Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model

Seung Min Kim; Hee Sun Sim; H. Stanley Kim; Ho Ki Shim; Young Kyung Yoon; Jeong Yeon Kim; Yun Sun Park; Dae Won Park; Jang Wook Sohn; Min Ja Kim

doi:10.3947/ic.2010.42.1.23

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Kim, Sim, Kim, Shim, Yoon, Kim, Park, Park, Sohn, and Kim: Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model

Original Article

Infection & Chemotherapy

Infection & Chemotherapy 2010; 42(1): 23-29.

Published online: 28 February 2010

DOI: https://doi.org/10.3947/ic.2010.42.1.23

Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model

Seung Min Kim¹, Hee Sun Sim³, H. Stanley Kim¹, Ho Ki Shim³, Young Kyung Yoon², Jeong Yeon Kim², Yun Sun Park², Dae Won Park^2,³, Jang Wook Sohn^2,³, Min Ja Kim^1,^2,³

¹BK21 Program of Graduate School, Korea University College of Medicine, Seoul, Korea.

²Division of Infectious Diseases, Korea University College of Medicine, Seoul, Korea.

³Research Institute of Emerging Infectious Diseases, Korea University, Seoul, Korea.

Corresponding Author: Min Ja Kim, M.D.,Ph.D. Division of Infectious Diseases, Korea University Medical Center, 126-1, 5ka, Anam-Dong, Seongbuk-gu, Seoul 136-705, Korea. Tel: +82-2-920-5685, Fax: +82-2-920-5616, macropha@korea.ac.kr

Received 23 October 2009 Accepted 21 December 2009

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. After L. pneumophila is inhaled through contaminated aerosols, it is phagocytized by alveolar macrophages, multiplies in a specialized phagosome approximately 10 h postinfection, and eventually leads to the death of host cells. Currently available diagnostic tests for Legionella pneumonia have some limitations. This study was conducted to find diagnostic biomarkers for Legionella pneumonia using virulence gene expression profiling in a murine experimental model.

Materials and Methods

A/J mice were intranasally inoculated with L. pneumophila serogroup 1, and lungs were harvested 4, 8, 24, and 48 h postinfection. The strain grown in buffered yeast extract broth was used as reference samples. Cy-dye labeled cDNA samples were prepared with total RNA from lungs or broth culture, and hybridized on the oligo-microarray slide containing 2,895 genes of L. pneumophila serogroup 1. Virulence gene expression patterns were analyzed using a MIDAS software from TIGR (www.tigr.org).

Results

Among a total of 332 virulence genes examined, 17 genes including sidA, lepB, the genes related to flagella assembly (fliR and fliP), LPS lipid A biosynthesis, and the enhanced entry protein EnhA were up-regulated at all four time points. We further confirmed by quantitative real-time reverse transcription PCR that the expression of fliP gene was highly expressed in lung tissue as well as in bronchoalveolar lavage fluids from the mouse infected with L. pneumophila serogroup 1.

Conclusions

Through gene expression analysis of L. pneumophila in a mouse model, several candidate biomarkers for diagnosing Legionnaires' disease could be identified.

Keywords: Legionella pneumophila, Diagnosis, Biomarker, cDNA microarray

Figures and Tables

Figure 1

Growth kinetics of Legionella pneumophila strain in lung tissue of A/J mouse and the number of genes with measurable expression by cDNA microarray analysis according to the postinfection time points. Total number of Legionella bacteria recovered from the lung was expressed as colony-forming units (cfu/lung). Number of gene analyzed was expressed as percentage of total number of genes examined.

Figure 2

Venn diagram showing number of virulence genes of Legionella pneumophila serogroup 1 expressed at postinfection 8, 24, 48 hr time points. Each of the 90, 87, and 86 genes were analyzed using microarray at 8, 24, 48 hrs after infection, respectively. Thirty-four genes were analyzed at all 3 time points.

Figure 3

Expression profiles of the highly up-regulated virulence genes of L. pneumophila serogroup 1 at postinfection 4, 8, 24 and 48 hr time points by using TIGR-MeV software. A total of 17 virulence genes were up-regulated at all time points. Each of the proteins was categorized according to the square with certain color, which presents the function of the protein.

Figure 4

Validation of microarray results on the fliP expression. The fliP gene expression profiles of microarray analysis and a quantitative real-time RT-PCR assay at postinfection 4 time points were compared. Microarray data was considered to be valid if the fold change calculated by the qRT-PCR was in the same direction as determined by microarray results.

Figure 5

Expression profiles of the highly down-regulated virulence genes of L. pneumophila serogroup 1 at postinfection 4, 8, 24 and 48 h time points by using TIGR-MeV software. A total of 18 virulence genes were down-regulated at all time points. Each of the squares having colors represents was categorized according to the functions of proteins. Each of the proteins was categorized according to the square with certain color, which presents function of the protein.

Figure 6

Gene expression of the fliP gene in BAL fluids collected from A/J mice at 24, 48, 72 hr after infection with Legionella pneumophila serogroup 1. Bars represent 2^-△△Ct values of the fliP in BAL fluids at each postinfection time point and the 2^-△△Ct Ct value using △△Ct confers the level of the fliP gene expression. The fold change was the highest value at postinfection 72 hr time point.

Table 1

The highly up-regulated genes among the virulence genes at all time points after infection of A/J mice with L. pneumophila serogroup 1

^*Mean values represent mean log₂ ratios (in vivo/in vitro) calculated from three replicates, ^†p.i., postinfection

Table 2

The highly down-regulated genes among the virulence genes at all time points after the A/J mice have been infected with L. pneumophila serogroup 1

^*Mean values represent mean log₂ ratios (in vivo/in vitro) calculated from three replicates, ^†p.i., postinfection

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