Imaging flow cytometry analysis of intracellular pathogens

Haridas, Viraga; Ranjbar, Shahin; Vorobjev, Ivan A.; Goldfeld, Anne E.; Barteneva, Natasha S.

doi:10.1016/j.ymeth.2016.09.007

Cited by 60 publications

(49 citation statements)

References 127 publications

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“…It has been previously reported that Acanthamoebae are able to reactivate VBNC Legionella pneumophila into an active pathogenic state . As shown in Figure A, VBNC cells cocultured within trophozoites appeared to revert to a culturable state, and that coculture with amoebae led to a 3‐log increase ( P < 0.0001) of culturable P. aeruginosa after 2 h. With slower kinetics, similar results were also obtained without amoeba due to the temperature upshift .…”

Section: Resultssupporting

confidence: 82%

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

et al. 2019

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Pseudomonas aeruginosa is a Gram‐negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod‐shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture‐based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species‐specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry

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Section: Resultssupporting

confidence: 82%

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

et al. 2019

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“…1d). The Bright Detail Similarity (BDS) score of colocalization between the uorescent signals of the SDCs and SARS-CoV-2 also showed a high degree of colocalization (generally, a BDS score of 2 or greater represents a high degree of overlap 68 ), especially in SDC3 and 4 transfectants ( Fig. 1d).…”

Section: ]) Colocalization Studies Revealed a Signi Cant Degree Of Cmentioning

confidence: 91%

“…Values of p<0.05 were accepted as signi cant 45,46 . During imaging ow cytometry, the IDEAS™ feature Bright Detail Similarity (BDS) was used to measure colocalization between two signals 68 The difference of SARS-CoV-2 internalization in A549 and K562 cells. WT A549 and K562 cells were incubated with heat-inactivated SARS-CoV-2 (at 1 MOI) for 18h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Contribution of syndecans to the cellular entry of SARS-CoV-2

Hudák

Szilák

Letoha

2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology significantly hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans facilitate the cellular entry of SARS-CoV-2. Among syndecans, syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake, yet overexpression of other isoforms, including the neuronal syndecan-3, also increased SARS-CoV-2 internalization. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus’s interactions with syndecans. Besides the polyanionic heparan sulfate chains - the established binding sites for several viruses - other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Among these inhibitors, a peptide compromising the spike protein’s heparin-binding PRRAR motif significantly reduced SARS-CoV-2 cellular uptake, highlighting the need to go beyond the ACE2 paradigm for developing efficient therapeutics against SARS-CoV-2. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offers molecularly precise, yet simple strategies in overcoming the complex nature of SARS-CoV-2 infection.

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“…Since last 10 years, this approach has been increasingly used to study aspects of red cell morphology (Samsel et al, ) and has been used in many hematological studies such as erythroid cell biology (Samsel & McCoy, ), in the megakaryocyte cell biology and pathology (Niswander, McGrath, Kennedy, & Palis, ), also in the assessment of projected surface area, volume and morphological changes in the stored red blood cells in the blood bank (Roussel et al, ). This method is also used in the study of the intracellular pathogen (Haridas, Ranjbar, Vorobjev, Goldfeld, & Barteneva, ). Mantel and colleagues used IFC to study erythrocyte‐derived microvesicles in human malaria infections (Mantel et al, ).…”

Section: Introductionmentioning

confidence: 99%

Applications of imaging flow cytometry in the diagnostic assessment of red cell membrane disorders

Dalal

Devendra

et al. 2019

Cytometry Part B Clinical

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Background: Red cell membranopathies refers to phenotypically and morphologically heterogeneous disorders. High throughput imaging flow cytometry (IFC) combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy to produce high content image analysis with quantitative analysis. We have evaluated the applications of IFC to examine both the morphology as well as fluorescence signal intensity in red cell membranopathies.Methods: Fluorescence intensity of eosin-5-maleimide (EMA) labeled red cells was measured for diagnosis of RBC membrane protein defect on Amnis ImageStreamX followed by Image analysis on IDEAS software to study features such as circularity and shape ratio.Results: The hereditary spherocytosis (HS) group showed significantly decreased MFI (52,800 ± 9,100) than normal controls (81,100 ± 4,700) (p < .05) whereas non-HS showed 78,300 ± 9,900. The shape ratio of hereditary elliptocytosis (HE) was significantly higher (43.8%) than normal controls (14.6%). The circularity score is higher in HS (64.15%) than the normal controls (44.3%) whereas the circularity score was very less in HE (10%) due to the presence of elliptocytes. Conclusions:The advantages of the IFC over standard flow cytometry is its ability to provide high-content image analysis and measurement of parameters such as circularity and shape ratio allow discriminating red cell membranopathies (HS and HE) due to variations in shape and size. It could be a single, effective, and rapid IFC test for detection and differentiation of red cell membrane disorders in hematology laboratories where an IFC is available. K E Y W O R D S hereditary spherocytosis, imaging flow cytometry, red blood cell

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Imaging flow cytometry analysis of intracellular pathogens

Cited by 60 publications

References 127 publications

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

Contribution of syndecans to the cellular entry of SARS-CoV-2

Applications of imaging flow cytometry in the diagnostic assessment of red cell membrane disorders

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