Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response

Abstract

Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in L. longipalpis LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance.

Keywords: Lutzomyia longipalpis; antiviral response; embryonic LL5 cells; exoproteome; interferon-like response; poly (I:C).

Figure 1

Secretome networks showing predicted functional linkages between identified proteins. (A) Interaction map of the whole LL5 double-stranded RNA (dsRNA)-transfected cell exoproteome; (B) interactome map of the secreted proteins with positive variation of ≥2-fold change. The STRING interactome maps were generated using default settings (medium confidence of 0.4, with 7 criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). Proteins were represented as nodes and their functional links were defined by solid lines. The thickness of the lines signifies the level of confidence of the reported association. The non-connected nodes were suppressed. No additional interplay proteins were added to the networks. The protein symbols and node colors are listed in Table S2.

Figure 2

LL5 cells protein secretion behavior in response to dsRNA treatment. (A) Variation of relative protein abundance in the whole exoproteome of LL5-transfected cells at 24 and 48 h post-transfection; (B) secretion profile behavior of proteins with amount variation of ≥2-fold change at 24-h; (C) secretion profile behavior of proteins with amount variation of ≥2-fold change at 48-h. White squares represent the proteins’ relative amount variation at 24-h after transfection. Black squares represent the proteins’ relative amount variation at 48-h after transfection. Squares in the same point of the X-axis represent the same protein. Numbers below graphs (B,C) represent proteins listed in corresponding inset.

Figure 3

Transcriptional profile of genes involved in antiviral response. (A) Genes with correlated transcriptional and protein secretion profiles on mass spectrometry at 24 h or 48 h post-transfection; (B) genes with non-correlated transcriptional and protein secretion profiles on mass spectrometry on mass spectrometry at 24 h or 48 h post-transfection. Y-axis represents fold change in gene expression relative to the mock-transfected group (horizontal traced line) normalized to the rp49 reference gene. The X-axis represents gene name (Table 1) and samples collected at different time points post-transfection. Quantification was normalized relative to the house keeping gene rp49, and relative gene expression expressed as fold change was calculated relative to the mock group. Bars represent mean with standard error (SEM) of two biological replicates. Tables represent protein fold change corresponding to each gene name. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

Comparison of transcript and secreted protein abundance. Mass spectrometry expression values (log2 ratio) of LL5 cell-secreted proteins collected at 24 h (A) or 48 h (B) post transfection were plotted on X-axis; mRNA levels obtained from qPCR (log2 ratio) of LL5 cells collected at 6 h (black circles), 12 h (red squares), 24 h (green triangles), and 48 h (blue diamonds) post-transfection were plotted on the Y-axis. Lines represent linear regression.