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

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.

Heme-Peroxidase 2, a Peroxinectin-Like Gene, Regulates Bacterial Homeostasis in Anopheles stephensi Midgut

“…Phyre 2 software analyses revealed the presence of 47% alpha helices in the secondary structure of AsHPX2 protein (Supplementary Figure S2) indicating that it is a globular protein as suggested by others (Pace and Scholtz, 1998;Kajla et al, 2016b). In addition, this protein also contains 10 heme-binding sites, 13 substrate-binding sites, three calcium-binding sites, and three homodimer interface sites (Supplementary Figure S2) in a way similar to other insect heme-peroxidases (Soudi et al, 2012;Kajla et al, 2016b;Bailey et al, 2017;Choudhury et al, 2019;Kakani et al, 2019). Three-dimensional structure of AsHPX2 protein (TM-score 0.70 ± 0.12) revealed the presence of catalytic quartet (Thr 212 , Ala 430 , Gly 602 , and Arg 669 ), which is positioned toward the protein surface.…”

“…Furthermore, the 5 upstream region of AsHPX2 gene was analyzed by JASPAR and MatInspector software to identify putative transcription factor binding motifs (TFBM) in its promoter region. Our analyses indicated the binding sites for some major transcription factors such as, GATA (pnr), Rel1, AP-1 (a Jun/Fos dimer) and Broad complex (Br-C) (Supplementary Figure S1) that indicated a tissue-specific immune role of AsHPX2 in a way similar to other insect genes (Senger et al, 2006;Zhu et al, 2007;Garver et al, 2013;Kajla et al, 2016b;Zakovic and Levashina, 2017;Kakani et al, 2019).…”

“…The 5 upstream region of AsHPX2 gene was analyzed by Augustus and JASPAR/MatInspector software to mark the putative promoter and regulatory sequences, respectively as before (Cartharius et al, 2005;Stanke et al, 2008;Mathelier et al, 2014;Kajla et al, 2016b;Kakani et al, 2019). The search criteria for these analyses were restricted to the transcription factors from insect family with minimum 80% similarity.…”

“…Our analyses of Anopheles stephensi genome revealed the presence of 18 heme-peroxidases that participate in important biological functions (Kajla et al, 2016b;Choudhury et al, 2019;Kakani et al, 2019). In this study, we characterized An.…”

Apolipophorin-III Acts as a Positive Regulator of Plasmodium Development in Anopheles stephensi

“…First-strand cDNA was synthesized from total RNA using QuantiTect reverse transcription kit (Qiagen). Expression profile of different genes was carried through semi-quantitative real time PCR using SYBR green supermix in an IQ5 multicolor real-time PCR detection system (Bio-Rad) where ribosomal protein subunit S7 mRNA was used as internal loading control as described before (Salazar et al, 1993; Kajla et al, 2016b). Primer pairs used for different genes were following, S7-Fwd: 5′-GGCGATCATCATCTACGT-3′ and S7-Rev: 5′-GTAGCTGCTGCAAACTTCGG-3′, AsApoLp-III Fwd: 5′-AGCCCAATTTCTTCCAGACC-3′, and AsApoLpIII Rev: 5′-CGGTTGCTTCAGCTCGTT-3′.…”