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CalY is a major virulence factor and a biofilm matrix protein

Thomas Candela¹, Annette Fagerlund², Christophe Buisson¹

¹Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, 78350, France.

Molecular Microbiology

|December 15, 2018

Abstract:

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

Insights

Bacillus thuringiensis CalY protein acts as a cell-surface adhesin in early growth and forms extracellular amyloid fibers in biofilms. This bifunctional protein is crucial for bacterial adhesion and virulence.

Area of Science:

Microbiology
Structural Biology
Biochemistry

Background:

Extracellular biofilm matrices often contain amyloid fibers.
In Bacillus cereus, TasA and CalY proteins form these amyloid fibers.
The function of CalY in Bacillus thuringiensis is not fully understood.

Purpose of the Study:

To investigate the dual function of CalY in Bacillus thuringiensis.
To determine the role of CalY in bacterial adhesion and biofilm formation.
To elucidate the mechanism behind CalY's localization shift.

Main Methods:

Immunodetection to locate CalY.
Gene deletion studies (calY, sipW) to assess protein function.
Adhesion assays with HeLa cells and Galleria mellonella hemocytes.
Virulence assays in insect larvae.
Analysis of protein polymerization in vitro.

Main Results:

CalY is located on the bacterial cell surface in early stationary phase.
CalY deletion significantly reduces adhesion to host cells and virulence.
CalY shifts to the extracellular medium in mid-stationary phase and biofilms, forming fibers.
SipW signal peptidase activity influences CalY localization.
CalY polymerization requires cell-surface components.

Conclusions:

CalY is a bifunctional protein in Bacillus thuringiensis.
CalY functions as a cell-surface adhesin early in growth.
CalY contributes to biofilm structure by forming extracellular fibers later in growth.
CalY's localization and function are regulated by SipW and cell-surface interactions.

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