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Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, Rickettsia typhi.

Dreher-Lesnick SM, Ceraul SM, Rahman MS, Azad AF - BMC Microbiol. (2008)

Bottom Line: A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database.The microarray results were validated with quantitative real time RT-PCR.Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, 660 W, Redwood Street, Room HH324B, Baltimore, MD 21201, USA. sdreh001@umaryland.edu

ABSTRACT

Background: The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. Rickettsia typhi, the etiologic agent of murine typhus, is a strictly intracellular gram negative alpha-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, Xenopsylla cheopis. Thus, R. typhi must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing R. typhi to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37 degrees C to 25 degrees C, mimicking the two different host temperatures in vitro.

Results: Temperature-responsive genes belonged to multiple functional categories including among others, transcription, translation, posttranslational modification/protein turnover/chaperones and intracellular trafficking and secretion. A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database. The microarray results were validated with quantitative real time RT-PCR.

Conclusion: This microarray screen identified various genes that were differentially expressed upon a shift in temperature from 37 degrees C to 25 degrees C. Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.

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Validation of microarray data and comparison with real time qRT-PCR data. (A) Comparison of real time (white bars) and microarray (black bars) fold change results for 14 select R. typhi genes listed in Additional file 6. Fold change ratios represent the difference in transcript abundance/signal for these genes post temperature shift from 37°C to 25°C. (B) Correlation analysis of microarray and real time transcript measurements for the 14 select R. typhi genes mentioned above. The real time qRT-PCR log2 values were plotted against the microarray log2 values. The correlation coefficient (R2) between the two datasets is 0.8227.
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Figure 2: Validation of microarray data and comparison with real time qRT-PCR data. (A) Comparison of real time (white bars) and microarray (black bars) fold change results for 14 select R. typhi genes listed in Additional file 6. Fold change ratios represent the difference in transcript abundance/signal for these genes post temperature shift from 37°C to 25°C. (B) Correlation analysis of microarray and real time transcript measurements for the 14 select R. typhi genes mentioned above. The real time qRT-PCR log2 values were plotted against the microarray log2 values. The correlation coefficient (R2) between the two datasets is 0.8227.

Mentions: Real time quantitative RT-PCR was used to validate our microarray screen. Temperature shift studies were repeated in triplicate, and R. typhi RNA was isolated as described (see Materials and Methods). Transcription data were obtained for 15 genes representing upregulated, downregulated and unchanged genes (Figure 2). While the fold change values obtained from our real time analysis showed some variation compared to the fold change values obtained in our arrays, the general trends remain consistent (Figure 2A). Log fold change of relative transcript abundance between the two temperature conditions was calculated and plotted against the average log fold change values obtained from the arrays (Figure 2B). We found a positive correlation (R2 = 0.8227) between the values obtained from the two techniques. The real time qRT-PCR analysis thus supports the trends observed in our array results, but demonstrates a need to verify transcription trends for individual genes during follow up studies.


Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, Rickettsia typhi.

Dreher-Lesnick SM, Ceraul SM, Rahman MS, Azad AF - BMC Microbiol. (2008)

Validation of microarray data and comparison with real time qRT-PCR data. (A) Comparison of real time (white bars) and microarray (black bars) fold change results for 14 select R. typhi genes listed in Additional file 6. Fold change ratios represent the difference in transcript abundance/signal for these genes post temperature shift from 37°C to 25°C. (B) Correlation analysis of microarray and real time transcript measurements for the 14 select R. typhi genes mentioned above. The real time qRT-PCR log2 values were plotted against the microarray log2 values. The correlation coefficient (R2) between the two datasets is 0.8227.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335108&req=5

Figure 2: Validation of microarray data and comparison with real time qRT-PCR data. (A) Comparison of real time (white bars) and microarray (black bars) fold change results for 14 select R. typhi genes listed in Additional file 6. Fold change ratios represent the difference in transcript abundance/signal for these genes post temperature shift from 37°C to 25°C. (B) Correlation analysis of microarray and real time transcript measurements for the 14 select R. typhi genes mentioned above. The real time qRT-PCR log2 values were plotted against the microarray log2 values. The correlation coefficient (R2) between the two datasets is 0.8227.
Mentions: Real time quantitative RT-PCR was used to validate our microarray screen. Temperature shift studies were repeated in triplicate, and R. typhi RNA was isolated as described (see Materials and Methods). Transcription data were obtained for 15 genes representing upregulated, downregulated and unchanged genes (Figure 2). While the fold change values obtained from our real time analysis showed some variation compared to the fold change values obtained in our arrays, the general trends remain consistent (Figure 2A). Log fold change of relative transcript abundance between the two temperature conditions was calculated and plotted against the average log fold change values obtained from the arrays (Figure 2B). We found a positive correlation (R2 = 0.8227) between the values obtained from the two techniques. The real time qRT-PCR analysis thus supports the trends observed in our array results, but demonstrates a need to verify transcription trends for individual genes during follow up studies.

Bottom Line: A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database.The microarray results were validated with quantitative real time RT-PCR.Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, 660 W, Redwood Street, Room HH324B, Baltimore, MD 21201, USA. sdreh001@umaryland.edu

ABSTRACT

Background: The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. Rickettsia typhi, the etiologic agent of murine typhus, is a strictly intracellular gram negative alpha-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, Xenopsylla cheopis. Thus, R. typhi must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing R. typhi to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37 degrees C to 25 degrees C, mimicking the two different host temperatures in vitro.

Results: Temperature-responsive genes belonged to multiple functional categories including among others, transcription, translation, posttranslational modification/protein turnover/chaperones and intracellular trafficking and secretion. A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database. The microarray results were validated with quantitative real time RT-PCR.

Conclusion: This microarray screen identified various genes that were differentially expressed upon a shift in temperature from 37 degrees C to 25 degrees C. Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.

Show MeSH
Related in: MedlinePlus