced proliferation of B-CLL cells of this homology subset, whereas proliferation of control B-CLL cells was not induced. In conclusion, we identified a novel B-CLL homology subset, expressing highly similar BCRs with specificity for yeast mannan. This study shows for the first time that a hypermutated B-CLL homology subset expresses functional BCRs with high-affinity towards a pathogen, which are able to transduce signals that support tumour cell growth. Figure 1: Recombinant IgM of a novel B-CLL subset stain yeast (A) Subset-IgM stain Zymosan by flow cytometry. (B) Staining of yeast in infected cervical smears. Left: Control-IgM. Right: Subset-IgM. Arrows indicate yeast (in hyphal form). Figure 1:. Recombinant IgM of a novel B-CLL subset stain yeast . / (A) Subset-IgM stain Zymosan by flow cytometry. (B) Staining of yeast in infected cervical smears. Left: Control-IgM. Right: Subset-IgM. Arrows indicate yeast (in hyphal form). Disclosures: No relevant conflicts of interest to declare.

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Roles of Cholesteryl-α-Glucoside Transferase and Cholesteryl Glucosides in Maintenance of Helicobacter pylori Morphology, Cell Wall Integrity, and Resistance to Antibiotics

mBio ◽

10.1128/mbio.01523-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 10

Author(s):

Majjid A. Qaria ◽

Naveen Kumar ◽

Arif Hussain ◽

Shamsul Qumar ◽

Sankara N. Doddam ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

Helicobacter Pylori ◽

Risk Factor ◽

Peptic Ulcer ◽

Cell Wall Integrity ◽

Content Type ◽

Enzymatic Function ◽

H Pylori ◽

Type Strains

ABSTRACT Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world’s population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori, and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori, their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains (Hp26695Δ421 and Hp76Δ421) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics. IMPORTANCE Helicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics.

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Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry

Journal of Immunological Methods ◽

10.1016/j.jim.2006.06.004 ◽

2006 ◽

Vol 314 (1-2) ◽

pp. 90-102 ◽

Cited By ~ 23

Author(s):

M. Martínez-Esparza ◽

A. Sarazin ◽

N. Jouy ◽

D. Poulain ◽

T. Jouault

Keyword(s):

Saccharomyces Cerevisiae ◽

Flow Cytometry ◽

Cell Wall ◽

Candida Albicans ◽

Comparative Analysis ◽

Wall Surface

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Mechanism of antimicrobial activity of honeybee (Apis mellifera) venom on Gram-negative bacteria: Escherichia coli and Pseudomonas spp.

AMB Express ◽

10.1186/s13568-021-01214-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Izlem Haktanir ◽

Maria Masoura ◽

Fani Th Mantzouridou ◽

Konstantinos Gkatzionis

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Cell Wall ◽

Membrane Permeability ◽

Metabolic Activity ◽

Mechanism Of Action ◽

Cell Injury ◽

Bacterial Species ◽

Atp Depletion ◽

Common Mechanism

AbstractHoneybee venom (Apitoxin, BV), a secretion substance expelled from the venom gland of bees, has being reported as antimicrobial against various bacterial species; however, the mechanism of action remains uncharacterized. In this study, the antibacterial activity of BV was investigated on hygiene indicator Escherichia coli and the environmental pathogen and spoilage bacterial species, Pseudomonas putida and Pseudomonas fluorescens. An array of methods was combined to elucidate the mode of action of BV. Viability by culture on media was combined with assessing cell injury with flow cytometry analysis. ATP depletion was monitored as an indicator to metabolic activity of cells, by varying BV concentration (75, 225and 500 µg/mL), temperature (25 $$^\circ \complement $$ ∘ ∁ and 37 $$^\circ \complement $$ ∘ ∁ ), and time of exposure (0 to 24 h). Venom presented moderate inhibitory effect on E. coli by viability assay, caused high membrane permeability and significant ATP loss where the effect was increased by increased concentration. The viability of P. putida was reduced to a greater extent than other tested bacteria at comparable venom concentrations and was dictated by exposure time. On the contrary, P. fluorescens appeared less affected by venom based on viability; however, flow cytometry and ATP analysis highlighted concentration- and time-dependent effect of venom. According to Transmission Electron Microscopy results, the deformation of the cell wall was evident for all species. This implies a common mechanism of action of the BV which is as follows: the cell wall destruction, change of membrane permeability, leakage of cell contents, inactivation of metabolic activity and finally cell death.

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Flow Cytometry as a Tool To Determine the Effects of Cell Wall-Active Antibiotics on Vancomycin-Susceptible and -ResistantEnterococcus faecalisStrains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00970-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 395-398 ◽

Cited By ~ 6

Author(s):

Françoise Chau ◽

Agnès Lefort ◽

Samira Benadda ◽

Vincent Dubée ◽

Bruno Fantin

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

Membrane Potential ◽

Confocal Microscopy ◽

Enterococcus Faecalis ◽

Resistant Strains

ABSTRACTFlow cytometry and confocal microscopy were used to study the effects of vancomycin, daptomycin, telavancin, and PA1409, a new investigational vancomyquine, on the morphology, membrane potential, and permeability of glycopeptide-susceptible and -resistantEnterococcus faecalisstrains. Daptomycin exerted the most pronounced effects on bacterial size and bacterial permeability against susceptible and resistant strains.

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The aminoalkylindole, BML-190, negatively regulates chitosan synthesis via the cAMP/PKA1 pathway in Cryptococcus neoformans

10.1101/751172 ◽

2019 ◽

Author(s):

Brian T. Maybruck ◽

Woei C. Lam ◽

Charles A. Specht ◽

Ma Xenia G. Ilagan ◽

Maureen J. Donlin ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

Cryptococcus Neoformans ◽

Cannabinoid Receptors ◽

Biological Activities ◽

Screening Method ◽

Signal Transduction Pathway ◽

Mammalian Host ◽

Camp Pathway ◽

Specific Proteins

AbstractCryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immune-compromising conditions. Current antifungals are suboptimal to treat this disease, therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall, is required for survival in the mammalian host, and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis.To identify these compounds we developed a robust and novel cell-based flow cytometry screening method to identify small molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans. We used flow cytometry-based counter and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits.One of the confirmed hits that reduced chitosan content was the aminoalkylindole, BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein coupled receptor, Gpr4, and via the cAMP/PKA signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis, and could lead to potential novel therapeutics against C. neoformans.ImportanceCryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungus from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of cryptococcal cells wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans. Two of these compounds were validated using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan in C. neoformans, and validates that this screening approach could be used to find potential antifungal agents.

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Optimization of staining with SYTO 9/propidium iodide: interplay, kinetics and impact on Brevibacillus brevis

BioTechniques ◽

10.2144/btn-2020-0036 ◽

2020 ◽

Vol 69 (2) ◽

pp. 88-98

Author(s):

Ying Deng ◽

Lili Wang ◽

Yujia Chen ◽

Yan Long

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

Physicochemical Properties ◽

Environmental Monitoring ◽

Propidium Iodide ◽

Food Industry ◽

Actual Performance ◽

Brevibacillus Brevis ◽

Syto 9

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Here we characterized the effects of these two dyes on Brevibacillus brevis under optimized staining. We found that SYTO 9 entered cells continuously while PI tended to adhere to the cell wall before entering the cell. In addition, results showed that a high amount of the dyes altered the physicochemical properties of membranes, improving their breakthrough. These results provide new perspectives and ideas for improving the characterization of bacterial viability using flow cytometry.

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The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans

mBio ◽

10.1128/mbio.02264-19 ◽

2019 ◽

Vol 10 (6) ◽

Author(s):

Brian T. Maybruck ◽

Woei C. Lam ◽

Charles A. Specht ◽

Ma. Xenia G. Ilagan ◽

Maureen J. Donlin ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

Protein Kinase ◽

Cyclic Amp ◽

Cryptococcus Neoformans ◽

Cannabinoid Receptors ◽

Biological Activities ◽

Screening Method ◽

Mammalian Host ◽

Content Type

ABSTRACT Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans. We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans. Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.

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