J Bacteriol Virol.  2015 Sep;45(3):200-214. 10.4167/jbv.2015.45.3.200.

Transcriptional Profiling of an Attenuated Salmonella Typhimurium ptsI Mutant Strain Under Low-oxygen Conditions using Microarray Analysis

• Lim S
• Han A
• Kim D
• Seo HS

• KMID: 2068735
• DOI: http://doi.org/10.4167/jbv.2015.45.3.200

Abstract

Salmonella causes a wide variety of diseases ranging from mild diarrhea to severe systemic infections, such as like typhoid fever, in multiple organisms, ranging from mice to humans. A lack of ptsI, which encodes the first component of phosphoenolpyruvate (PEP) : carbohydrate phosphotransferase system (PTS), is known to cause Salmonella Typhimurium attenuation; however, the mechanisms behind this have not yet been elucidated. In this study, a DNA microarray was performed to determine why the virulence of ptsI mutants is attenuated under low-oxygen conditions in which the ptsI expression is enhanced. Of 106 down-regulated genes, the most repressed were pdu and tdc genes, which are required for propanediol utilization and threonine and serine metabolism, respectively. In addition, half the flagellar genes were down-regulated in the ptsI mutant strain. Because pdu genes are induced during infection and Tdc products and flagella-mediated motility are necessary for the invasion of S. Typhimurium, the invasive ability of ptsI mutants was examined. We found that ptsI mutation reduced the ability of S. Typhimurium to invade into host cells, suggesting that reduced expression of the pdu, tdc, and flagellar genes is involved in the attenuation of ptsI mutants.

Keyword

Salmonella Typhimurium; PTS; Flagella; Invasion

MeSH Terms

Animals
Diarrhea
Flagella
Humans
Metabolism
Mice
Microarray Analysis*
Oligonucleotide Array Sequence Analysis
Phosphoenolpyruvate
Salmonella typhimurium*
Salmonella*
Serine
Threonine
Typhoid Fever
Virulence
Phosphoenolpyruvate
Serine
Threonine

Figure

• 

  Figure 1. Distribution of differentially expressed genes. The genes whose expression was changed in the ptsI mutant were grouped into COG functional categories. The bars (black, down-regulated; and white, up-regulated) represent the number of genes in each COG category.

• 

  Figure 2. mRNA levels of ulaC (STM2344) in mlc mutants. Bacterial cultures were grown statically to mid-log phase. After total RNA isolation, qRT-PCR analysis was performed to determine ptsG and ulaC transcript levels. mRNA levels of each gene in the wild type were arbitrarily set to 1 (white bars), and then the relative expression of each mlc mutant gene (black bars) was determined by normalizing the mRNA levels to this value. Error bars indicate the standard deviations obtained in three independent experiments performed in duplicate.

• 

  Figure 3. The fold change for the pdu and tdc genes. Bars represent the expression ratio (ptsI− to wt) for each gene, which was derived from the log2-transformed microarray hybridization results.

• 

  Figure 4. The fold change for the flagella and chemotaxis genes. Bars represent the expression ratio (ptsI− to wt) for each gene, which was derived from the log2-transformed microarray hybridization results.

• 

  Figure 5. Defects in flagella biosynthesis in ptsI mutants. In all bar graphs, white and black bars represent the wild-type (WT) and ptsI mutant (ptsI−) mutant strains, respectively. (A) Validation of microarray results for five flagellar genes. mRNA levels were measured through qRT-PCR, and the mRNA levels of each gene in WT were set to 1. Error bars indicate the standard deviations (SD) obtained in three independent experiments performed in duplicate. (B) WT and ptsI− were compared for motility. Bar graphs show the mean halo diameters (zones of motility) ± SDs obtained from a quantitative motility assay performed in triplicate. The zone of motility was measured at 7 and 14 h after inoculation. (C) Salmonella invasion into HEp-2 epithelial and RAW 264.7 macrophage cells. The mammalian cells infected by WT and ptsI− were lysed 2 h after infection and dilutions of the suspension were plated on LB agar medium to determine CFU. Data are presented as percentages of CFU of WT. Values are the means ± SDs obtained in three independent experiments performed in duplicate.

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