Mip inhibitors, SF235 (Seufert et al., 2016) and AN_CH_37 (Scheuplein et al., 2023) were synthesized as described previously (Australian Patent Application No. PCT/AU2023/050201). Inhibitors were solubilized in DMSO.

Recombinant B. pseudomallei Mip (Bps Mip; strain K96243) was produced and purified as described previously (Norville et al., 2011a). The peptidyl-prolyl cis/trans isomerase assay was conducted as described previously (Norville et al., 2011b; Begley et al., 2014; Scheuplein et al., 2023) with modifications. Briefly, 20 nM recombinant Bps Mip protein was pre-incubated for 6 min at 10°C with SF235, AN_CH_37 or rapamycin at a final concentration of 400 nM or equivalent volume of DMSO in 1.2 ml 35 mM HEPES buffer (pH 7.8) with succinyl-Ala-Phe-Pro-Phe-p-nitroanilide (10 mg/ml stock; Bachem #4016001). Chymotrypsin (Merck #C4129) was added to the cuvette at a final concentration of 0.8 mg/ml and mixed. Hydrolysis of the substrate was measured at 390 nm using a Shimadzu 100 UV/visible spectrophotometer at 2 s intervals for 15 min. Three independent runs were performed for each protein and inhibitor/DMSO combination. The inhibition constant (K_i ) of SF235 and AN_CH_37 against Bps Mip were determined as previously described (Iwasaki et al., 2022; Debowski et al., 2023).

J774A.1 murine macrophages were seeded into a 24-well tissue culture treated plate (Thermofisher, #NUN142475) at a concentration of 4 × 10⁵ cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with a final concentration of 1% (v/v) GlutaMAX (Gibco, Life Technologies) and 10% (v/v) heat-inactivated fetal calf serum (Gibco, Life Technologies) and incubated for 20 h at 37°C with 5% CO₂ to achieve a confluent monolayer of approximately 1 × 10⁶ cells/well. B. pseudomallei strain K96243 (Dstl, UK) was grown overnight at 37°C for 18 h and adjusted in Leibovitz L-15 medium (Gibco, Life Technologies) supplemented with 1% (v/v) GlutaMAX and 10% (v/v) heat-inactivated fetal calf serum to an absorbance at 580 nm between 0.35 and 0.4 using a PLP Colorimeter. This culture was serially diluted in L-15 medium to achieve a multiplicity of infection (MOI) of approximately 10:1. Prior to infection of cells, B. pseudomallei was treated with 50 µM of each inhibitor or DMSO alone for 1 h at 37°C. J774A.1 cells were infected with treated B. pseudomallei for 30 min at 37°C. To determine the exact starting inoculum at the time of infection, bacteria were further serially diluted and plated on LB agar. Post-infection, bacteria were aspirated off the cell monolayer, and infected cells were gently washed three times with phosphate-buffered saline (PBS; autoclaved and filter sterilized; Life Technologies) and then incubated with L-15 medium containing 1 mg/ml kanamycin for a further 30 min at 37°C to kill extracellular bacteria. The supernatant was removed, and infected cells were then incubated with L-15 medium containing 250 μg/ml kanamycin with 50 µM of appropriate inhibitor or DMSO for 4 or 24 h at 37°C.

Cells were stored in RNAProtect Cell Reagent (QIAGEN, #76526) and stored at 4°C overnight or -80°C for long-term storage. Cells from duplicate wells were combined, pelleted and RNA was extracted using the RNeasy Micro Plus kit (QIAGEN, #74034) as per the manufacturer’s protocol. RNA quantity and quality was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA integrity was assessed by Azenta using an RNA ScreenTape (Agilent). Details of each of sample including QC data are provided in Supplementary Table 1. Four independent experiments were conducted to obtain four biological replicates for each treatment. Library preparation and RNA sequencing was conducted by Azenta. Briefly, libraries were sequenced using a 1× 150-bp single end rapid run on an Illumina HiSeq platform. To remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed using Trimmomatic (v0.30) (Bolger et al., 2014). Remaining sequence reads were mapped to the Mus musculus mm10 reference genome using Hisat2 (v2.0.1) (Kim et al., 2019). Raw data obtained from RNA sequencing were deposited as FASTQ and gene abundance data in Gene Expression Omnibus (GEO) database (accession number: GSE244127).

Gene expression analysis was conducted by Azenta. Briefly, gene expression calculation was performed using published methods (Mortazavi et al., 2008), and the fragments per kilo bases per million reads (FPKM) were calculated. Gene expression results across all samples are available on the GSE244127_all.fpkm_anno.txt.gz file on the GEO database. Expression levels of all genes under different experimental conditions were compared by FPKM profiles. Gene differential analysis was performed using DESeq2 (v1.6.3) and EdgeR (v3.4.6) (Anders and Huber, 2010; Love et al., 2014). The results from EdgeR analysis were further analyzed to determine genes with significant differential expression according to the criteria of absolute fold change greater than 1.5 or less than -1.5 and adjusted p value (FDR) less than 0.05. For KEGG pathway analysis, in-house scripts (Azenta) were used to enrich significant differential expression genes. Principal component analysis (PCA) plots were generated using the R stats package (v4.3.1). To account for potential batch effects, batch correction was performed using linear models for microarray analysis (limma; v3.56.2) (Ritchie et al., 2015).

At 24 h post-infection with B. pseudomallei, macrophage cell monolayers were lysed with MilliQ water (1 mL per well of a 24-well tissue culture plate). The lysed macrophage cells were serially diluted to 10^-4 (total volume of 1 mL) in PBS before being plated onto LB agar for bacterial enumeration. Colonies were counted after two days incubation at 37°C. Results are presented as the means of seven independent experiments. All statistical analyses were carried out using the Wilcoxon test in Graph-Pad Prism version v8.3.1.

B. pseudomallei-induced cytotoxicity was assessed through measurement of lactate dehydrogenase (LDH) in cell culture supernatants 24 h post-infection. LDH release was measured using the Roche Cytotoxicity Detection Kit (Roche, Sigma Aldrich #11644793001) as per the manufacturer’s instructions. All results are presented as the means of eight independent experiments. Infected cells treated with DMSO were included as controls. To express the results as the percentage of wildtype cytotoxicity, the LDH activity in SF235 and AN_CH_37 treated wells were compared to that in wells infected with B. pseudomallei treated with DMSO. Spontaneous release (LDH release following incubation of uninfected cells with the corresponding inhibitor or DMSO) was subtracted from the final values. All statistical analyses were carried out using the Mann-Whitney U test in Graph-Pad Prism version v8.3.1.

Broth microdilutions were tested against three concentrations of SF235 and AN_CH_37 as described previously (Bzdyl et al., 2019; Scheuplein et al., 2023) with modifications. B. pseudomallei K96243 was incubated overnight in Luria-Bertani (LB) broth at 37°C with agitation. Overnight cultures were diluted 1:100 in fresh LB broth and were incubated at 37°C for 2 h with agitation. Compounds were diluted across a 96-well plate in LB broth with a final volume of 100 µL at 100 µM. Vehicle control wells were also included with an equivalent volume of DMSO for each concentration (1% DMSO). Following bacterial incubation, 100 µL of B. pseudomallei was added to the compound/DMSO containing medium to give a final compound concentration of 50 µM and incubated statically at 37°C for 24 h. B. pseudomallei only wells were also included. The optical density of plates was read at 590 nm using a spectrophotometer (Bio-Rad Xmark). Background absorbance of media with inhibitor was subtracted from all wells. Results are expressed as the percentage of growth relative to the unexposed B. pseudomallei control wells using the equation below:

Greater than 20% growth was considered viable. Results are presented as the means of three independent experiments. All statistical analyses were carried out using the Wilcoxon test in Graph-Pad Prism version v8.3.1.

IL-1β, IL-6, TNFα and MCP-1/CCL2 were detected using standardized sandwich ELISAs in cell culture supernatants from J774A.1 cells 24 hours following infection with B. pseudomallei following the protocol described above. Cytokine measurements on supernatants from uninfected J774A.1 cells treated with DMSO were also performed. Cytokines were quantitated using Invitrogen uncoated ELISAs (Thermo Fisher Scientific #88-7013-86, 88-7391-88, 88-7324-86, 88-7064-86) as per the manufacturer’s instructions. Results were determined by measuring absorbance at 450 nm and 570 nm using a Bio-Rad Xmark plate reader. The values of 570 nm were subtracted from those of 450 nm and unknown values were interpolated into standard curves in Prism v8.3.1 using the second order polynomial equation. Results are presented as the means of eight independent experiments. All statistical analyses were carried out using Wilcoxon test in Graph-Pad Prism version v8.3.1.

SF235 and AN_CH_37 are pipecolic acid derivatives that contain the pipecolic sulphonamide and an amide-based linker to a pyridyl unit. In contrast to SF235, AN_CH_37 contains an additional benzyl moiety which has been shown to increase stability and potency [Figure 1A; (Scheuplein et al., 2023)]. To verify that SF235 and AN_CH_37 target and directly inhibit the Mip protein in B. pseudomallei (Bps Mip), the inhibition of the PPIase activity of recombinant Bps Mip was initially evaluated in a protease-coupled assay by measuring cis-trans isomerization of the tetrapeptide Suc-Ala-Phe-Pro-Phe-p-nitroanilide. These studies demonstrated that at a concentration of 400 nM, AN_CH_37 inhibited the activity of Bps Mip at a greater rate than SF235 (Figure 1B). The PPIase activity of Bps Mip was completely abolished by the known PPIase inhibitor rapamycin. Further analysis revealed the K_i values of SF235 and AN_CH_37 to be 290 ± 60 (Iwasaki et al., 2022) and 180 ± 30 nM, respectively. We have previously determined the Ki of rapamycin against Bps Mip to be 3 ± 2 nM (Norville et al., 2011a).

The murine macrophage J774A.1 cell line has been utilized to study the innate immune response to bacterial pathogens (Cross et al., 2004; Yu et al, 2010). Indeed, in in vitro models of melioidosis, we and others have demonstrated that B. pseudomallei infects, survives and replicates intracellularly in these cells (Miyagi et al, 1997; Harley et al., 1998; Sim et al., 2009; Rasko and Sperandio, 2010; Norville et al., 2011b; Srinon et al., 2013; Ritchie et al., 2015; Reimer et al., 2016; Seufert et al., 2016; Pandey et al., 2017; Bzdyl et al., 2019; Sangsri et al., 2020; Scheuplein et al., 2023). Despite this, the inflammatory response following B. pseudomallei infection has not been studied. In this respect, J774A.1 cells were used to characterize the global host gene expression response to B. pseudomallei and to determine whether inhibition of the virulence factor Mip results in modifications of the inflammatory responses to infection. To do this, we firstly performed transcriptional analysis on cells infected with B. pseudomallei K96243 in the presence of the vehicle control DMSO and compared to that of uninfected cells treated with DMSO. We then evaluated the global transcript profiles of cells infected with B. pseudomallei in the presence of SF235 or AN_CH_37 and compared it to that of B. pseudomallei infected cells treated with DMSO. These analyses were performed four hours following infection by RNA-sequencing to characterize the early stages of infection. The RNA quality and data alignment statistics are shown in Supplementary Table 1. RNA extracted from cells 8, 12, and 24 hours post-infection resulted in insufficient quantity and/or quality of RNA required for RNA-seq in B. pseudomallei infected samples and thus not used for downstream analyses (data not shown).

Visualization of overall similarities between the global transcriptional expression patterns four hours post-infection by Principal Component Analysis (PCA) showed complete separation between uninfected macrophages treated with DMSO and B. pseudomallei infected cells on the first principal component (Figure 2A). After removing batch effects (Supplementary Figure 1), a distinction in infected cells treated with AN_CH_37 compared to DMSO and SF235 was evident on the second principal component (Figure 2A). 1229 upregulated and 1086 downregulated differentially expressed genes (DEGs) were identified in B. pseudomallei infected cells with DMSO compared to uninfected cells according to the criteria of absolute fold change greater or less than 1.5 and -1.5, respectively, and a false discovery rate (FDR) of less than 0.05 (Figure 2B; Table 1; Supplementary Table 2). When comparing the effect of the Mip inhibitors on the response to B. pseudomallei infection under the same statistical analysis conditions, we observed no DEGs with SF235 treatment (Supplementary Table 3), while treatment with AN_CH_37 resulted in 16 upregulated and 12 downregulated differentially expressed genes (Figure 2C; Table 2; Supplementary Table 5).

Examination of the top significantly expressed genes following B. pseudomallei infection revealed upregulation of a vast array of immune associated genes, with il1a, il1b and cxcl2 constituting the most strikingly upregulated DEGs in infected macrophages compared to uninfected cells based on fold change (Table 1). At this time point, tlr2 was also highly upregulated (fold change = 7.3; FDR = 3.58 x 10^-76). The complete list of differentially expressed genes is provided in Supplementary Table 2. To facilitate biological interpretation of the large number of genes altered in expression upon infection, pathway functional enrichment analysis was conducted using KEGG (Kyoto Encyclopedia of Genes and Genomes). The KEGG enrichment analysis is represented in Figure 3 in which the top 30 pathways that were most significantly enriched are presented. Enrichment was assessed using “RichFactor” which was defined as the ratio of the number of genes differentially expressed in the pathway to the total number of genes in the pathway. Thus, the larger the RichFactor, the greater the degree of enrichment observed. Specifically, this analysis showed significant enrichment of the ‘TNF signaling pathway’ (adjusted p value = 1.41 x 10^-18) with 36 (7.35%) DEGs within this pathway (Supplementary Table 4). ‘Cytokine-cytokine receptor interaction’, ‘IL-17 signaling’, ‘NOD-like receptor signaling’ and ‘Toll-like receptor signaling’ (adjusted p values of 9.90 x 10^-10, 8.29 x 10^-9, 5.95 x 10^-6 and 6.02 x 10^-6, respectively) constituted the next most significant immune-related pathways identified.

To determine the effect of treatment with Mip inhibitors on differential gene expression of J774A.1 cells infected with B. pseudomallei, we identified DEGs comparing SF235 or AN_CH_37 infected cells with DMSO treatment only. Although SF235 treatment did not reveal any DEG with an FDR less than 0.05 (Supplementary Table 3), treatment with AN_CH_37 resulted in significant downregulation of il1b (fold change = -5.3; FDR = 0.03) and upregulation of a deubiquitinating enzyme usp17le (fold change = 10.6; FDR = 0.02). The list of the top differentially expressed genes is shown in Table 2 with the complete list provided in Supplementary Table 5.

According to the transcriptional evaluation, B. pseudomallei induces the expression of innate immune genes and key inflammatory pathways early in infection, and AN_CH_37 treatment downregulates some of these genes, particularly il1b. To validate this, the production of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 were measured in cell culture supernatants using ELISA 24 hours post-infection (Figures 4A–C). B. pseudomallei induced IL-1β, TNF-α and IL-6 production as anticipated, which in the presence of AN_CH_37 was significantly decreased (p< 0.01). On the other hand, treatment with SF235 resulted in a reduction in IL-1β only (p< 0.01). To investigate the potentially protective role of the chemokine, monocyte chemotactic protein-1 (MCP-1), we measured MCP-1 in the cell culture supernatants and found that B. pseudomallei infection resulted in a significant reduction in MCP-1, whereas MCP-1 production was higher with SF235 or AN_CH_37 treatment (p< 0.05; Figure 4D).

To examine cell death and B. pseudomallei-induced cell cytotoxicity, LDH was measured in cell culture supernatants in the presence and absence of Mip inhibitors. As reported previously, SF235 (Iwasaki et al., 2022) and for the first time, AN_CH_37 significantly reduced LDH release following B. pseudomallei infection (p< 0.001; Figure 4E). The cytotoxicity of Mip inhibitors alone, to macrophage cells, were low (mean 14.42% for SF235 and 12.54% for AN_CH_37; Supplementary Figure 2). When bacteria were enumerated at the same time point, a significant difference in the number of intracellular bacteria was seen with AN_CH_37 (p< 0.05) but not SF235, demonstrating reduced survival and/or replication in macrophage cells following treatment with AN_CH_37 (Figure 4F). Neither SF235 nor AN_CH_37 treatment reduced planktonic bacterial growth (Supplementary Figure 3).