scholarly journals Fungicidal Mechanisms of Cathelicidins LL-37 and CATH-2 Revealed by Live-Cell Imaging

2014 ◽  
Vol 58 (4) ◽  
pp. 2240-2248 ◽  
Author(s):  
Soledad R. Ordonez ◽  
Ilham H. Amarullah ◽  
Richard W. Wubbolts ◽  
Edwin J. A. Veldhuizen ◽  
Henk P. Haagsman
Keyword(s):  
Antifungal Activity ◽  
Cell Membrane ◽  
Live Cell Imaging ◽  
Fungal Pathogens ◽  
Cell Imaging ◽  
Mechanisms Of Action ◽  
Live Cell ◽  
Energy Status ◽  
Fungal Cell ◽  
Content Type

ABSTRACTAntifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5).Candida albicanswas used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides killC. albicansrapidly. CATH-2 is the most active peptide and killsC. albicanswithin 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to theC. albicanscell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreasesC. albicanscell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

scholarly journals F-Actin Dynamics in Neurospora crassa

2010 ◽  
Vol 9 (4) ◽  
pp. 547-557 ◽  
Author(s):  
Adokiye Berepiki ◽  
Alexander Lichius ◽  
Jun-Ya Shoji ◽  
Jens Tilsner ◽  
Nick D. Read
Keyword(s):  
Neurospora Crassa ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Content Type ◽  
Actin Cables ◽  
Germ Tubes

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

scholarly journals Single-Inclusion Kinetics of Chlamydia trachomatis Development

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Travis J. Chiarelli ◽  
Nicole A. Grieshaber ◽  
Anders Omsland ◽  
Christopher H. Remien ◽  
Scott S. Grieshaber
Keyword(s):  
Chlamydia Trachomatis ◽  
Mathematical Models ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Host Cells ◽  
Developmental Cycle ◽  
Cell Type ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

scholarly journals Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

2014 ◽  
Vol 197 (5) ◽  
pp. 807-818 ◽  
Author(s):  
Morten Kjos ◽  
Rieza Aprianto ◽  
Vitor E. Fernandes ◽  
Peter W. Andrew ◽  
Jos A. G. van Strijp ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Pneumococcal Infection ◽  
Live Cell ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Mouse Tissue ◽  
Content Type

Streptococcus pneumoniaeis a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imagingS. pneumoniaestrain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used forin situimaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.

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