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Wolbachia infections in Anopheles gambiae cells: transcriptomic characterization of a novel host-symbiont interaction.

Hughes GL, Ren X, Ramirez JL, Sakamoto JM, Bailey JA, Jedlicka AE, Rasgon JL - PLoS Pathog. (2011)

Bottom Line: We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes.Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts.The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.

View Article: PubMed Central - PubMed

Affiliation: The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

ABSTRACT
The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.

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Validation of microarray data in cell culture and whole mosquitoes.A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.
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ppat-1001296-g002: Validation of microarray data in cell culture and whole mosquitoes.A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.

Mentions: To gauge the accuracy of the microarray data, we selected a subset of genes to validate by quantitative real-time PCR from cell culture. Eight genes, (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin11, Filamin, TEP3) with varying expression profiles, regulated by both Wolbachia strains were evaluated. These genes spanned a variety of functional classes including defensive and immune genes that may be relevant to Plasmodium infection and potential Wolbachia-mediated reproductive phenotypes. qPCR results corroborated the array data and had a positive linear correlation (R2 = 0.9595) when comparing the log2 values using both gene expression techniques (Figure 2A).


Wolbachia infections in Anopheles gambiae cells: transcriptomic characterization of a novel host-symbiont interaction.

Hughes GL, Ren X, Ramirez JL, Sakamoto JM, Bailey JA, Jedlicka AE, Rasgon JL - PLoS Pathog. (2011)

Validation of microarray data in cell culture and whole mosquitoes.A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040664&req=5

ppat-1001296-g002: Validation of microarray data in cell culture and whole mosquitoes.A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.
Mentions: To gauge the accuracy of the microarray data, we selected a subset of genes to validate by quantitative real-time PCR from cell culture. Eight genes, (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin11, Filamin, TEP3) with varying expression profiles, regulated by both Wolbachia strains were evaluated. These genes spanned a variety of functional classes including defensive and immune genes that may be relevant to Plasmodium infection and potential Wolbachia-mediated reproductive phenotypes. qPCR results corroborated the array data and had a positive linear correlation (R2 = 0.9595) when comparing the log2 values using both gene expression techniques (Figure 2A).

Bottom Line: We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes.Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts.The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.

View Article: PubMed Central - PubMed

Affiliation: The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

ABSTRACT
The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.

Show MeSH
Related in: MedlinePlus