Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health

Abstract

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

Fig 1. Adherence of EcN mini-Tn5 mutants to Caco-2 cells.

A subset of mutants recovered from biofilm screens with disruptions in genes predicted to be involved in generation of surface tstructures, were assessed for their ability to attach to Caco-2 cells in in vitro co-culture models. Caco-2 cell monolayers (~80% confluence) were exposed to bacterial suspensions from mid-log-phase cultures at an MOI of 1:1 for 4 h at 37°C, 5% CO₂. Genes disrupted in mutants tested are noted in parentheses and details can be found in Table 1. Data are expressed as the mean of three replicates, and error bars show SE of the mean. Significant differences between attachment of EcN WT and mutants is indicated by ** (P ≤ 0.01) or **** (P <0.0001).

Fig 2. Initial characterisation of kfiB and kfiC deletion mutants.

Isogenic mutants deleted for the K5 capsule biosynthesis genes kfiB (EcNΔkfiB) or kfiC (EcNΔkfiC) were assessed to confirm attenuation of capsule production, ensure gene deletions did not impact expression of downstream genes, and assess impact on biofilm formation and Caco-2 adherence. A) Loss of capsule production was confirmed using the ΦK5 bacteriophage sensitivity assay, in which cells lacking a K5 capsule are resistant. Images show results from soft ager overlay plates for strains exposed to 10⁶ to 10⁴ pfu/ml of bacteriophage, in which phage replication is manifest as plaques or clearing of the confluent bacterial growth. EcN WT—E. coli Nissle 1917 wild-type; MG1655—E. coli K12 control strain naturally lacking a K5 capsule; EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant. B) Expression of associated genes in the kfi gene cluster were assessed in deletion mutants using RT-PCR, and figures indicate gene found to be active in each mutant. C) The impact of kfiB or kfiC gene deletion on the ability of EcN to form biofilms on abiotic surfaces was assessed using the CV biofilm assay, as originally used to screen mini-Tn5 mutants, and compared to the levels of WT biofilm formation. Data shows absorbance readings obtained following elution of CV stain from biofilms. D) The impact of kfiB or kfiC gene deletion on the ability of EcN to adhere to cultured Caco-2 cells was assessed using a co-culture system as for Fig. 1. EcN WT—E. coli Nissle 1917 wild-type; EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant. All figures show the mean of three replicate experiments, and error bars show SE of the mean. Significant differences between WT EcN and mutants is indicated by ** (P ≤ 0.01) or **** (P <0.0001).

Fig 3. Impact of E. coli Nissle capsule deficient mutants on Caco-2 cell health.

Owing to differences in adherence to Caco-2 cells, the impact of kfiB or kfiC gene deletion, and the K5 capsule on cell health was investigated using the co-culture system. A) The impact of capsule deficient mutants and wild-type strains on apoptosis was assessed by measuring caspase 3/7 activity after exposure to E. coli strains (MOI 10:1, bacteria:Caco-2) using the Caspase-Glo 3/7 luminescent assay. Cells treated with camptothecin (0.1 M) served as positive controls for apoptosis. Activity measured in relative light units (RLUs) and expressed as a % difference in activity observed in untreated Caco-2 controls. B) Cytotoxic effects of capsule deficient mutants were assessed by measuring the release of Lactate Dehydrogenase (LDH) from Caco-2 cells, after exposure to E. coli strains (MOI 10:1). LDH in media was quantified using the CytoTox 96 colorimetric assay (OD 490nm), and readings normalised to values obtained from complete lysates of untreated Caco-2 cells (100% LDH). Differences in LDH release between treatments was expressed as the % difference in normalised LDH release observed in media from untreated Caco-2 cells. C) The expression of COX-2 in lysates from treated or untreated Caco-2 cells was determined by Western blotting using anti-COX-2 antibody, followed by densitometry of developed films. Caco-2 cells were treated with E. coli cells from mid-log phase cultures at an MOI of 10:1, LPS, or human TNF-α; the latter treatments serving as positive controls for COX-2 expression. Quantities of COX-2 protein were normalised to densitometry readings from GAPDH. The chart provides normalised COX-2 densitometry readings, as the mean of three independent experiments, and error bars SE of the mean. Images show example blots for COX-2 and GAPDH. D) Changes in the actin cytoskeleton and nuclear morphology were assessed by double staining of treated and control cells with phalloidin red (F-actin) and DAPI (DNA). Stained cells were viewed using confocal laser microscopy, and images shown are representative of replicate experiments. Images were processed only to normalise brightness, contrast, and saturation. Data represent the means of 4 replicate experiments, and error bars show SE of the mean. EcN WT—E. coli Nissle 1917 wild-type; MG1655—E. coli K12 control strain naturally lacking a K5 capsule; EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant; Cmpt—Camptothecin (0.1 M). Significant differences to untreated controls are indicate by: * (P ≤ 0.05); ** (P ≤ 0.01); *** (P <0.001); **** (P <0.0001).

Fig 4. Cell morphology and formation of aggregates in EcN wild-type and K5 capsule mutants.

The potential for alterations in cell-cell interaction, and the general cell morphology of EcN WT and capsule mutants was examined by phase contrast microscopy. 16h cultures were examined directly by phase contrast microscopy, to assess cell morphology and the potential for cellular aggregation in EcN WT and capsule mutants. 9 fields of view were selected at random for each slide examined, and subsequently reviewed as a collection to identify the pervading features. Images provide representative examples from assessment of each strain. EcN WT—E. coli Nissle 1917 wild-type; KpsT-Tn5—EcN JNBF17 mini-Tn5 mutant disrupted in the K5 kpsT gene (Table 1); EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant. Images are at 40× magnification and represent entire fields of view.