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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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Activation of SRPN6 midgut expression by E. cloacae.(A) Time course of An. stephensi SRPN6 mRNA expression after feeding of E. cloacae (1×106/ml; open bars) or P. berghei (green bars) as determined by qRT-PCR using ribosomal protein S7 (rpS7) for normalization. Values are reported in fold change relative to expression before feeding (0 h). (B) Immunolocalization of SRPN6 in midguts of mosquitoes fed with buffer (left panels) or E. cloacae (right panels). Guts were dissected 6 h after feeding, opened up into sheets, fixed and the protein detected with an anti-SRPN6 antibody. The inserts show higher magnifications of the areas within the squares. (C) Western blot analysis of SRPN6 protein expression after feeding with buffer alone (B) or at 6 and 24 h after E. cloacae ingestion, as indicated. Recombinant SRPN6 protein was used as a positive control (Rec). The blot was stripped and re-probed with an anti-actin antibody as a loading control (lower panel).
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pone-0062937-g003: Activation of SRPN6 midgut expression by E. cloacae.(A) Time course of An. stephensi SRPN6 mRNA expression after feeding of E. cloacae (1×106/ml; open bars) or P. berghei (green bars) as determined by qRT-PCR using ribosomal protein S7 (rpS7) for normalization. Values are reported in fold change relative to expression before feeding (0 h). (B) Immunolocalization of SRPN6 in midguts of mosquitoes fed with buffer (left panels) or E. cloacae (right panels). Guts were dissected 6 h after feeding, opened up into sheets, fixed and the protein detected with an anti-SRPN6 antibody. The inserts show higher magnifications of the areas within the squares. (C) Western blot analysis of SRPN6 protein expression after feeding with buffer alone (B) or at 6 and 24 h after E. cloacae ingestion, as indicated. Recombinant SRPN6 protein was used as a positive control (Rec). The blot was stripped and re-probed with an anti-actin antibody as a loading control (lower panel).

Mentions: To better understand the mechanism of AsSRPN6 induction by gram-negative bacteria, we compared the time course of AsSRPN6 expression with that of P. berghei, which served as a positive control. Bacterial induction of AsSRPN6 was rapid as mRNA abundance and protein expression reached peak levels at ∼6 h after bacteria feeding (Figure 3A and 3C). In contrast, peak expression after a P. berghei-infected blood meal occurred at ∼24 h (Figure 3A). This difference is likely explained by the timing of the physical interactions between the inducing agent and the midgut epithelium. Following bacteria feeding, the contact between bacteria and the midgut epithelium likely occurs soon after ingestion. However, the ingestion of a blood meal triggers the formation of the peritrophic matrix, a chitinous acellular layer that completely surrounds the blood bolus, physically separating it from the midgut epithelium [25]. Not until Plasmodium ookinetes differentiate, traverse the peritrophic matrix, and invade the midgut epithelium are the presumed signals leading to AsSRPN6 activation initiated.


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

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

Activation of SRPN6 midgut expression by E. cloacae.(A) Time course of An. stephensi SRPN6 mRNA expression after feeding of E. cloacae (1×106/ml; open bars) or P. berghei (green bars) as determined by qRT-PCR using ribosomal protein S7 (rpS7) for normalization. Values are reported in fold change relative to expression before feeding (0 h). (B) Immunolocalization of SRPN6 in midguts of mosquitoes fed with buffer (left panels) or E. cloacae (right panels). Guts were dissected 6 h after feeding, opened up into sheets, fixed and the protein detected with an anti-SRPN6 antibody. The inserts show higher magnifications of the areas within the squares. (C) Western blot analysis of SRPN6 protein expression after feeding with buffer alone (B) or at 6 and 24 h after E. cloacae ingestion, as indicated. Recombinant SRPN6 protein was used as a positive control (Rec). The blot was stripped and re-probed with an anti-actin antibody as a loading control (lower panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643921&req=5

pone-0062937-g003: Activation of SRPN6 midgut expression by E. cloacae.(A) Time course of An. stephensi SRPN6 mRNA expression after feeding of E. cloacae (1×106/ml; open bars) or P. berghei (green bars) as determined by qRT-PCR using ribosomal protein S7 (rpS7) for normalization. Values are reported in fold change relative to expression before feeding (0 h). (B) Immunolocalization of SRPN6 in midguts of mosquitoes fed with buffer (left panels) or E. cloacae (right panels). Guts were dissected 6 h after feeding, opened up into sheets, fixed and the protein detected with an anti-SRPN6 antibody. The inserts show higher magnifications of the areas within the squares. (C) Western blot analysis of SRPN6 protein expression after feeding with buffer alone (B) or at 6 and 24 h after E. cloacae ingestion, as indicated. Recombinant SRPN6 protein was used as a positive control (Rec). The blot was stripped and re-probed with an anti-actin antibody as a loading control (lower panel).
Mentions: To better understand the mechanism of AsSRPN6 induction by gram-negative bacteria, we compared the time course of AsSRPN6 expression with that of P. berghei, which served as a positive control. Bacterial induction of AsSRPN6 was rapid as mRNA abundance and protein expression reached peak levels at ∼6 h after bacteria feeding (Figure 3A and 3C). In contrast, peak expression after a P. berghei-infected blood meal occurred at ∼24 h (Figure 3A). This difference is likely explained by the timing of the physical interactions between the inducing agent and the midgut epithelium. Following bacteria feeding, the contact between bacteria and the midgut epithelium likely occurs soon after ingestion. However, the ingestion of a blood meal triggers the formation of the peritrophic matrix, a chitinous acellular layer that completely surrounds the blood bolus, physically separating it from the midgut epithelium [25]. Not until Plasmodium ookinetes differentiate, traverse the peritrophic matrix, and invade the midgut epithelium are the presumed signals leading to AsSRPN6 activation initiated.

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