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Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi.

Eappen AG, Smith RC, Jacobs-Lorena M - PLoS ONE (2013)

Bottom Line: Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium.Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota.We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.

ABSTRACT
Successful development of Plasmodium in the mosquito is essential for the transmission of malaria. A major bottleneck in parasite numbers occurs during midgut invasion, partly as a consequence of the complex interactions between the endogenous microbiota and the mosquito immune response. We previously identified SRPN6 as an immune component which restricts Plasmodium berghei development in the mosquito. Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium. Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota. We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

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SRPN6 is differentially induced by bacteria in the mosquito midgut.(A) Bacteria (1×106/ml of buffer; 2,000 bacteria assuming ingested volume of 2 µl) or the indicated component were fed to An. stephensi mosquitoes and their midguts were dissected 6 h later. Total RNA (3 µg) was analyzed by Northern blot using a 32P-labeled SRPN6 cDNA probe (upper panel). The blot was then stripped and hybridized with mitochondrial rRNA probe as a loading control (lower panel). Samples are identified above each lane as follows. U: unfed control; B: buffer-fed; Lp: E. coli LPS (10 mg/ml); Ec: Enterobacter cloacae; St: Salmonella typhimurium; Sm: Serratia marcescens; Pa: Pseudomonas aeruginosa; Ml: Micrococcus luteus; Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Pb: P. berghei-infected blood, analyzed 24 h after feeding (positive control). (B) Expression of gambicin, a mosquito anti-microbial peptide. Gambicin transcript abundance was analyzed by semi-quantitative RT-PCR (upper panel) using ribosomal protein S7 (rpS7) mRNA expression as a loading control (lower panel). RNA templates are the same as those used in panel (A).
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pone-0062937-g001: SRPN6 is differentially induced by bacteria in the mosquito midgut.(A) Bacteria (1×106/ml of buffer; 2,000 bacteria assuming ingested volume of 2 µl) or the indicated component were fed to An. stephensi mosquitoes and their midguts were dissected 6 h later. Total RNA (3 µg) was analyzed by Northern blot using a 32P-labeled SRPN6 cDNA probe (upper panel). The blot was then stripped and hybridized with mitochondrial rRNA probe as a loading control (lower panel). Samples are identified above each lane as follows. U: unfed control; B: buffer-fed; Lp: E. coli LPS (10 mg/ml); Ec: Enterobacter cloacae; St: Salmonella typhimurium; Sm: Serratia marcescens; Pa: Pseudomonas aeruginosa; Ml: Micrococcus luteus; Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Pb: P. berghei-infected blood, analyzed 24 h after feeding (positive control). (B) Expression of gambicin, a mosquito anti-microbial peptide. Gambicin transcript abundance was analyzed by semi-quantitative RT-PCR (upper panel) using ribosomal protein S7 (rpS7) mRNA expression as a loading control (lower panel). RNA templates are the same as those used in panel (A).

Mentions: Previous experiments have demonstrated that SRPN6 expression is induced by Plasmodium ookinete invasion of the mosquito midgut [19], [22] and sporozoite invasion of the salivary glands [20]. To investigate whether bacteria also play a role in AsSRPN6 activation, we measured changes in gene expression after feeding An. stephensi mosquitoes with different species of bacteria (Figure 1A). These experiments revealed that the intensity of AsSRPN6 expression is differentially regulated by different bacterial species. AsSRPN6 expression was strongly induced by most gram-negative bacteria examined, while gram-positive bacteria produced much weaker expression. These results agree with previous results that SRPN6 was primarily induced by gram-negative bacteria [9]. Interestingly, AsSRPN6 expression was independent of the anti-microbial protein (AMP) gambicin [23], which is strongly expressed in the mosquito midgut and induced by most agents, including LPS and gram-positive bacteria but not by buffer or P. berghei parasites (Figure 1B). These observations suggest that multiple pathways may contribute to the midgut immune response to produce specific responses toward endogenous gram-negative bacteria and invading pathogens.


Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi.

Eappen AG, Smith RC, Jacobs-Lorena M - PLoS ONE (2013)

SRPN6 is differentially induced by bacteria in the mosquito midgut.(A) Bacteria (1×106/ml of buffer; 2,000 bacteria assuming ingested volume of 2 µl) or the indicated component were fed to An. stephensi mosquitoes and their midguts were dissected 6 h later. Total RNA (3 µg) was analyzed by Northern blot using a 32P-labeled SRPN6 cDNA probe (upper panel). The blot was then stripped and hybridized with mitochondrial rRNA probe as a loading control (lower panel). Samples are identified above each lane as follows. U: unfed control; B: buffer-fed; Lp: E. coli LPS (10 mg/ml); Ec: Enterobacter cloacae; St: Salmonella typhimurium; Sm: Serratia marcescens; Pa: Pseudomonas aeruginosa; Ml: Micrococcus luteus; Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Pb: P. berghei-infected blood, analyzed 24 h after feeding (positive control). (B) Expression of gambicin, a mosquito anti-microbial peptide. Gambicin transcript abundance was analyzed by semi-quantitative RT-PCR (upper panel) using ribosomal protein S7 (rpS7) mRNA expression as a loading control (lower panel). RNA templates are the same as those used in panel (A).
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Related In: Results  -  Collection

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pone-0062937-g001: SRPN6 is differentially induced by bacteria in the mosquito midgut.(A) Bacteria (1×106/ml of buffer; 2,000 bacteria assuming ingested volume of 2 µl) or the indicated component were fed to An. stephensi mosquitoes and their midguts were dissected 6 h later. Total RNA (3 µg) was analyzed by Northern blot using a 32P-labeled SRPN6 cDNA probe (upper panel). The blot was then stripped and hybridized with mitochondrial rRNA probe as a loading control (lower panel). Samples are identified above each lane as follows. U: unfed control; B: buffer-fed; Lp: E. coli LPS (10 mg/ml); Ec: Enterobacter cloacae; St: Salmonella typhimurium; Sm: Serratia marcescens; Pa: Pseudomonas aeruginosa; Ml: Micrococcus luteus; Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Pb: P. berghei-infected blood, analyzed 24 h after feeding (positive control). (B) Expression of gambicin, a mosquito anti-microbial peptide. Gambicin transcript abundance was analyzed by semi-quantitative RT-PCR (upper panel) using ribosomal protein S7 (rpS7) mRNA expression as a loading control (lower panel). RNA templates are the same as those used in panel (A).
Mentions: Previous experiments have demonstrated that SRPN6 expression is induced by Plasmodium ookinete invasion of the mosquito midgut [19], [22] and sporozoite invasion of the salivary glands [20]. To investigate whether bacteria also play a role in AsSRPN6 activation, we measured changes in gene expression after feeding An. stephensi mosquitoes with different species of bacteria (Figure 1A). These experiments revealed that the intensity of AsSRPN6 expression is differentially regulated by different bacterial species. AsSRPN6 expression was strongly induced by most gram-negative bacteria examined, while gram-positive bacteria produced much weaker expression. These results agree with previous results that SRPN6 was primarily induced by gram-negative bacteria [9]. Interestingly, AsSRPN6 expression was independent of the anti-microbial protein (AMP) gambicin [23], which is strongly expressed in the mosquito midgut and induced by most agents, including LPS and gram-positive bacteria but not by buffer or P. berghei parasites (Figure 1B). These observations suggest that multiple pathways may contribute to the midgut immune response to produce specific responses toward endogenous gram-negative bacteria and invading pathogens.

Bottom Line: Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium.Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota.We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.

ABSTRACT
Successful development of Plasmodium in the mosquito is essential for the transmission of malaria. A major bottleneck in parasite numbers occurs during midgut invasion, partly as a consequence of the complex interactions between the endogenous microbiota and the mosquito immune response. We previously identified SRPN6 as an immune component which restricts Plasmodium berghei development in the mosquito. Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium. Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota. We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

Show MeSH
Related in: MedlinePlus