Lipoprotein Lpp and L, D-transpeptidases regulate the master regulator of virulence AggR in EAEC

Abstract

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks, and sporadic diarrhea in industrialized and developing countries. Regulation of virulence factors in EAEC is mediated by the master regulator AggR, an AraC/XylS family member controlling the expression of more than 44 genes associated with metabolism and virulence. Although the AggR regulon is well-characterized, the mechanism and upstream signaling cascades that regulate its activation are poorly understood. This study demonstrates that Lpp (Braun's lipoprotein) and L, D-transpeptidases are required for AggR activation. We found that deletion lpp in EAEC resulted in the downregulation of more than 100 genes involved in transport, metabolism, and virulence. Among the genes, fourteen transcriptional factors, including AggR, were differentially expressed in 042Δlpp. Our findings also showed that Lpp anchoring to the peptidoglycan is a requisite for AggR-activation. Hence, chemical inhibition or genetic deletion of L, D-transpeptidases encoding genes involved in the crosslink of Lpp to the peptidoglycan abolished AggR activation. Moreover, the 042Δlpp mutant exhibited reduced biofilm formation on abiotic surfaces and reduced colonization of human intestinal colonoids. This is the first study to demonstrate the tight regulation of the AraC/XylS transcriptional regulator AggR, essential in EAEC virulence and intestinal colonization by components of the bacterial cell envelope.

Fig. 1

Lpp controls AggR expression. The expression of AggR-regulated Aap and AafA was determined by SDS-PAGE and WB in periplasmic (Panel A) and membrane fractions (Panel B). Transcriptional levels of lpp (panel C), aggR (Panel D), and aap (Panel E) were evaluated in 042, 042Δlpp, 042ΔlppΔaatD, and 042ΔlppΔaap by qRT-PCR. As negative controls, 042Δlpp (Panel C), 042ΔaggR (Panel D), and 042Δaap (Panel E) were included in our analysis. Asterisks indicate significant differences by ANOVA ( ***P < 0.0001).

Fig. 2

L, D- transpeptidases are required for AggR expression. Five L, D- transpeptidases were identified in the EAEC 042 strain. Single or double mutations in L, D- transpeptidases were generated by lambda red. AggR expression was determined by qRT-PCR in 042, 042ΔaggR, 042ΔaatD∆ybiS, 042ΔaatDΔynhG, 042ΔaatDΔerfK, 042ΔaatDΔynhGΔybiS and 042ΔaatDΔynhGΔerfK (Panel A). Periplasmic fractions were isolated and analyzed by SDS-PAGE and WB using an anti-Aap polyclonal antibody (Panel B). Asterisks indicate significant differences by ANOVA (**, P < 0.01; ***, P < 0.001, ****, P < 0.0001).

Fig. 3

Analysis of Lpp regulon. EAEC042 and 042Δlpp were analyzed by RNAseq. 102 differentially expressed genes are represented in the volcano graph of Panel A. The genes were grouped into six major categories (Panel B): membrane/secreted proteins (Panel C), metabolic proteins (Panel D), transporter systems (Panel E), transcriptional factors (Panel F), and AggR-regulated virulence factors (Panel G).

Fig. 4

Validation of transcriptomic database by qRT-PCR. The RNAseq data was validated by qRT-PCR. lpp (Panel A), AggR-regulated genes (aatP, aatA, aatB, aatC, aatD, aggR, and aafA) (Panels B-H), membrane/secreted proteins (orf1180, orf3550 and flu) (Panels I-K), and transcriptional factors nac and aggR (Panels G, L) were selected based on their relevance in virulence and metabolism. The transcriptional levels of each gene were quantitated in 042 (green bars), 042Δlpp (blue bars), and 042Δlpp(pLpp) (black bars). Expression levels for each queried gene were normalized to the constitutively expressed cat gene in EAEC042. Data are representative of at least three independent experiments. Asterisks indicate significant difference by ANOVA (*, P < 0.05; **, P < 0.01, ***, P < 0.001, ****P < 0.0001).

Fig. 5

Lpp impairs biofilm formation on abiotic surfaces. 042, 042Δlpp, 42ΔaafA, and 042Δlpp(pLpp) strains were grown in a 96-well plate in DMEM-high glucose for eight hours. The bacterial biofilm was stained with crystal violet (Panel A). The cell-bound crystal violet was dissolved in 96% ethanol and measured at OD 600 ƞm in a microplate reader (Panel B). Asterisks indicate significant differences by ANOVA (**, P < 0.01, ****P < 0.0001).

Fig. 6

Lpp modulates colonization of EAEC 042 in human colonoids. Human colonoids were infected with 042 (Panels A, D), 042Δlpp (Panels B, E) or 042Δlpp(pLpp) (Panels C, F). Anti-actin antibodies (for the human colonoid in red) and anti-044 antibodies (for the bacteria in green) were used in the experiment. The relative number of bacteria was measured in randomly selected Z-stack microscopic fields by ImageJ software (Panel G). Representative confocal 3D ( Panels A-C) and 2D (D-F) images from three independent samples are shown. Asterisks indicate significant differences by ANOVA (*, P < 0.05; **, P < 0.01).

Fig. 7

AggR regulation diagram. Lipoprotein Lpp and L, D-transpeptidases regulate the master regulator of virulence AggR in EAEC promoting biofilm formation and bacterial intestinal colonization.