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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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E. cloacae-mediated inhibition of P. falciparum development is reversed by SRPN6 silencing.(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on An. stephensi midgut samples after feeding with a P. falciparum gametocyte/E. cloacae (106/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) An. stephensi mosquitoes were fed on a P. falciparum gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a P. falciparum gametocyte/E. cloacae (106/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of Enterobacter feeding or dsRNA treatment are shown below each sample. P-values are denoted by asterisks (** = P<0.01; *** = P<0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one P. falciparum oocyst, although the results are not significant when analyzed by Chi-squared analysis.
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pone-0062937-g005: E. cloacae-mediated inhibition of P. falciparum development is reversed by SRPN6 silencing.(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on An. stephensi midgut samples after feeding with a P. falciparum gametocyte/E. cloacae (106/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) An. stephensi mosquitoes were fed on a P. falciparum gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a P. falciparum gametocyte/E. cloacae (106/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of Enterobacter feeding or dsRNA treatment are shown below each sample. P-values are denoted by asterisks (** = P<0.01; *** = P<0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one P. falciparum oocyst, although the results are not significant when analyzed by Chi-squared analysis.

Mentions: The efficiency of dsRNA-mediated silencing of AsSRPN6 was evaluated by RT-PCR (Figure 5A). Similar to our experiments in Figure 4, the addition of E. cloacae significantly inhibited parasite development in both the dsGFP and dsSRPN6 (Figure 5B), although we cannot distinguish between the effects of E. cloacae feeding and dsRNA treatment. However, there was a significant increase in the number of P. falciparum oocysts that develop in the dsSRPN6 mosquitoes when compared to the dsGFP controls (Figure 5B). Silencing of AsSRPN6 expression also resulted in an increased prevalence of infection (Figure 5C). This would suggest that the loss of AsSRPN6 function following dsRNA-silencing partially rescues the inhibitory effects of E. cloacae that limit parasite growth. While other immune components are likely triggered by E. cloacae, our data suggest that inhibition of parasite development is partially mediated by AsSRPN6 function.


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

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

E. cloacae-mediated inhibition of P. falciparum development is reversed by SRPN6 silencing.(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on An. stephensi midgut samples after feeding with a P. falciparum gametocyte/E. cloacae (106/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) An. stephensi mosquitoes were fed on a P. falciparum gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a P. falciparum gametocyte/E. cloacae (106/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of Enterobacter feeding or dsRNA treatment are shown below each sample. P-values are denoted by asterisks (** = P<0.01; *** = P<0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one P. falciparum oocyst, although the results are not significant when analyzed by Chi-squared analysis.
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pone-0062937-g005: E. cloacae-mediated inhibition of P. falciparum development is reversed by SRPN6 silencing.(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on An. stephensi midgut samples after feeding with a P. falciparum gametocyte/E. cloacae (106/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) An. stephensi mosquitoes were fed on a P. falciparum gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a P. falciparum gametocyte/E. cloacae (106/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of Enterobacter feeding or dsRNA treatment are shown below each sample. P-values are denoted by asterisks (** = P<0.01; *** = P<0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one P. falciparum oocyst, although the results are not significant when analyzed by Chi-squared analysis.
Mentions: The efficiency of dsRNA-mediated silencing of AsSRPN6 was evaluated by RT-PCR (Figure 5A). Similar to our experiments in Figure 4, the addition of E. cloacae significantly inhibited parasite development in both the dsGFP and dsSRPN6 (Figure 5B), although we cannot distinguish between the effects of E. cloacae feeding and dsRNA treatment. However, there was a significant increase in the number of P. falciparum oocysts that develop in the dsSRPN6 mosquitoes when compared to the dsGFP controls (Figure 5B). Silencing of AsSRPN6 expression also resulted in an increased prevalence of infection (Figure 5C). This would suggest that the loss of AsSRPN6 function following dsRNA-silencing partially rescues the inhibitory effects of E. cloacae that limit parasite growth. While other immune components are likely triggered by E. cloacae, our data suggest that inhibition of parasite development is partially mediated by AsSRPN6 function.

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