Systematical Analysis of the Protein Targets of Lactoferricin B and Histatin-5 Using Yeast Proteome Microarrays

Abstract

Antimicrobial peptides (AMPs) have potential antifungal activities; however, their intracellular protein targets are poorly reported. Proteome microarray is an effective tool with high-throughput and rapid platform that systematically identifies the protein targets. In this study, we have used yeast proteome microarrays for systematical identification of the yeast protein targets of Lactoferricin B (Lfcin B) and Histatin-5. A total of 140 and 137 protein targets were identified from the triplicate yeast proteome microarray assays for Lfcin B and Histatin-5, respectively. The Gene Ontology (GO) enrichment analysis showed that Lfcin B targeted more enrichment categories than Histatin-5 did in all GO biological processes, molecular functions, and cellular components. This might be one of the reasons that Lfcin B has a lower minimum inhibitory concentration (MIC) than Histatin-5. Moreover, pairwise essential proteins that have lethal effects on yeast were analyzed through synthetic lethality. A total of 11 synthetic lethal pairs were identified within the protein targets of Lfcin B. However, only three synthetic lethal pairs were identified within the protein targets of Histatin-5. The higher number of synthetic lethal pairs identified within the protein targets of Lfcin B might also be the reason for Lfcin B to have lower MIC than Histatin-5. Furthermore, two synthetic lethal pairs were identified between the unique protein targets of Lfcin B and Histatin-5. Both the identified synthetic lethal pairs proteins are part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex that regulates gene expression via histone modification. Identification of synthetic lethal pairs between Lfcin B and Histatin-5 and their involvement in the same protein complex indicated synergistic combination between Lfcin B and Histatin-5. This hypothesis was experimentally confirmed by growth inhibition assay.

Keywords: Histatin-5; Lactoferricin B (Lfcin B); antifungal activity; antimicrobial peptides (AMPs); proteome microarray; synergy.

Figure 1

The schematic diagram of this study. Yeast proteome microarrays were fabricated from the ~5800 yeast proteins which were individually expressed and purified from yeast. To identify the protein targets, the fabricated yeast proteome microarrays were probed with biotinylated Lfcin B and Histatin-5, individually. Followed by the probing of DyLight 650 labeled streptavidin and DyLight 550 labeled anti-GST antibody on the yeast proteome microarrays to detect the signal of biotinylated AMPs bound to their specific binding partners and the amount of relative protein on proteome microarray, respectively. The identified hits of Lfcin B and Histatin-5 were systematically analyzed for GO and synthetic lethal pairs by using several online bioinformatics databases (DAVID tools and Synthetic Lethality).

Figure 2

The representative yeast proteome microarray images and the representative hits of Lfcin B and Histatin-5 on yeast proteome microarrays. Images show the microarray and the enlarge hit image of Lfcin B and Histatin-5. Red and green color denotes the signal from DyLight 650 labeled streptavidin and DyLight 550 labeled anti-GST antibody, respectively. Red spots represent the signal of Lfcin B and Histatin-5 on yeast proteome microarrays. (A) Triplicate microarray images probed with biotinylated Lfcin B. The position of the representative 5 hits image is shown on the microarray. (B) Representative enlarged hits images of Lfcin B. (C) Triplicate microarray images probed with biotinylated Histatin-5. (D) Representative enlarged hits images of Histatin-5.

Figure 2

The representative yeast proteome microarray images and the representative hits of Lfcin B and Histatin-5 on yeast proteome microarrays. Images show the microarray and the enlarge hit image of Lfcin B and Histatin-5. Red and green color denotes the signal from DyLight 650 labeled streptavidin and DyLight 550 labeled anti-GST antibody, respectively. Red spots represent the signal of Lfcin B and Histatin-5 on yeast proteome microarrays. (A) Triplicate microarray images probed with biotinylated Lfcin B. The position of the representative 5 hits image is shown on the microarray. (B) Representative enlarged hits images of Lfcin B. (C) Triplicate microarray images probed with biotinylated Histatin-5. (D) Representative enlarged hits images of Histatin-5.

Figure 3

Unique and common hits of Lfcin B and Histatin-5 identified from yeast proteome microarrays. The protein targets of Lfcin B and Histatin-5 identified from yeast proteome microarrays are 140 and 137, respectively. These identified hits of Lfcin B and Histatin-5 were categorized as unique and common hits. Unique protein targets that are present only in Lfcin B are 63 hits whereas the unique protein targets that are present only in Histatin-5 are 60 hits. 77 protein targets were common to both Lfcin B and Histatin-5.

Figure 4

Enrichment in biological process of Lfcin B and Histatin-5 hits obtained from yeast proteome microarrays. The significant enrichment categories in biological process of Lfcin B and Histatin-5 of yeast-binding proteins (dotted line indicates the p-value of 0.05).

Figure 5

Functional enrichment of Lfcin B and Histatin-5 hits obtained from yeast proteome microarrays. The significant enrichment categories of Lfcin B and Histatin-5 of yeast-binding proteins. (A) Enrichment in molecular function (B) Enrichment in cellular component (dotted line indicates the p-value of 0.05).

Figure 6

Functional enrichment of Lfcin B hits obtained from E. coli proteome microarray and yeast proteome microarrays. The comparison of enrichment categories of Lfcin B target proteins from yeast and E. coli proteomes. (A) Enrichment in biological process (B) Enrichment in molecular function and (C) Enrichment in cellular component (dotted line indicates the p-value of 0.05).

Figure 7

Synthetic lethality and enlarge protein images of SPT8, RAD6, and HFI1 on yeast proteome microarray. The enlarged protein images of two synthetic lethality pairs identified between the unique hits of Lfcin B and Histatin-5 as well as the synthetic lethality pair within the protein targets of Lfcin B.

Figure 8

Growth inhibition effect of individual and combination of Lfcin B and Histatin-5 on yeast. Yeast was grown without AMPs and in the presence of individual and combination of Lfcin B (15 µg/mL) and Histatin-5 (20 µg/mL). Significant inhibition in growth was observed with the combination of Lfcin B and Histstin-5. The combinational inhibition of Lfcin B and Histatin was significantly lower than the expected value of Lfcin B and Histatin-5 combination calculated from the individual inhibition of Lfcin B and Histatin-5, concluding the synergy combination between Lfcin B and Histatin-5.