Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects.

Yanyan Sun,Linlin Yang,Dandan Li,Camilo Ayra-Pardo,Lunguang Yao,Denis J Wright,Yushan Ding,Chaoliang Leng,Yunchao Kan,Huili Qiao,Lianet Rodríguez-Cabrera,Siliang Huang,Courtney J Robinson

doi:10.1128/spectrum.00604-21

Abstract

ABSTRACTThe spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter’s role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to ‘virulence priming’. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830.IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects’ fate, the response of Bt to hemolymph and the latter’s role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

Highlights

IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood, where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia
In this study, we used tandem mass tag (TMT)-based quantitative proteomics to identify a set of Bt kurstaki strain HD73 (Btk) proteins whose synthesis rate changed in response to Bo. mori hemolymph and have called it the Btk hemolymph “stimulon.” The Gene ontology (GO) classifications showed that differentially accumulated proteins (DAPs) were enriched mainly in glutamate metabolism, transketolase activity, and transmembrane transport
Our experimental design was not intended to investigate Btk proteome alterations during bacterial cell multiplication within the insect blood but rather alterations occurring after exposure of bacterial cells to small quantities of hemolymph, which simulates the early stages of invasion when the first bacterial cells have crossed the midgut epithelial barrier

Summary

Introduction

IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia. Humoral responses include melanization, clotting, and secretion of antimicrobial peptides (AMPs) and complement-like proteins to immobilize and kill the intruders [27] The activation of both responses relies on the recognition of pathogen-associated molecular patterns (PAMPs) on the microbial surface by pattern recognition receptors [26, 28]. The expression of InhA1 is dependent on Spo0A;P, which in turn represses PlcR [34, 35] Another Bt metalloprotease not regulated by PlcR, the cell surface-associated camelysin (CalY), was recently shown to play critical roles in both bacterial adhesions to insect cells (adhesin activity) and in biofilm formation and is required for Bt’s full virulence to insects by either ingestion or intrahemocoelic injection routes [36,37,38]

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