DOI: 10.5061/dryad.tht76hf8w
title: Bulk RNA seq of macrophages isolated from Kmo morphant or control morphant zebrafish larvae infected with <em>Salmonella Typhimurium</em>

description: The kynurenine pathway of tryptophan degradation has been implicated in
 various diseases, including cancer, neurodegenerative disorders, and
 infectious diseases. A key branchpoint in this pathway is production of
 the metabolite 3-hydroxy-kynurenine (3-HK) by the enzyme kynurenine
 3-monooxygenase (Kmo). We recently found that administration of exogenous
 3-HK promotes survival to Salmonella Typhimurium infection in zebrafish
 larvae by restricting bacterial expansion in macrophages via a systemic
 mechanism that targets kainate sensitive glutamate receptor (KAR) ion
 channels. Here, we show that endogenous production of 3-HK by Kmo,
 likewise, is required for defense against systemic Salmonella Typhimurium
 infection in vivo and that loss of endogenous production of 3-HK impairs
 macrophage microbicial activity, resulting in increased bacterial
 expansion. Mechanistically, 3-HK acts by antagonizing KARs to promote
 lysosome acidification and subsequent control of bacterial burden.
 Finally, we establish a novel link between activity at KARs and lysosomal
 acidification in macrophages, revealing a novel regulatory role for KARs
 in promoting macrophage microbicidal activity and a novel mechanism though
 which 3-HK promotes control of bacterial infection.
description: Bulk RNA sequencing transcriptomes of macrophages isolated from 3
 days post-fertilization zebrafish larvae. Fluorescently labeled cells were
 isolated in bulk from a single cell suspension obtained by enzymatic
 digestion of groups consisting of 5 homozygous
 Tg(<em>mpeg1:mCherry</em>) transgenic animals (7-13 biological
 replicates/condition) injected with either the translation-blocking
 <em>kmo</em> morpholino (Kmo MO) or standard control
 morpholino (Ctrl MO). cDNA libraries were constructed from a range of
 50-800 collected cells with an in-house adaptation of the Smart-seq2
 protocol in combination with the Nextera XT Library preparation kit
 (Illumina, Inc., San Diego, CA). Library size and concentration were
 evaluated using the TapeStation 2200 system (Agilent) and a Qubit
 fluorometer (Invitrogen) before sequencing. Samples were multiplexed and
 pair end sequences were generated with NovaSeq SP sequencing system
 (Illumina) generating 5.5e10^7 reads per sample. Samples were
 demultiplexed and FASTQ files representing each sample were generated. Any
 remaining adapter sequences were removed using skewer (0.2.2) and FASTQ
 files were assessed for quality control using FASTQC (0.11.5). Reads were
 aligned to the Ensembl zebrafish reference genome GRCz11 using Hisat2
 (2.1.0) and counts were quantified using HTSeq-Count (0.12.4).
 Differential gene expression analysis was performed using DESeq2 (1.38.3).
 Gene ontology analyses were performed using gene set enrichment analysis
 (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
 analyses with an FDR below 0.05 using gseKEGG from ClusterProfiler
 (v4.6.2).
description: # Bulk RNA seq of macrophages isolated from Kmo morphant or control
 morphant zebrafish larvae infected with *Salmonella Typhimurium* Dataset
 DOI: [10.5061/dryad.tht76hf8w](10.5061/dryad.tht76hf8w) ## Description of
 the data and file structure For the article: Kmo restricts *Salmonella* in
 macrophages by promoting lysosomal acidification through kainate receptor
 antagonism in a whole organism infection model. Bulk RNA sequencing of
 macrophages isolated from either kmo morphant or control morphant
 zebrafish larvae at 3 days post-fertilization infected intravenously with
 *Salmonella* Typhimurium or uninfected. The data shows that infected kmo
 morphant macrophages have suppression of lysosomal genes compared to
 infected control morphant macrophages using GSEA KEGG. For each biological
 replicate, five zebrafish larvae were dissociated together and macrophages
 sorted using FACS, with macrophages labelled using the transgenic line
 Tg(*mpeg1.1:mCherry*) and *Salmonella* with GFP. cDNA libraries were
 prepared with Smart-Seq2 protocol in combination with Nextera XT Library
 preparation kit (Illumina Inc). Samples were multiplexed and pair end
 sequences were generated with NovaSeq SP sequencing system (Illumina).
 Gene set enrichment analysis was performed with ClusterProfiler (v.4.0)
 with a FDR &lt;0.05 using the Kyoto Encyclopedia of Genes and Genomes
 terms. ### Files and variables #### File: ctrl_uninf_BR_1_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive control morphant
 larvae. Biological replicate 1, read 1. #### File:
 ctrl_uninf_BR_7_R1.fastq.gz **Description:** Macrophages from uninfected,
 naive control morphant larvae. Biological replicate 7, read 1. #### File:
 ctrl_uninf_BR_7_R2.fastq.gz **Description:** Macrophages from uninfected,
 naive control morphant larvae. Biological replicate 7, read 2. #### File:
 ctrl_inf_BR_1_R1.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 1, read 1. ####
 File: ctrl_uninf_BR_2_R2.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 2, read 2.
 #### File: ctrl_inf_BR_2_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 2, read 1.
 #### File: ctrl_uninf_BR_1_R2.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 1, read 2.
 #### File: ctrl_inf_BR_1_R2.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 1, read 2.
 #### File: ctrl_uninf_BR_2_R1.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 2, read 1.
 #### File: ctrl_uninf_BR_3_R1.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 3, read 1.
 #### File: ctrl_uninf_BR_3_R2.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 3, read 2.
 #### File: ctrl_inf_BR_3_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 3, read 1.
 #### File: ctrl_inf_BR_2_R2.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 2, read 2.
 #### File: ctrl_uninf_BR_4_R1.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 4, read 1.
 #### File: kmo_inf_BR_1_R1.fastq.gz **Description:** Infected macrophages
 from infected Kmo morphant larvae. Biological replicate 1, read 1. ####
 File: ctrl_inf_BR_3_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 3, read 2. ####
 File: ctrl_uninf_BR_5_R1.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 5, read 1
 #### File: ctrl_uninf_BR_5_R2.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 5, read 2.
 #### File: ctrl_inf_BR_5_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 5, read 1.
 #### File: ctrl_inf_BR_4_R2.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 4, read 2.
 #### File: ctrl_uninf_BR_4_R2.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 4, read 2.
 #### File: ctrl_inf_BR_4_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 4, read 1.
 #### File: ctrl_uninf_BR_6_R1.fastq.gz **Description:** Macrophages from
 uninfected, naive control morphant larvae. Biological replicate 6, read 1.
 #### File: ctrl_inf_BR_6_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 6, read 1.
 #### File: ctrl_inf_BR_8_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 8, read 1.
 #### File: ctrl_inf_BR_6_R2.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 6, read 2.
 #### File: kmo_inf_BR_1_R2.fastq.gz **Description:** Infected macrophages
 from infected Kmo morphant larvae. Biological replicate 1, read 2. ####
 File: ctrl_inf_BR_9_R1.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 9, read 1. ####
 File: ctrl_inf_BR_5_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 5, read 2. ####
 File: ctrl_inf_BR_7_R1.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 7, read 1. ####
 File: kmo_inf_BR_2_R2.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 2, read 2. #### File:
 ctrl_inf_BR_9_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 9, read 2. ####
 File: ctrl_inf_BR_7_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 7, read 2. ####
 File: ctrl_inf_BR_8_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 8, read 2. ####
 File: kmo_inf_BR_2_R1.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 2, read 1. #### File:
 ctrl_uninf_BR_6_R2.fastq.gz **Description:** Macrophages from uninfected,
 naive control morphant larvae. Biological replicate 6, read 2. #### File:
 kmo_inf_BR_8_R2.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 8, read 2. #### File:
 kmo_inf_BR_9_R2.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 9, read 2. #### File:
 kmo_inf_BR_5_R2.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 5, read 2. #### File:
 ctrl_inf_BR_13_R2.fastq.gz **Description:** Infected macrophages from
 infected control morphant larvae. Biological replicate 13, read 2. ####
 File: ctrl_inf_BR_10_R1.fastq.gz **Description:** Infected macrophages
 from infected control morphant larvae. Biological replicate 10, read 1.
 #### File: ctrl_inf_BR_11_R1.fastq.gz **Description:** Infected
 macrophages from infected control morphant larvae. Biological replicate
 11, read 1. #### File: kmo_inf_BR_10_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 10,
 read 1. #### File: kmo_inf_BR_6_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 6,
 read 2. #### File: kmo_inf_BR_3_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 3,
 read 1. #### File: kmo_inf_BR_12_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 12,
 read 1. #### File: ctrl_inf_BR_10_R2.fastq.gz **Description:** Infected
 macrophages from infected control morphant larvae. Biological replicate
 10, read 2. #### File: kmo_inf_BR_4_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 4,
 read 2. #### File: kmo_inf_BR_11_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 11,
 read 2. #### File: kmo_inf_BR_12_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 12,
 read 2. #### File: kmo_inf_BR_8_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 8,
 read 1. #### File: kmo_inf_BR_7_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 7,
 read 1. #### File: kmo_inf_BR_9_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 9,
 read 1. #### File: kmo_inf_BR_10_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 10,
 read 2. #### File: kmo_inf_BR_13_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 13,
 read 1. #### File: kmo_inf_BR_13_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 13,
 read 2. #### File: ctrl_inf_BR_13_R1.fastq.gz **Description:** Infected
 macrophages from infected control morphant larvae. Biological replicate
 13, read 1. #### File: kmo_inf_BR_5_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 5,
 read 1. #### File: ctrl_inf_BR_11_R2.fastq.gz **Description:** Infected
 macrophages from infected control morphant larvae. Biological replicate
 11, read 2. #### File: kmo_inf_BR_6_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 6,
 read 1. #### File: kmo_inf_BR_11_R1.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 11,
 read 1. #### File: kmo_inf_BR_3_R2.fastq.gz **Description:** Infected
 macrophages from infected Kmo morphant larvae. Biological replicate 3,
 read 2. #### File: ctrl_inf_BR_12_R1.fastq.gz **Description:** Infected
 macrophages from infected control morphant larvae. Biological replicate
 12, read 1. #### File: kmo_uninf_BR_1_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 1, read 1. #### File: kmo_uninf_BR_4_R2.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 4, read 2. #### File: kmo_uninf_BR_3_R2.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 3, read 2.  #### File: kmo_uninf_BR_2_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 2, read 1. #### File: kmo_uninf_BR_3_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 3, read 1. #### File: kmo_uninf_BR_1_R2.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 1, read 2. #### File: kmo_uninf_BR_4_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 4, read 1. #### File: kmo_uninf_BR_7_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 7, read 1. #### File: kmo_uninf_BR_2_R2.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 2, read 2. #### File: kmo_uninf_BR_5_R1.fastq.gz
 **Description:** Macrophages from uninfected, naive Kmo morphant larvae.
 Biological replicate 5, read 1. #### File: kmo_inf_BR_7_R2.fastq.gz
 **Description:** Infected macrophages from infected Kmo morphant larvae.
 Biological replicate 7, read 2. #### File: ctrl_inf_BR_12_R2.fastq.gz
 **Description:** Infected macrophages from infected control morphant
 larvae. Biological replicate 12, read 2. #### File:
 kmo_inf_BR_4_R1.fastq.gz **Description:** Infected macrophages from
 infected Kmo morphant larvae. Biological replicate 4, read 1. #### File:
 kmo_uninf_BR_5_R2.fastq.gz **Description:** Macrophages from uninfected,
 naive Kmo morphant larvae. Biological replicate 5, read 2. #### File:
 kmo_uninf_BR_6_R1.fastq.gz **Description:** Macrophages from uninfected,
 naive Kmo morphant larvae. Biological replicate 6, read 1. #### File:
 kmo_uninf_BR_7_R2.fastq.gz **Description:** Macrophages from uninfected,
 naive Kmo morphant larvae. Biological replicate 7, read 2. #### File:
 kmo_uninf_BR_6_R2.fastq.gz **Description:** Macrophages from uninfected,
 naive Kmo morphant larvae. Biological replicate 6, read 2. #### File:
 exposed_ctrl_kmo.zip **Description:** Macrophages from infected control or
 Kmo morphant larvae that are exposed but not actively infected. FASTQ
 files from biological replicates 1-7 with reads 1 and 2 from control
 morphant and Kmo morphant replicates. #### File: Metadata.csv
 **Description:** Table with metadata used for sample analysis. #####
 Variables * sample_name: Complete sample name, including condition,
 infection status, and biological replicate. * infection: Infection status
 of the macrophage. Uninf: uninfected, naive. Inf: actively infected. Exp:
 exposed, not actively infected from infected larvae. * condition:
 Morpholino treatment. Ctrl: control morphant larvae. Kmo: Kmo morphant
 larvae. * combined_condition: Infection status combined with morpholino
 treatment. Ctrl uninf: uninfected macrophage from control morphant larvae.
 Kmo uninf: uninfected macrophage from Kmo morphant larvae. Kmo inf:
 actively infected macrophage from Kmo morphant larvae. Kmo exp: exposed
 macrophage from Kmo morphant larvae. Ctrl inf: actively infected
 macrophage from control morphant larvae. Ctrl exp: exposed macrophage from
 control morphant larvae. * date: Date that macrophages were sorted using
 FACS. 11.19: sorted on 11.19.21. 11.12: sorted on 11.12.21  * br: Complete
 sample name. Same as Sample Name. * replicate: Biological replicate
 number.  #### File: Counts.csv **Description:** Raw count data table.
 ENSEMBL gene names in rows and sample names in columns. ##### Variables *
 gene_name: ENSEMBL gene labels. * ctrl_uninf_BR1: Control morphant,
 uninfected, biological replicate 1. * ctrl_uninf_BR2: Control morphant,
 uninfected, biological replicate 2. * ctrl_uninf_BR3: Control morphant,
 uninfected, biological replicate 3. * ctrl_uninf_BR4: Control morphant,
 uninfected, biological replicate 4. * ctrl_uninf_BR5: Control morphant,
 uninfected, biological replicate 5. * ctrl_uninf_BR6: Control morphant,
 uninfected, biological replicate 6. * ctrl_uninf_BR7: Control morphant,
 uninfected, biological replicate 7. * kmo_uninf_BR1: Kmo morphant,
 uninfected, biological replicate 1. * kmo_uninf_BR2: Kmo morphant,
 uninfected, biological replicate 2. * kmo_uninf_BR3: Kmo morphant,
 uninfected, biological replicate 3. * kmo_uninf_BR4: Kmo morphant,
 uninfected, biological replicate 4. * kmo_uninf_BR5: Kmo morphant,
 uninfected, biological replicate 5. * kmo_uninf_BR6: Kmo morphant,
 uninfected, biological replicate 6. * kmo_uninf_BR7: Kmo morphant,
 uninfected, biological replicate 7. * kmo_inf_BR10: Kmo morphant,
 infected, biological replicate 10. * kmo_inf_BR11: Kmo morphant, infected,
 biological replicate 11. * kmo_inf_BR12: Kmo morphant, infected,
 biological replicate 12. * kmo_inf_BR13: Kmo morphant, infected,
 biological replicate 13. * kmo_inf_BR1: Kmo morphant, infected, biological
 replicate 1. * kmo_inf_BR2: Kmo morphant, infected, biological replicate
 2. * kmo_inf_BR3: Kmo morphant, infected, biological replicate 3. *
 kmo_inf_BR4: Kmo morphant, infected, biological replicate 4. *
 kmo_inf_BR5: Kmo morphant, infected, biological replicate 5. *
 kmo_inf_BR6: Kmo morphant, infected, biological replicate 6. *
 kmo_inf_BR7: Kmo morphant, infected, biological replicate 7. *
 kmo_inf_BR8: Kmo morphant, infected, biological replicate 8. *
 kmo_inf_BR9: Kmo morphant, infected, biological replicate 9. *
 kmo_exp_BR1: Kmo morphant, exposed, biological replicate 1. *
 kmo_exp_BR2: Kmo morphant, exposed, biological replicate 2. *
 kmo_exp_BR3: Kmo morphant, exposed, biological replicate 3. *
 kmo_exp_BR4: Kmo morphant, exposed, biological replicate 4. *
 kmo_exp_BR5: Kmo morphant, exposed, biological replicate 5. *
 kmo_exp_BR6: Kmo morphant, exposed, biological replicate 6. *
 kmo_exp_BR7: Kmo morphant, exposed, biological replicate 7. *
 ctrl_inf_BR10: Control morphant, infected, biological replicate 10. *
 ctrl_inf_BR11: Control morphant, infected, biological replicate 11. *
 ctrl_inf_BR12: Control morphant, infected, biological replicate 12. *
 ctrl_inf_BR13: Control morphant, infected, biological replicate 13. *
 ctrl_inf_BR1: Control morphant, infected, biological replicate 1. *
 ctrl_inf_BR2: Control morphant, infected, biological replicate 2. *
 ctrl_inf_BR3: Control morphant, infected, biological replicate 3. *
 ctrl_inf_BR4: Control morphant, infected, biological replicate 4. *
 ctrl_inf_BR5: Control morphant, infected, biological replicate 5. *
 ctrl_inf_BR6: Control morphant, infected, biological replicate 6. *
 ctrl_inf_BR7: Control morphant, infected, biological replicate 7. *
 ctrl_inf_BR8: Control morphant, infected, biological replicate 8. *
 ctrl_inf_BR9: Control morphant, infected, biological replicate 9. *
 ctrl_exp_BR1: Control morphant, exposed, biological replicate 1. *
 ctrl_exp_BR2: Control morphant, exposed, biological replicate 2. *
 ctrl_exp_BR3: Control morphant, exposed, biological replicate 3. *
 ctrl_exp_BR4: Control morphant, exposed, biological replicate 4. *
 ctrl_exp_BR5: Control morphant, exposed, biological replicate 5. *
 ctrl_exp_BR6: Control morphant, exposed, biological replicate 6. *
 ctrl_exp_BR7: Control morphant, exposed, biological replicate 7. ##
 Code/software No specific software required. ## Access information Other
 publicly accessible locations of the data: * None Data was derived from
 the following sources: * Data derived directly from Illumina NovaSeq SP
 sequencer and is not processed.