Antibiofilm agents with therapeutic potential against enteroaggregative Escherichia coli

Abstract

Background: Enteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation.

Methodology: We used EAEC strains, 042 and MND005E in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of 25 other EAEC strains. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit.

Principal findings: In all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30-85% while inhibiting growth by under 10%. Hits exhibited potent antibiofilm activity at concentrations at least 10-fold lower than those reported for nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800.

Conclusions: This study identified five compounds, not previously described as anti-adhesins or Gram-negative antibacterials, with significant EAEC antibiofilm activity. Molecule, MMV687800 targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches against EAEC infection.

Fig 1

(a-b) Preliminary screen outcome of 400 drug-like molecules in Pathogen Box as a measure of percentage biofilm inhibition against growth inhibitions at 5μM for (a) EAEC strain 042 and (b) EAEC strain MND500E. Each compound except the antibiofilm hits is represented by a blue diamond. Compounds in the top portion of the scatterplot (above the horizontal red line) show >30% biofilm inhibition activity and those to the left of the vertical line additionally inhibit growth by <10%. The number of initial hit candidates (colored red) was six for 042 in (a) and three for MND005E in (b). (c) Hit progression cascade leading to 5 validated EAEC biofilm non growth inhibiting compounds. A total of 8 hits were obtained (One hit inhibited biofilms without inhibiting growth in both strains), but only five (validated hits) reproducibly inhibit biofilms in EAEC. (d) Biofilm inhibition (left) and growth inhibition (right) of hit compounds and nitazoxanide against (042). The black dots represent drug-free controls while the ash dots represent outcomes with drug treatment. Overall, five hits inhibited biofilm formation by over 30% while inhibiting growth by under 10%. NTZ showed activity but not within the range of these cut-offs. Bars represent the median for replicates in all cases.

Fig 2. Chemistry of validated 042 and MND005E hits: structures, and molecular descriptors.

Data provided by MMV and PubChem: At 5 μM test concentration, all five hits outperformed NTZ with regards to biofilm inhibition in the two EAEC strains tested. Only one of the five hits (MMV688990) exhibited antibiofilm activity on the two strains tested.

Fig 3. Hits did not significantly alter growth kinetics of EAEC strains at the various concentrations tested over 8 h: Growth kinetics of EAEC 042 with different concentrations of a) MMV688978, b) MMV687800, c) MMV688990, d) Growth kinetics of MND005E with different concentrations of MMV688990.

Error bars represent the standard deviation among replicates.

Fig 4. Regression analysis showing the relationship between percentage biofilm inhibition and drug concentrations by: a) MMV688978, (b) MMV687800, (c) MMV688990.

Hits demonstrated concentration dependent biofilm inhibition and were active (meeting >30% biofilm inhibition cut-off) at concentrations as low as 2.5 μM.

Fig 5. Time course assay of 042 biofilm by 3 hits at 5 μM shows inhibition is high at the earlier time points but highest at the later phase in: (a) MMV688978 (b) MMV687800 (c) MMV688990.

Error bars represent the range of replicates.

Fig 6. Effect of hits (grey) on biofilm formation (black) in E. coli 042 and 042 mutants: (a) MMV687800 (b) MMV687696 (c) MMV688978 (d) MMV688990.

042 is the prototypical EAEC strain from Peru, 3.1.14: 042ΔaafA, SB1: 042Δhra, LV1: 042Δaap, LV2; 042ΔaapΔhra1 and LTW1: 042ΔaapΔaafA. Biofilm inhibition in wild type strain 042, and non-aap mutant SB1 was highly significant (* = P < 0.05), this was in contrast with inhibition in aap mutants LV1and LWT1 (P > 0.05) indicating aap is a likely target for MMV687800. Statistical comparison was achieved between treated (grey) and untreated (black) biofilms formed by each strain using student’s T-test, and bars represent the median of three replicates.

Fig 7. Biofilm inhibition of E. coli 042, 042 mutants and aap complimented strains by MMV687800.

Biofilm formation (black) and inhibition by hit (grey). 042 is the prototypical EAEC strain from Peru, LV1: 042Δaap, LV2; 042ΔaapΔhra1 and LTW1: 042ΔaapΔaafA. LV1(pDAK24), LV2(pDAK24), and LTW1(pDAK24) are complement strains of LV1, LV2 and LTW1 mutants with aap inserts. Biofilm was significantly inhibited (* = P < 0.05) in wild type strain, and in aap complemented strain, LV1(pDAK24) compared to aap mutants (P > 0.05). This fulfils Molecular Koch’s postulates and confirms aap’s involvement in biofilm inhibition by MMV687800. Statistical comparison was achieved between treated and untreated controls for each strain using student T-test, and bars represent medians of replicates.

Fig 8

(a-b) Concentration dependent biofilm inhibition in E. coli 042, aap mutants and aap complimented strains by MMV687800. (a) Biofilm inhibition in aap mutants were not concentration dependent compared to wild type strain 042 and non-aap mutant SB1 (b) Inhibition of biofilms in aap complimented strains was concentration dependent and similar to those of wild type strain 042. 042 is the prototypical EAEC strain from Peru, LV1: 042Δaap, LV2; 042ΔaapΔhra1 and LTW1: 042ΔaapΔaafA. LV1(pDAK24), LV2(pDAK24), and LTW1(pDAK24) are complement strains of LV1, LV2 and LTW1 mutants with aap inserts.

Fig 9

(a) Biofilm dismantling in 042 after 2 and 18 hs of adding MMV687800 or MMV688990 onto an 8 h biofilm. Horizontal bars are medians independent data points plotted for each drug concentration (b) Concentration dependence of preformed biofilm dismantling 2 hs after drug addition onto 8 hour 042 biofilms by MMV687800 (R² = 0.6801), MMV688990 (R² = 0.8048), MMV688978 (R² = 0.9051), MMV687696 (R² = 0.9593) and in MND005E by MMV000023 (R² = 0.6757).

Fig 10. Average binding energy of 5 hits and NTZ to 20 conformers of Aap (kcal/mol).

MMV687696 and MMV687800 demonstrated the highest binding affinity for the 20 Aap conformers. Error bars represent the range of values documented, with outliers depicted as dots.