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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 inhibits P. falciparum development in An. stephensi.(A) Mosquitoes were fed on a P. falciparum gametocyte culture mixed either with medium alone (control) or with E. cloacae (+Ec; at a final concentration of 1×106/ml). After 8 days, oocyst numbers were counted by mercurochrome staining of dissected midgut samples and the data were pooled from four independent experiments. Median oocyst numbers are depicted by the red line and the P-value was determined using a Mann–Whitney U test. The total numbers (n) of mosquitoes analyzed are denoted below each sample. The percentage of mosquitoes containing at least one P. falciparum oocyst (or prevalence of infection) is shown in (B). Samples were analyzed by Chi-squared analysis to determine significance. P-values are denoted by asterisks (* = P<0.05; *** = P<0.001).
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pone-0062937-g004: E. cloacae inhibits P. falciparum development in An. stephensi.(A) Mosquitoes were fed on a P. falciparum gametocyte culture mixed either with medium alone (control) or with E. cloacae (+Ec; at a final concentration of 1×106/ml). After 8 days, oocyst numbers were counted by mercurochrome staining of dissected midgut samples and the data were pooled from four independent experiments. Median oocyst numbers are depicted by the red line and the P-value was determined using a Mann–Whitney U test. The total numbers (n) of mosquitoes analyzed are denoted below each sample. The percentage of mosquitoes containing at least one P. falciparum oocyst (or prevalence of infection) is shown in (B). Samples were analyzed by Chi-squared analysis to determine significance. P-values are denoted by asterisks (* = P<0.05; *** = P<0.001).

Mentions: Previous reports have demonstrated that the presence of bacteria within the mosquito midgut greatly influences Plasmodium development [8], [9], [12], and that field isolates of an Enterobacter species have a profound effect on P. falciparum development [8]. Our results (Figures 1 and 3) show that E. cloacae strongly induces AsSRPN6 expression (and other components of the mosquito immune response) in the mosquito midgut. To determine the effects of our Enterobacter strain on parasite development, we fed An. stephensi mosquitoes on a P. falciparum gametocyte culture mixed with E. cloacae bacteria [8]. The bacteria caused a dramatic decrease in the number of parasites that developed to the oocyst stage, as compared to control mosquitoes (Figure 4A). In addition, there was a significant decrease in prevalence (percent mosquitoes that were infected) that accompanied this reduction in oocyst numbers (Figure 4B). To determine whether this decrease is due to interference of Plasmodium development prior to midgut invasion similar to an Enterobacter strain described in Cirimotich et al.[8], we measured the effect of bacteria on the formation of mature ookinetes. As shown in Table 1, the presence of bacteria up to a concentration of 106/ml had no effect on the development of P. falciparum gametocytes to mature ookinetes in the mosquito midgut. These results suggest that E. cloacae interferes with ookinete invasion and traversal of the midgut or alternatively, with the differentiation of ookinetes into oocysts. This is in contrast with the pre-invasion phenotype described for a natural Enterobacter isolate [8], thus highlighting the differences in the inhibitory mechanisms against Plasmodium development even between bacteria of the same genus. However, considering that these experiments were performed in different mosquito species, we cannot rule out that the observed differences are due to differences in how An. stephensi and An. gambiae respond to bacteria rather than differences between Enterobacter species.


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 inhibits P. falciparum development in An. stephensi.(A) Mosquitoes were fed on a P. falciparum gametocyte culture mixed either with medium alone (control) or with E. cloacae (+Ec; at a final concentration of 1×106/ml). After 8 days, oocyst numbers were counted by mercurochrome staining of dissected midgut samples and the data were pooled from four independent experiments. Median oocyst numbers are depicted by the red line and the P-value was determined using a Mann–Whitney U test. The total numbers (n) of mosquitoes analyzed are denoted below each sample. The percentage of mosquitoes containing at least one P. falciparum oocyst (or prevalence of infection) is shown in (B). Samples were analyzed by Chi-squared analysis to determine significance. P-values are denoted by asterisks (* = P<0.05; *** = P<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643921&req=5

pone-0062937-g004: E. cloacae inhibits P. falciparum development in An. stephensi.(A) Mosquitoes were fed on a P. falciparum gametocyte culture mixed either with medium alone (control) or with E. cloacae (+Ec; at a final concentration of 1×106/ml). After 8 days, oocyst numbers were counted by mercurochrome staining of dissected midgut samples and the data were pooled from four independent experiments. Median oocyst numbers are depicted by the red line and the P-value was determined using a Mann–Whitney U test. The total numbers (n) of mosquitoes analyzed are denoted below each sample. The percentage of mosquitoes containing at least one P. falciparum oocyst (or prevalence of infection) is shown in (B). Samples were analyzed by Chi-squared analysis to determine significance. P-values are denoted by asterisks (* = P<0.05; *** = P<0.001).
Mentions: Previous reports have demonstrated that the presence of bacteria within the mosquito midgut greatly influences Plasmodium development [8], [9], [12], and that field isolates of an Enterobacter species have a profound effect on P. falciparum development [8]. Our results (Figures 1 and 3) show that E. cloacae strongly induces AsSRPN6 expression (and other components of the mosquito immune response) in the mosquito midgut. To determine the effects of our Enterobacter strain on parasite development, we fed An. stephensi mosquitoes on a P. falciparum gametocyte culture mixed with E. cloacae bacteria [8]. The bacteria caused a dramatic decrease in the number of parasites that developed to the oocyst stage, as compared to control mosquitoes (Figure 4A). In addition, there was a significant decrease in prevalence (percent mosquitoes that were infected) that accompanied this reduction in oocyst numbers (Figure 4B). To determine whether this decrease is due to interference of Plasmodium development prior to midgut invasion similar to an Enterobacter strain described in Cirimotich et al.[8], we measured the effect of bacteria on the formation of mature ookinetes. As shown in Table 1, the presence of bacteria up to a concentration of 106/ml had no effect on the development of P. falciparum gametocytes to mature ookinetes in the mosquito midgut. These results suggest that E. cloacae interferes with ookinete invasion and traversal of the midgut or alternatively, with the differentiation of ookinetes into oocysts. This is in contrast with the pre-invasion phenotype described for a natural Enterobacter isolate [8], thus highlighting the differences in the inhibitory mechanisms against Plasmodium development even between bacteria of the same genus. However, considering that these experiments were performed in different mosquito species, we cannot rule out that the observed differences are due to differences in how An. stephensi and An. gambiae respond to bacteria rather than differences between Enterobacter species.

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