NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

ABSTRACT Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The ΔcnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the ΔcnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the ΔcnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the ΔcnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

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How Can a Histidine Kinase Respond to Mechanical Stress?

Frontiers in Microbiology ◽

10.3389/fmicb.2021.655942 ◽

2021 ◽

Vol 12 ◽

Author(s):

Linda J. Kenney

Keyword(s):

Gene Expression ◽

Shear Stress ◽

Osmotic Stress ◽

Mechanical Stress ◽

Histidine Kinase ◽

Blood Stream ◽

Membrane Tension ◽

Histidine Kinases ◽

Inner Membrane ◽

Physical Forces

Bacteria respond to physical forces perceived as mechanical stress as part of their comprehensive environmental sensing strategy. Histidine kinases can then funnel diverse environmental stimuli into changes in gene expression through a series of phosphorelay reactions. Because histidine kinases are most often embedded in the inner membrane, they can be sensitive to changes in membrane tension that occurs, for example, in response to osmotic stress, or when deformation of the cell body occurs upon encountering a surface before forming biofilms, or inside the host in response to shear stress in the kidney, intestine, lungs, or blood stream. A summary of our recent work that links the histidine kinase EnvZ to mechanical changes in the inner membrane is provided and placed in a context of other bacterial systems that respond to mechanical stress.

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High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

Plants ◽

10.3390/plants9030304 ◽

2020 ◽

Vol 9 (3) ◽

pp. 304 ◽

Cited By ~ 1

Author(s):

Alexander Hofmann ◽

Sophia Müller ◽

Thomas Drechsler ◽

Mareike Berleth ◽

Katharina Caesar ◽

...

Keyword(s):

Osmotic Stress ◽

Stress Responses ◽

Histidine Kinase ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Cd Spectroscopy ◽

Single Step ◽

Signaling Mechanism ◽

Wide Range

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

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Group III Histidine Kinase Is a Positive Regulator of Hog1-Type Mitogen-Activated Protein Kinase in Filamentous Fungi

Eukaryotic Cell ◽

10.1128/ec.4.11.1820-1828.2005 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1820-1828 ◽

Cited By ~ 82

Author(s):

Akira Yoshimi ◽

Kaihei Kojima ◽

Yoshitaka Takano ◽

Chihiro Tanaka

Keyword(s):

Osmotic Stress ◽

Filamentous Fungi ◽

Histidine Kinase ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Group Iii ◽

Positive Regulator ◽

Mitogen Activated Protein ◽

In The Wild

ABSTRACT We previously reported that the group III histidine kinase Dic1p in the maize pathogen Cochliobolus heterostrophus is involved in resistance to dicarboximide and phenylpyrrole fungicides and in osmotic adaptation. In addition, exposure to the phenylpyrrole fungicide fludioxonil led to improper activation of Hog1-type mitogen-activated protein kinases (MAPKs) in some phytopathogenic fungi, including C. heterostrophus. Here we report, for the first time, the relationship between the group III histidine kinase and Hog1-related MAPK: group III histidine kinase is a positive regulator of Hog1-related MAPK in filamentous fungi. The phosphorylation pattern of C. heterostrophus BmHog1p (Hog1-type MAPK) was analyzed in wild-type and dic1-deficient strains by Western blotting. In the wild-type strain, phosphorylated BmHog1p was detected after exposure to both iprodione and fludioxonil at a concentration of 1 μg/ml. In the dic1-deficient strains, phosphorylated BmHog1p was not detected after exposure to 10 μg/ml of the fungicides. In response to osmotic stress (0.4 M KCl), a trace of phosphorylated BmHog1p was found in the dic1-deficient strains, whereas the band representing active BmHog1p was clearly detected in the wild-type strain. Similar results were obtained for Neurospora crassa Os-2p MAPK phosphorylation in the mutant of the group III histidine kinase gene os-1. These results indicate that group III histidine kinase positively regulates the activation of Hog1-type MAPKs in filamentous fungi. Notably, the Hog1-type MAPKs were activated at high fungicide (100 μg/ml) and osmotic stress (0.8 M KCl) levels in the histidine kinase mutants of both fungi, suggesting that another signaling pathway activates Hog1-type MAPKs in these conditions.

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The Eukaryotic Two-Component Histidine Kinase Sln1p RegulatesOCH1via the Transcription Factor, Skn7p

Molecular Biology of the Cell ◽

10.1091/mbc.01-09-0434 ◽

2002 ◽

Vol 13 (2) ◽

pp. 412-424 ◽

Cited By ~ 69

Author(s):

Sheng Li ◽

Susan Dean ◽

Zhijian Li ◽

Joe Horecka ◽

Robert J. Deschenes ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Osmotic Stress ◽

Binding Site ◽

Histidine Kinase ◽

Response Regulators ◽

Hog Pathway ◽

Receiver Domain ◽

Osmotic Response ◽

Two Component

The yeast “two-component” osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with acis-acting element identified upstream ofOCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.

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A Fungus-Specific Ras Homolog Contributes to the Hyphal Growth and Virulence of Aspergillus fumigatus

Eukaryotic Cell ◽

10.1128/ec.4.12.1982-1989.2005 ◽

2005 ◽

Vol 4 (12) ◽

pp. 1982-1989 ◽

Cited By ~ 61

Author(s):

Jarrod R. Fortwendel ◽

Wei Zhao ◽

Ruchi Bhabhra ◽

Steven Park ◽

David S. Perlin ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Growth Control ◽

Biomass Accumulation ◽

Hyphal Growth ◽

Pathogenic Fungi ◽

Total Biomass ◽

Gene Encoding ◽

Directional Growth ◽

Solid Media ◽

Hyphal Morphology

ABSTRACT The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ∼65% survival compared to ∼10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

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