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P1/s1, a 3â€™-nucleotidase/nuclease, allows Leishmania to circumvent innate immune response mechanisms

ABSTRACT: 3â€™-nucleotidases/nucleases, distinct class I nucleases of protozoan parasites, play a pivotal role in extracellular purine salvage. As Leishmania are purine auxotroph and lack de novo synthesis, ectoenzymes facilitating nucleotide and nucleic acid cleavage are indispensable for subsequent uptake. Employing quantitative proteomics, we identified a class I nuclease p1/s1 cluster in L. major that comprises enzymes exhibiting dual 3â€™-nucleotidase and endonuclease activity. Expression of these enzymes is induced upon miltefosine or staurosporine treatment and was specifically detected in stationary phase, but not in logarithmic phase promastigotes. After confirming secretion of p1/s1, ecto-enzymatic activity was detected on parasites and in the culture supernatant. Viable null mutants deficient for the p1/s1 cluster were only obtained when a diCre-based inducible knockout system was applied, whereas direct deletion approaches were lethal. The viable knockout strains exhibited significantly reduced 3â€™-nucleotidase/nuclease activity. Notably, these parasites adapted by compensatory enrichment of various alternative purine salvage proteins on the proteomic level. Furthermore, both enzymatic functions implied mechanisms of host-pathogen interactions to facilitate infection establishment: Utilizing 3â€™-nucleotidase activity, Leishmania generate extracellular adenosine to suppress inflammatory cytokine secretion from macrophages and reduce lymphocyte proliferation in a human primary cell model. The presence of ecto-nucleases also allowed these parasites to degrade and survive neutrophil extracellular traps, a potent first-line innate immune mechanism in pathogen defense. In summary, our integrative approach combining proteomics, immunological and genome editing methods expands current knowledge about Leishmania major 3â€™-nucleotidases/nucleases. By offering new insights into the diverse involvements in host-pathogen interactions, we highlight p1/s1 as pivotal infection factor and potential drug target.

ORGANISM(S): Leishmania Major

SUBMITTER: Ger van Zandbergen

PROVIDER: PXD066928 | JPOST Repository | Tue May 05 00:00:00 GMT+01:00 2026

REPOSITORIES: jPOST

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			Action	DRS
	F241222_005_S4-A1_1_16300.d.zip	Other
	F241222_008_S4-A4_1_16303.d.zip	Other
	F241222_011_S4-A7_1_16306.d.zip	Other
	F241222_014_S4-A10_1_16309.d.zip	Other
	F241222_017_S4-D1_1_16312.d.zip	Other

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Project description:To accelerate previous RNA structure probing approaches, which focus on analyzing one RNA sequence at a time, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing to identify single-stranded RNA (ssRNA) regions from fragments generated by nuclease P1, which is specific for single-stranded nucleic acids. In the accompanying study, we show that we can accurately and simultaneously map ssRNA regions in multiple non-coding RNAs with known structure in experiments probing the entire mouse nuclear transcriptome. We carried out probing in two cell types to assess reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed. We examined mouse nuclear RNA from two cell types: undifferentiated embryonic stem cells (UNDIFF) and cells differentiated into neural precursors (D5NP). For each cell type, nuclear RNA was purified and deproteinized, denatured, and refolded in vitro, from which we prepared three barcoded samples: "nuclease" (RNA partially digested with P1 ssRNA-specific nuclease, yielding 5'-PO4/3'-OH end chemistry at each cleavage site), "control" (control for "nuclease" sample to idenfity endogenous 5'-PO4/3'-OH), and "PNK" (same as "control" but followed by a polynucleotide kinase treatment to convert 5'-OH/3'-cyclic-phosphate ends to clonable 5'-PO4/3'-OH ends). Resulting RNA fragments were cloned using the SOLiD Small RNA Expression Kit (SREK) protocol, which ligates linkers only to 5'-PO4/3'-OH containing RNA, enriching for clones of products resulting from P1 cleavage in "nuclease" sample and selecting against random degradation. Two cell types, three treatments each, thus resulted in six barcoded samples total (barcodes 01, 02, 04, 05, 07, 08). Four other barcoded samples were prepared for separate experiments not used in our study (barcodes 03, 06, 09, 10), so their preparation is not described here. The total run of ten barcodes was done on the ABI SOLiD3 platform and a custom algorithm (FragSeq v0.0.1) was used to compute "cutting scores" (as described in our paper) that show ssRNA regions in hundreds of ncRNAs.

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P1/s1, a 3â€™-nucleotidase/nuclease, allows Leishmania to circumvent innate immune response mechanisms

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