A hemolytic-uremic syndrome-associated strain O113:H21 Shiga toxin-producing Escherichia coli specifically expresses a transcriptional module containing dicA and is related to gene network dysregulation in Caco-2 cells

Abstract

Shiga toxin-producing (Stx) Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some of these strains may cause hemolytic uremic syndrome (HUS). The molecular mechanism underlying this capacity and the differential host cell response to HUS-causing strains are not yet completely understood. In Brazil O113:H21 strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here we conducted comparative gene co-expression network (GCN) analyses of two O113:H21 STEC strains: EH41, reference strain, isolated from HUS patient in Australia, and Ec472/01, isolated from cattle feces in Brazil. These strains were cultured in fresh or in Caco-2 cell conditioned media. GCN analyses were also accomplished for cultured Caco-2 cells exposed to EH41 or Ec472/01. Differential transcriptome profiles for EH41 and Ec472/01 were not significantly changed by exposure to fresh or Caco-2 conditioned media. Conversely, global gene expression comparison of both strains cultured in conditioned medium revealed a gene set exclusively expressed in EH41, which includes the dicA putative virulence factor regulator. Network analysis showed that this set of genes constitutes an EH41 specific transcriptional module. PCR analysis in Ec472/01 and in other 10 Brazilian cattle-isolated STEC strains revealed absence of dicA in all these strains. The GCNs of Caco-2 cells exposed to EH41 or to Ec472/01 presented a major transcriptional module containing many hubs related to inflammatory response that was not found in the GCN of control cells. Moreover, EH41 seems to cause gene network dysregulation in Caco-2 as evidenced by the large number of genes with high positive and negative covariance interactions. EH41 grows slowly than Ec472/01 when cultured in Caco-2 conditioned medium and fitness-related genes are hypoexpressed in that strain. Therefore, EH41 virulence may be derived from its capacity for dysregulating enterocyte genome functioning and its enhanced enteric survival due to slow growth.

Fig 1. Workflow of gene co-expression network analyses for STEC strains and Caco-2 cells.

(A) Network analysis for STEC strains. Two comparative analyses were done to investigate if enterocyte soluble mediators modify global gene expression after bacterial growth in Caco-2-conditioned (C) medium for 3h: i) EH41 in C medium X EH41 in F medium; ii) Ec472/01 in C medium X Ec472/01 in F medium. Another comparative analysis was done to assess global gene expression differences between EH41 and Ec472/01 in C medium. (B) Network analyses for Caco-2 cells after 3h of interaction with EH41 or Ec472/01. Two comparative analyses were done: Caco-2 exposed to EH41 X Caco-2 control and Caco-2 exposed to Ec472/01 X Caco-2 control.

Fig 2. Differential gene expression profiles for STEC strains.

Venn diagram analysis for DE and EE genes obtained from two comparisons: EH41 in C medium X EH41 in F medium or Ec472/01 in C medium X Ec472/01 in F medium.

Fig 3. Functional profile analyses of DE and EE transcripts obtained from the comparison of EH41 X Ec472/01 in C medium.

Pie charts show: (A) the DE transcripts set; (B) the EE transcripts in EH41; (C) the EE transcripts in Ec472/01. Functional categories are identified by roman numerals as follows: I, metabolic process; II, chaperone; III, fimbrin/ outer membrane protein; IV, ion transport/ protein transport; V, transcription; VI, qin prophage/ prophage; VII, acid resistance; VIII, transposase; IX, putative protein. The number of genes belonging to a particular functional category is indicated between parentheses in each slice. Transcripts described as hypothetical or unknown proteins are not represented in this figure.

Fig 4. DE and EE gene co-expression networks (GCNs) for STEC strains.

GCNs for EH41 and Ec472/01 are depicted in (A) and (B) respectively. The most relevant hubs (high number of gene-gene links) are graphically represented by the proportionally larger nodes. Positive or negative Pearson’s coefficients—indicating positive or inverse covariation between gene-pairs—are represented by gray or red lines respectively. Nodes in red or green indicate hyper or hypo expressed genes, respectively. Circle or diamond nodes indicate DE or EE genes respectively. Node borders in blue indicate hub genes; node borders in yellow indicate common hubs between the two GCNs.

Fig 5. Hierarchical structure of the EH41 network.

Node hierarchical arrangement represents the main direction within a network (A). In (B) and (C) the EE transcriptional module is displayed in detail. Links in red represent the first node connections, centered in dicA (B) or in dicC (C). Nodes in red or green indicate hyper or hypo expressed genes, respectively. Circle or diamond nodes indicate DE or EE genes respectively. Node borders in blue indicate hub genes; node borders in yellow indicate common hubs between EH41 and Ec472/02 GCNs.

Fig 6. DE gene co-expression network (GCN) for Caco-2 cells after interaction with STEC strains.

GCNs for Caco-2 interacting with EH41 or with Ec472/01 are shown in (A) and (B) respectively. Hubs are graphically represented by the proportionally larger nodes. Positive or negative Pearson’s coefficients are indicated by gray or red lines respectively. Nodes in red or green indicate hyper or hypo expressed genes, respectively. Blue node border indicates a hub gene; yellow node border indicates a common hub between the two GCNs.

Fig 7. DE gene co-expression network (GCN) for Caco-2 cells control group.

Caco2-control network was constructed considering all DE genes obtained from two comparisons: Caco-2 with EH41 X Caco-2 control and Caco-2 with Ec472/01 X Caco-2 control. Hubs (square nodes) are graphically represented by the proportionally larger nodes. Positive or negative Pearson’s coefficient is indicated by gray or red lines respectively. Nodes in blue, pink or purple indicate, respectively: DE genes present only in Caco-2 interacting with EH41, or with Ec472/01 or common for the two groups. Node borders in green or red indicate hypo or hyper expressed genes in the control group.

Fig 8. SEM visualization of Caco-2 cells after 3h of interaction with STEC strains.

The images depict the Caco-2 cells control (A) and cells interacting with EH41 (B) or Ec472/01 (C).