scholarly journals Opsonin-free, real-time imaging of Cryptococcus neoformans capsule during budding

2018 ◽  
Author(s):  
Hugo Costa Paes ◽  
Stefânia de Oliveira Frazão ◽  
Camila Pereira Rosa ◽  
Patrícia Albuquerque ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Numerical Aperture ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Interference Microscopy ◽  
Cell Physiology ◽  
Charge Coupled Device ◽  
India Ink ◽  
Induction Process ◽  
Virulence Attribute

AbstractCryptococcus neoformans is a unicellular fungal pathogen that causes meningoencephalitis, killing hundreds of thousands of patients each year. Its most distinctive characteristic is a polysaccharide capsule that envelops the whole cell. It is the major virulence attribute and the antigen for serologic diagnosis. We have developed a method for easy observation of the capsule and its growth dynamics using the cell-separation reagent Percoll and differential interference contrast (DIC) microscopy. Percoll suspension is far less disruptive of cell physiology than methods relying on antibody binding to the capsule, and measurements made with it are equivalent with India ink. Time-lapse microscopy observations using this method suggest that during budding, a dividing cell can regulate whether the capsule polysaccharide it produces is deposited on the capsule of the bud or on its own. This observation has important implications for our understanding of the C. neoformans capsule induction process during budding.List of abbreviations and acronymsCSFCerebrospinal fluidDICDifferential interference microscopyNANumerical apertureCCDCharge-coupled deviceMMMinimal mediumCIMCO2-independent mediumMOPS3-Morpholinopropane-1-sulfonic acidSDStandard deviation

scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

scholarly journals Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

2013 ◽  
Vol 79 (7) ◽  
pp. 2294-2301 ◽  
Author(s):  
Konstantinos P. Koutsoumanis ◽  
Alexandra Lianou
Keyword(s):  
Kinetic Parameters ◽  
Single Cells ◽  
Growth Curves ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Initial Population ◽  
Bacterial Cells ◽  
Stochastic Growth ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTConventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells ofSalmonella entericaserotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations withN0of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

scholarly journals Cell-to-cell heterogeneity in Escherichia coli stress response originates from pulsatile expression and growth

2020 ◽  
Author(s):  
Nadia M. V. Sampaio ◽  
Caroline M. Blassick ◽  
Jean-Baptiste Lugagne ◽  
Mary J. Dunlop
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Response ◽  
Single Cell ◽  
Phenotypic Variability ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Heterogeneity ◽  
Temporal Expression ◽  
Functional Consequences

AbstractCell-to-cell heterogeneity in gene expression and growth can have critical functional consequences, such as determining whether individual bacteria survive or die following stress. Although phenotypic variability is well documented, the dynamics that underlie it are often unknown. This information is critical because dramatically different outcomes can arise from gradual versus rapid changes in expression and growth. Using single-cell time-lapse microscopy, we measured the temporal expression of a suite of stress response reporters in Escherichia coli, while simultaneously monitoring growth rate. In conditions without stress, we found widespread examples of pulsatile expression. Single-cell growth rates were often anti-correlated with gene expression, with changes in growth preceding changes in expression. These pulsatile dynamics have functional consequences, which we demonstrate by measuring survival after challenging cells with the antibiotic ciprofloxacin. Our results suggest that pulsatile expression and growth dynamics are common in stress response networks and can have direct consequences for survival.

scholarly journals Efficient microbial colony growth dynamics quantification with ColTapp, an automated image analysis application

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Julian Bär ◽  
Mathilde Boumasmoud ◽  
Roger D. Kouyos ◽  
Annelies S. Zinkernagel ◽  
Clément Vulin
Keyword(s):  
Growth Dynamics ◽  
Time Lapse ◽  
Colony Growth ◽  
Lag Time ◽  
Automated Image Analysis ◽  
Clinical Samples ◽  
Clinical Settings ◽  
Bacterial Colony ◽  
Analysis Application ◽  
Population Survival

Abstract Populations of genetically identical bacteria are phenotypically heterogeneous, giving rise to population functionalities that would not be possible in homogeneous populations. For instance, a proportion of non-dividing bacteria could persist through antibiotic challenges and secure population survival. This heterogeneity can be studied in complex environmental or clinical samples by spreading the bacteria on agar plates and monitoring time to growth resumption in order to infer their metabolic state distribution. We present ColTapp, the Colony Time-lapse application for bacterial colony growth quantification. Its intuitive graphical user interface allows users to analyze time-lapse images of agar plates to monitor size, color and morphology of colonies. Additionally, images at isolated timepoints can be used to estimate lag time. Using ColTapp, we analyze a dataset of Staphylococcus aureus time-lapse images including populations with heterogeneous lag time. Colonies on dense plates reach saturation early, leading to overestimation of lag time from isolated images. We show that this bias can be corrected by taking into account the area available to each colony on the plate. We envision that in clinical settings, improved analysis of colony growth dynamics may help treatment decisions oriented towards personalized antibiotic therapies.

scholarly journals The Vacuolar Ca2+ Exchanger Vcx1 Is Involved in Calcineurin-Dependent Ca2+ Tolerance and Virulence in Cryptococcus neoformans

2010 ◽  
Vol 9 (11) ◽  
pp. 1798-1805 ◽  
Author(s):  
Lívia Kmetzsch ◽  
Charley Christian Staats ◽  
Elisa Simon ◽  
Fernanda L. Fonseca ◽  
Débora L. de Oliveira ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Cyclosporine A ◽  
Capsular Polysaccharide ◽  
Cytosolic Calcium ◽  
Content Type ◽  
Human Body Temperature ◽  
Life Threatening ◽  
High Concentrations ◽  
Capsule Size ◽  
Virulence Attribute

ABSTRACT Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca2+-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl2 lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.

scholarly journals Ordered Kinetochore Assembly in the Human-Pathogenic Basidiomycetous Yeast Cryptococcus neoformans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lukasz Kozubowski ◽  
Vikas Yadav ◽  
Gautam Chatterjee ◽  
Shreyas Sridhar ◽  
Masashi Yamaguchi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cryptococcus Neoformans ◽  
Budding Yeast ◽  
Genetic Material ◽  
Time Lapse ◽  
Content Type ◽  
Nuclear Envelope Breakdown ◽  
Kinetochore Assembly ◽  
Transmission Electron ◽  
Partial Opening

ABSTRACT Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

scholarly journals Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan

2004 ◽  
Vol 3 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Javier García-Rivera ◽  
Yun C. Chang ◽  
K. J. Kwon-Chung ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Missense Mutation ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Immunogold Electron Microscopy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Diameter ◽  
India Ink ◽  
Hydrolysis Of ◽  
Gene Deletion Mutant

ABSTRACT Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

scholarly journals Temporal Kinetics and Quantitative Analysis of Cryptococcus neoformans Nonlytic Exocytosis

2014 ◽  
Vol 82 (5) ◽  
pp. 2059-2067 ◽  
Author(s):  
Sabriya A. Stukes ◽  
Hillel W. Cohen ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Time Lapse ◽  
Host Cells ◽  
Type I ◽  
Phagocytic Cells ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Iii ◽  
Cell To Cell Transfer ◽  
Extracellular Environment

ABSTRACTCryptococcus neoformansis a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems.C. neoformanshas the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies ofC. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellularC. neoformansand identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P< 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection.

scholarly journals ColTapp, an automated image analysis application for efficient microbial colony growth dynamics quantification

2020 ◽  
Author(s):  
Julian Bär ◽  
Mathilde Boumasmoud ◽  
Roger Kouyos ◽  
Annelies S. Zinkernagel ◽  
Clément Vulin
Keyword(s):  
Growth Dynamics ◽  
Time Estimation ◽  
Time Lapse ◽  
Colony Growth ◽  
Lag Time ◽  
Phenotypic Heterogeneity ◽  
Automated Image Analysis ◽  
Bacterial Colony ◽  
Staphyloccocus Aureus ◽  
Analysis Application

AbstractPhenotypic heterogeneity occurs in a population of genetically identical bacteria due to stochastic molecular fluctuations and environmental variations. In extreme cases of phenotypic heterogeneity, a fraction of the bacterial population enters dormancy, and these metabolically inactive or non-dividing bacteria persist through most antibiotic challenges. These subpopulations of persister cells are difficult to study in patient samples. However, the proportion of persisters in a sample can be accessed by physically separating bacteria on a plate measuring the time until colonies become visible as dormant bacteria resume growth later than their active counterparts and form smaller colonies.Here, we present ColTapp (Colony Time-lapse app), an application dedicated to bacterial colony growth quantification, freely available for download together with its MATLAB source code or as a MacOS/Windows executable. ColTapp’s intuitive graphical user interface allows users without prior coding knowledge to analyze endpoint or time-lapse images of colonies on agar plates. Colonies are detected automatically, and their radius can be tracked over time. Downstream analyses to derive colony lag time and growth rate are implemented.We demonstrate here the applicability of ColTapp on a dataset of Staphyloccocus aureus colony time-lapse images. Colonies on dense plates reached saturation early, biasing lag time estimation from endpoint images. This bias can be reduced by considering the area available to each colony on a plate.By facilitating the analysis of colony growth dynamics in clinical settings, this application will enable a new type of diagnostics, oriented towards personalized antibiotic therapies.

scholarly journals Kriptokokal meningitis: Aspek klinis dan diagnosis laboratorium

2012 ◽  
Vol 1 (1) ◽  
Author(s):  
Efrida Efrida ◽  
Desi Ekawati
Keyword(s):  
Enzyme Immunoassay ◽  
Cryptococcus Neoformans ◽  
Opportunistic Infection ◽  
Cryptococcal Meningitis ◽  
Clinical Symptoms ◽  
Cell Mediated Immunity ◽  
India Ink ◽  
Aids Pandemic ◽  
Direct Microscopic Examination ◽  
Common Opportunistic Infection

Abstrak Kriptokokosis merupakan infeksi yang disebabkan oleh jamur Cryptococcus neoformans, infeksi ini secara luas ditemukan di dunia dan umumya dialami oleh penderita dengan sistem imun yang rendah. Munculan klinis terutama adalah meningitis dan meningoensefalitis yang dikenal dengan kriptokokal meningitis. Sejalan dengan infeksi HIV yang menjadi pandemi, kriptokokosis sebagai infeksi oportunistik juga semakin berkembang di dunia. Kriptokokal meningitis merupakan infeksi oportunistik kedua paling umum yang terkait dengan AIDS di Afrika dan Asia Selatan dengan kejadian kriptokokosis 15%-30% ditemukan pada pasien dengan AIDS. Tanpa pengobatan dengan antifungal yang spesifik, mortalitas dilaporkan 100% dalam dua minggu setelah munculan klinis kriptokokosis dengan meningoensefalitis pada populasi terinfeksi HIV. Di Indonesia, sebelum pandemi AIDS kasus kriptokokosis jarang dilaporkan. Sejak tahun 2004, seiring dengan pertambahan pasien terinfeksi HIV, Departemen Parasitologi FKUI mencatat peningkatan insidensi kriptokokal meningitis pada penderita AIDS yaitu sebesar 21,9%. Faktor yang terkait dengan virulensi Cryptococcus neoformans adalah adanya kapsul polisakarida, produksi melanin dan sifat thermotolerance. Imunitas yang dimediasi oleh sel memiliki peranan penting dalam pertahanan pejamu terhadap Cryptococcus. Pemeriksaan laboratorium penunjang untuk diagnosis adalah pemeriksaan mikroskopis langsung menggunakan tinta India, deteksi antigen, metode enzyme immunoassay, kultur, dan metode molekular. Kata kunci: kriptokokal meningitis, Cryptococcus neoformans,infeksi oportunistik Abstract Cryptococcosis is an infection caused by Cryptococcus neoformans, that is widely found worldwide and generally experienced by patients with immunodeficiency. Meningitis and meningoencephalitis is the major clinical symptoms in cryptococcal meningitis. Coincide with the pandemic of HIV infection, cryptococcosis as an opportunistic infection is also growing in the world. Cryptococcal meningitis is the second most common opportunistic infection associated with AIDS in Africa and South Asia with the incidence of cryptococcosis is 15% -30% found in patients with AIDS. Without specific antifungal treatment, 100% mortality reported within two weeks after clinical cryptococcosis with meningoencephalitis in HIV-infected population. In Indonesia, before the AIDS pandemic, cryptococcosis cases are rarely reported. Since 2004, by the increasing HIV-infected patients. Department of Parasitology Faculty of Medicine, Indonesian University also reported an increase incidence of cryptococcal meningitis in AIDS patients that is about 21.9%. Associated factors with virulence of Cryptococcus neoformans is the polysaccharide capsule, melanin production and thermotolerance. Cell-mediated immunity has an important role in host defense against Cryptococcus. Laboratory tests for cryptococcosis diagnosis is direct microscopic examination using India ink, antigen detection, enzyme immunoassay, culture, and molecular methods. Keywords: cryptococcal meningitis, Cryptococcus neoformans, opportunistic infection

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