Naegleria fowleri encodes a diverse repertoire of small RNAs.

(A) Schematic showing Naegleria fowleri-secreted EV small RNA identification pipeline. (B) Top 3 most prevalent precursor and mature microRNAs identified in whole cell small RNA sequencing with miRDeep2. (C) Top 4 most prevalent tRNAs identified in secreted EV small RNA sequencing with tRNAscan-SE. (D) Top 6 highly prevalent small RNAs in secreted EV RNAs identified manually via read-stacking with IGV. SmallRNA-5 is a tRNA fragment of s68.tRNA2-Glu. The 2 most prevalent small RNAs were identified first with Shortstack and confirmed manually with IGV. RNA secondary structures were generated with RNAfold v2.5.1(33). Schematic in panel A was generated with (BioRender).

© 2025, Russell et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Detection of smallRNA-1 and smallRNA-2 in the EVs of various pathogenic and non-pathogenic free-living amoebae.

(A) Standard curve for smallRNA-1 assay (consisting of 675 standards across 43 qPCR plates) was used to calculate copy number for qPCR reactions. (B) smallRNA-1 detection in RNA of EVs extracted via ultracentrifugation across 7 different clinical isolates of N. fowleri. Cq values ranged from 10.5-16.3. (C) Standard curve for smallRNA-2 assay used to calculate copy number. (D) smallRNA-2 detection in RNA of EVs extracted via ultracentrifugation across 4 different isolates of N. fowleri. Cq values ranged from 13.2-16.3. (E) smallRNA-1 detection in RNA of amoebae EVs extracted via extraction reagent from various species of free-living amoebae. For FBS and non-Naegleria species Cq values ranged from 33 to no signal. For Naegleria species Cq values ranged from 14.1-27.4. Each data point and/or bar in panels B-D consists of 3 technical replicates in RT-qPCR assay. Statistical significance was determined for panel E using One-way ANOVA test in GraphPad Prism v10.0.0 (GraphPad, La Jolla, CA, USA).

Detection of smallRNA-1 via RT-qPCR in N. fowleri media (A), intact trophozoites (B), and media with amoebae alone or feeding on Vero cells (D).

(A) N. fowleri Villa Jose strain (VJ) amoebae were washed, diluted to final concentrations (1 – 10,000 amoebae), and cultured for 24h. Then cell suspensions were centrifuged and media extracted for quantitation of smallRNA-1. (Cq range: 24.8 to no signal.) (B) Trophozoites of N. fowleri were washed, centrifuged and RNA was extracted from amoebae pellets. smallRNA-1 was detected in amoebae diluted from 10,000 to 1 amoeba (Cq range for 1 amoeba: 29-35.5). (C) Schematic showing experimental set-up for assay to assess detection of smallRNA-1 in media from amoebae cultured (alone), from Vero cells (alone), and in cultures of amoebae feeding on Vero cells. Mixture of media from amoebae and Vero cultured alone were used as a control to assess if Vero cell-conditioned media affected the smallRNA-1 quantification. For each condition cells were incubated for 24 h before RNA extraction. (D) smallRNA-1 levels detected in media extracted from wells cultured for 24h (as shown in C) containing: 1-VJ feeding on Vero cell monolayer (Cq range: 19-22), 2-axenically cultured VJ (Cq range: 24.9-27.4), 3-axenic VJ media mixed with Vero media (Cq range: 23.1-25.9), 4-Vero media (Cq range: 41.8 to no signal). Each data point in panels A, B and D represents a single well in a 96-well plate, or a single amoeba pellet with 3 technical replicates in RT-qPCR assay. Statistical significance was determined for panel D using Unpaired T tests in GraphPad Prism v10.0.0 (GraphPad, La Jolla, CA, USA). Schematic in panel C was generated with (BioRender).

© 2025, Russell et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Detection of smallRNA-1 and -2 in the plasma of PAM-infected mice at the end-stage of infection.

(A) Schematic showing infection process, plasma extraction via cardiac puncture followed by RNA extraction and RT-qPCR. (B) Assaying for smallRNA-1 in plasma of mice infected by 6 different N. fowleri clinical isolates provided 100% positivity in our assay compared to the uninfected mouse and human plasma. (C) Assaying for smallRNA-2 provided similar results to smallRNA-1 albeit at slightly lower concentrations per µL of plasma. All N. fowleri-infected mice were confirmed positive for amoebae by culturing brains and observing amoebae. Each data point in panels B-D is representative of 3 technical replicates in RT-qPCR assay. Schematic in panel A was generated with (BioRender).

© 2025, Russell et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Detection of smallRNA-1 in the plasma, serum, and urine of PAM-infected mice at various timepoints post-infection.

(A) Schematic showing infection process followed by urine collection at various timepoints and blood collection at the end-stage of infection followed by RNA extraction and RT-qPCR. (B) A cohort of 64 mice was infected with 1,000 VJ amoebae (fed over Vero cells 7 times) and cohorts of 8 mice each were sacrificed at various timepoints to extract blood. Urine was extracted from mice throughout infection. Mean time to death was 116.6h post infection. (C) A cohort of 10 mice was infected with 5,000 Nf69 amoebae (fed over Vero cells 8 times) and urine was either collected before infection or at various timepoints post-infection with plasma being extracted with euthanasia. Mean time to death was 140h post infection. The infection status of each cohort for panels B-C was determined by culture positivity of mouse brains and is shown under graphs. Each data point in panels B-C is representative of 3 technical replicates in RT-qPCR assay. “Positive” signals (>35 copies/µL) were defined as Cq values of 19.2 to 35.1 for panel B and 17.1 to 35.1 for panel C. Schematic in panel A was generated with (BioRender).

© 2025, Russell et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Determination of sensitivity of smallRNA-1 assay in various human biofluids compared to water and detection in PAM-infected human cerebrospinal fluid and whole blood.

(A) SmallRNA-1 spike-ins into various biofluids indicate that CSF, plasma, and urine provide the most consistent results compared to H2O spike-ins, while serum seems to inhibit the assay. CSF, plasma, and serum were pooled from multiple individuals, whereas urine was tested from 8 individual samples. All human biofluid samples were de-identified and provided by AdventHealth Hospital, Orlando, Florida. (B) Detection of smallRNA-1 in PAM-infected (n=6), Acanthamoeba-infected (n=2), B. mandrillaris-infected (n=2), and uninfected human CSF (n=2) (Cq range for PAM-infected CSF: 24-30.8). (C) Detection of smallRNA-1 in N. fowleri-infected, uninfected, B. mandrillaris-infected, and Acanthamoeba spp.-infected human whole blood, convalescent plasma, and serum (Cq range for PAM-infected whole blood: 32.4-32.6). We adopted a “positivity” cut-off of <33 cycles for urine, and <35 cycles for other biofluids; equivalent to > ∼100 copies per µL of biofluid). Each data point is representative of 3 technical replicates in RT-qPCR assay.