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Identification and characterization of autotransporter proteins of Yersinia pestis KIM
Yihfen T Yen1, Aarthi Karkal, Mou Bhattacharya
1Department of Biology and Biochemistry, University of Houston, Texas 77204, USA.
Abstract:
Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.
Insights
This study identifies and characterizes ten autotransporter genes in Yersinia pestis, the bacterium causing plague. Researchers confirmed gene expression and localized key proteins, revealing their roles in bacterial virulence and potential as bioterrorism targets.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Yersinia pestis causes plague, a re-emerging infectious disease and potential bioterrorism threat.
- Autotransporters (ATs) are critical virulence factors in Gram-negative pathogens, facilitating protein translocation.
Purpose of the Study:
- To identify and characterize autotransporter genes in Yersinia pestis KIM.
- To investigate the expression, localization, and function of these ATs in Y. pestis pathogenicity.
Main Methods:
- In silico genomic screening for AT genes in Y. pestis KIM.
- RT-PCR to confirm gene expression.
- Cloning and expression in E. coli for protein secretion studies.
- Cell fractionation and protease accessibility assays for protein localization.
- Functional assays (hemagglutination, autoagglutination).
Main Results:
- Ten putative AT genes were identified in Y. pestis KIM, with two located on pathogenicity islands.
- Expression of all ten AT genes was confirmed by RT-PCR.
- YapA, YapC, YapG, YapK, and YapN were successfully cloned and expressed.
- YapA was secreted, while YapC, YapG, YapK, and YapN localized to the outer membrane.
- YapC, YapN, and YapK exhibited autoagglutination and hemagglutination activities, respectively.
Conclusions:
- This is the first comprehensive study of autotransporters in Yersinia pestis.
- Identified ATs are potential virulence factors contributing to Y. pestis pathogenicity.
- These findings provide insights into Y. pestis biology and potential therapeutic targets.
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