Martin, A. T. | bioRxiv Science

bioRxiv2024

A flow cytometric approach to identifying the relative abundance and functional capacities of hemocyte subsets in the American cockroach, Periplaneta americana (L.)

There is growing interest in insects as subjects for comparative immunological studies; however, very little has been done to wholistically characterize insect immune cells using modern techniques such as flow cytometry, and virtually no work of this kind has been done in Periplaneta americana. Here, we use an array of general molecular probes including fluorescent lectins, lysosomal indicators, and functional assays to distinguish and characterize insect immune cells (hemocytes) based on cell markers and functions. We have utilized fluorescent tracers of lysosomal content, ROS production, and phagocytosis, as well as microscopic examination of morphology and melanization to distinguish hemocyte types based on these functions. Our findings support the use of lectins as an additional means of separating at least three populations of cockroach immune cells coupled with morphological measurements such as size and complexity. Our results indicate that many functions are enriched in the more granular hemocyte population as these are the cells that undergo phagocytosis and melanization.

immunology↗

bioRxiv2021

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor

Leishmaniasis is a neglected tropical disease with diverse infection outcomes ranging from self-healing lesions, to progressive non-healing lesion, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to well controlled self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread of disease. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling of the primary amino acid sequence and protein crystal structures to identify a putative CXCL10-binding site on GP63. We found the putative CXCL10 binding site to be located in a region of gp63 under significant positive selection and that it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. We determined that the predicted binding site and adjacent positively selected amino acids are required for CXCL10 suppression by mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis allele and demonstrating impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panemensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles at the CXCL10 binding site, possibly due to balancing selection. Our results demonstrate how parasite genetic diversity can contribute to variation in the host immune response to Leishmania spp. infection that may play critical roles in the outcome of infection.

microbiology↗