NLP-31

Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.

Paracoccidioidomycosis is a systemic fungal infection affecting mainly Latin American countries that is caused by Paracoccidioides brasiliensis and Paracoccidioides lutzii. During the study of fungal pathogenesis, in vivo studies are crucial to understand the overall mechanisms involving the infection as well as to search for new therapeutic treatments and diagnosis. Caenorhabditis elegans is described as an infection model for different fungi species and a well-characterized organism to study the innate immune response. This study evaluates C. elegans as an infection model for Paracoccidioides spp. It was observed that both species do not cause infection in C. elegans, as occurs with Candida albicans, and one possible explanation is that the irregular size and shape of Paracoccidioides spp. difficult the ingestion of these fungi by the nematode. Besides this difficulty in the infection, we could observe that the simple exposition of C. elegans to Paracoccidioides species was able to trigger a distinct pattern of expression of antimicrobial peptide genes. The expression of cnc-4, nlpl-27 and nlp-31 was superior after the exposure to P. brasiliensis in comparison to P. lutzii (P < 0.05), and these findings demonstrate important differences regarding innate immune response activation caused by the two species of the Paracoccidioides genus.

Both plants and animals respond to infection by synthesizing compounds that directly inhibit or kill invading pathogens. We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. Expression of two of these peptides, NLP-29 and NLP-31, was differentially regulated by fungal and bacterial infection and was controlled in part by tir-1, which encodes an ortholog of SARM, a Toll-interleukin 1 receptor (TIR) domain protein. Inactivation of tir-1 by RNA interference caused increased susceptibility to infection. We identify protein partners for TIR-1 and show that the small GTPase Rab1 and the f subunit of ATP synthase participate specifically in the control of antimicrobial peptide gene expression. As the activity of tir-1 was independent of the single nematode Toll-like receptor, TIR-1 may represent a component of a previously uncharacterized, but conserved, innate immune signaling pathway.