MARTX Toxin-Stimulated Interplay between Human Cells and Vibrio vulnificus

Abstract

To understand toxin-stimulated host-pathogen interactions, we performed dual-transcriptome sequencing experiments using human epithelial (HT-29) and differentiated THP-1 (dTHP-1) immune cells infected with the sepsis-causing pathogen Vibrio vulnificus (either the wild-type [WT] pathogen or a multifunctional-autoprocessing repeats-in-toxin [MARTX] toxin-deficient strain). Gene set enrichment analyses revealed MARTX toxin-dependent responses, including negative regulation of extracellular related kinase 1 (ERK1) and ERK2 (ERK1/2) signaling and cell cycle regulation in HT-29 and dTHP-1 cells, respectively. Further analysis of the expression of immune-related genes suggested that the MARTX toxin dampens immune responses in gut epithelial cells but accelerates inflammation and nuclear factor κB (NF-κB) signaling in immune cells. With respect to the pathogen, siderophore biosynthesis genes were significantly more highly expressed in WT V. vulnificus than in the MARTX toxin-deficient mutant upon infection of dTHP-1 cells. Consistent with these results, iron homeostasis genes that limit iron levels for invading pathogens were overexpressed in WT V. vulnificus-infected dTHP-1 cells. Taken together, these results suggest that MARTX toxin regulates host inflammatory responses during V. vulnificus infection while also countering host defense mechanisms such as iron limitation.IMPORTANCEV. vulnificus is an opportunistic human pathogen that can cause life-threatening sepsis in immunocompromised patients via seafood poisoning or wound infection. Among the toxic substances produced by this pathogen, the MARTX toxin greatly contributes to disease progression by promoting the dysfunction and death of host cells, which allows the bacteria to disseminate and colonize the host. In response to this, host cells mount a counterattack against the invaders by upregulating various defense genes. In this study, the gene expression profiles of both host cells and V. vulnificus were analyzed by RNA sequencing to gain a comprehensive understanding of host-pathogen interactions. Our results suggest that V. vulnificus uses the MARTX toxin to subvert host cell immune responses as well as to oppose host counterattacks such as iron limitation.

Keywords: MARTX toxin; Vibrio vulnificus; dual-RNA sequencing; iron limitation; siderophore.

FIG 1

MARTX toxin-specific gene expression changes during infection. (A) Schematic representation of the experimental design. (B and C) Multidimensional scaling (MDS) plots of mock-treated, WT V. vulnificus-infected, and ΔrtxA1 V. vulnificus-infected HT-29 cells (B) or dTHP-1 cells (C). (D and E) MDS plots of WT and ΔrtxA1 V. vulnificus during HT-29 cell infection (D) or dTHP-1 cell infection (E).

FIG 2

Differentially expressed genes (DEGs) in V. vulnificus-infected HT-29 and dTHP-1 cells. (A) Common and specific DEGs in WT- or ΔrtxA1 V. vulnificus-infected HT-29 cells. (B) Gene sets enriched in the common DEGs shown in panel A. (C) Gene sets enriched in the DEGs specific for WT V. vulnificus-infected HT-29 cells. (D) Common and specific DEGs in WT- or ΔrtxA1 V. vulnificus-infected dTHP-1 cells. (E) Gene sets enriched in the common DEGs shown in panel D. (F) Gene sets enriched in the DEGs specific for WT V. vulnificus-infected dTHP-1 cells. Red vertical dotted lines indicate the cutoff P value for significance (<0.05).

FIG 3

Immune-related DEGs in HT-29 and dTHP-1 cells. (A) Immune-related DEGs in WT V. vulnificus-infected HT-29 cells compared with mock-treated cells. (B) Immune-related DEGs in ΔrtxA1 V. vulnificus-infected HT-29 cells compared with mock-treated cells. (C) Immune-related DEGs in common in or specific to WT- or ΔrtxA1 V. vulnificus-infected HT-29 cells. (D) Immune-related DEGs in the WT V. vulnificus-infected dTHP-1 cells compared with mock-treated cells. (E) Immune-related DEGs in ΔrtxA1 V. vulnificus-infected dTHP-1 cells compared with mock-treated cells. (F) Immune-related DEGs in common in or specific to either WT- or ΔrtxA1 V. vulnificus-infected dTHP-1 cells. Common upregulated immune genes in WT- and ΔrtxA1 mutant-infected dTHP-1 cells are shown in Table S2. (G) Some immune-related genes showed opposite expression patterns in the two types of host cells upon V. vulnificus infection. An asterisk (*) represents a statistically significant change in gene expression (|fold change| level of ≥1.5 and false-discovery rate [FDR] of <0.05).

FIG 4

DEGs in the V. vulnificus strains during host cell infection. (A and B) Enriched KEGG pathways in WT V. vulnificus compared with ΔrtxA1 V. vulnificus during infection of HT-29 cells (A) or dTHP-1 cells (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C and D) DEGs that were expressed at higher levels or at lower levels in WT V. vulnificus than in ΔrtxA1 V. vulnificus during infection of HT-29 cells (C) or dTHP-1 cells (D). CPM, counts per million mapped reads.

FIG 5

Vulnibactin biosynthesis genes are significantly more highly expressed in WT V. vulnificus than in ΔrtxA1 V. vulnificus during dTHP-1 cell infection. Genes belonging to the vulnibactin biosynthesis pathway were identified from the KEGG pathway database and previous studies (60, 79).

FIG 6

Proposed model for host-V. vulnificus interactions during infection. While the PAMP molecules of the pathogen trigger general immune responses in host cells, the MARTX toxin dysregulates immune-related genes in epithelial or immune cells to reduce inflammation or to enhance inflammation and NF-κB signaling, respectively. In response to this, host immune cells recalibrate the expression of iron homeostasis genes to minimize the iron available to the invading pathogen. Through the expression of siderophore biosynthetic genes, V. vulnificus overcomes this iron limitation. Host cells are eventually lysed by the pore-forming activity of the MARTX toxin, and the pathogen utilizes nutrients released by the lysed host cells to proliferate further. LPS, lipopolysaccharide.