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Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria - PubMed

  • ️Fri Jan 01 2016

Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

Mithilesh Kajla et al. Front Microbiol. 2016.

Abstract

Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito.

Keywords: Anopheles stephensi; HPX15; Plasmodium; innate immunity; midgut bacteria; mucin barrier; peroxidases; vectorial capacity.

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Figures

FIGURE 1
FIGURE 1

The kinetics of 16S rRNA levels in blood fed mosquito midguts. Mosquitoes were fed either on blood alone (controls) or supplemented with a mixture of M. luteus and E. coli bacteria (109 cells/ml blood). The relative levels of 16S rRNA were analyzed in the pool of mosquito midguts collected at different time intervals after the feeding and represented here in log10 scale. SF represents the 16S rRNA levels of sugar fed midguts.

FIGURE 2
FIGURE 2

Expression kinetics of heme peroxidases and pattern recognition receptor (PRR) in bacteria fed midguts. The kinetics of relative mRNA levels of AsHPX15, HPX8, or GNBP gene were analyzed in the blood alone (controls) or supplemented with a mixture of M. luteus and E. coli bacteria fed midguts. SF represents the mRNA levels of these genes in sugar fed midguts. Significant differences in the gene expressions between control and bacteria fed midguts are denoted by asterisks.

FIGURE 3
FIGURE 3

Expression of Toll and gambicin immune genes in bacteria fed Anopheles stephensi midguts. mRNA levels of midgut Toll (Toll precursor) or gambicin gene were analyzed at 6 and 24 h after feeding the blood alone (controls) or supplemented with a mixture of E. coli and M. luteus. Relative levels of mRNA are presented against the sugar fed midguts. Significant differences in the gene expression levels are denoted by an asterisk.

FIGURE 4
FIGURE 4

Expression of NOS and SOCS immune genes in bacteria fed A. stephensi midguts. Midgut mRNA levels of NOS or SOCS gene were analyzed at 6 and 24 h after feeding the blood alone (controls) or supplemented with a mixture of E. coli and M. luteus. Relative levels of mRNA are presented against the sugar fed midguts. Significant differences in the gene expression levels are shown by asterisks.

FIGURE 5
FIGURE 5

Relative levels of AsHPX15 mRNA and 16S rRNA in AsHPX15 silenced and bacteria fed midguts. Mosquitoes injected with dsLacZ (controls) or dsAsHPX15 (silenced) RNA were fed on bacteria supplemented blood and relative levels of AsHPX15 mRNA and 16S rRNA were analyzed in their midguts at different time intervals after feeding. Relative mRNA levels are presented against the sugar fed midguts. Significant differences are shown by asterisks.

FIGURE 6
FIGURE 6

Expression of classical immune pathway genes in AsHPX15 silenced and bacteria fed midguts. Mosquitoes injected with dsLacZ (controls) or dsAsHPX15 (silenced) RNA were fed on bacteria supplemented blood and relative mRNA levels of GNBP, Toll (Toll precursor) or gambicin gene were analyzed in their midguts after 6 and 24 h post feeding. Relative levels of mRNA are presented against the sugar fed midguts. Significant differences between dsLacZ and dsAsHPX15 samples are shown by an asterisk.

FIGURE 7
FIGURE 7

Relative levels of NOS and SOCS mRNA in AsHPX15 silenced and bacteria fed midguts. Mosquitoes injected with dsLacZ (controls) or dsAsHPX15 (silenced) RNA were fed on bacteria supplemented blood and relative levels of NOS or SOCS mRNA were analyzed in their midguts at different time intervals after feeding. Relative levels of mRNA are presented against the sugar fed midguts. Significant differences are denoted by an asterisk.

FIGURE 8
FIGURE 8

Expression of HPX8 gene in AsHPX15 silenced and bacteria fed midguts. Mosquitoes injected with dsLacZ (controls) or dsAsHPX15 (silenced) RNA were fed on bacteria supplemented blood and relative levels of HPX8 mRNA in their midguts were analyzed at different time points after feeding. Relative levels of mRNA are presented against the sugar fed midguts. Significant differences are shown by an asterisk.

FIGURE 9
FIGURE 9

Model of A. stephensi midgut immunity in the presence or absence of AsHPX15 gene. AsHPX15 catalyzes the formation of mucin layer that blocks the direct interaction of lumen bacteria or bacterial elicitors with midgut epithelium. The increased load of bacteria induces classical immune pathways such as, GNBP, Toll and an antibacterial heme peroxidase HPX8 to regulate bacterial numbers. AsHPX15 silencing reduces the formation of mucin barrier that allows direct interaction of lumen bacteria or their elicitors with midgut epithelium. In this condition, STAT pathway effector molecule NOS is induced to regulate the numbers of midgut bacteria.

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