Characterization of a novel AraC/XylS-regulated family of N-acyltransferases in pathogens of the order Enterobacterales

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 in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.

Fig 1. Untargeted metabolomic analysis (UMA) of 042 and 042aar.

EAEC 042 and 042aar derivatives were grown in DMEM high glucose for 6 h and processed for UMA by Liquid chromatography / mass spectrometry (LC/MS). Differential amounts of phosphatidylethanolamine (PE, PE-NMe, PE-NMe2) and lysophosphatidylethanolamine (LysoPE) lipid species were detected between these strains. The averaged intensities from 2 samples run in duplicates for six of the most abundant PE and LysoPE species are shown in Panel A (whole raw data for remaining species are shown in S1A and S1B Table). Unpaired T-test statistical analysis of individual PE and Lyso-PE data points normalized to wild type 042 from two independent experiments run in duplicate are shown in panel B (*, P < 0.01; **, P < 0.001). General lipid structure of phosphatidylethanolamine species (PE) and Lysophosphatidylethanolamine (LysoPE) species identified in the UMA are depicted in panel C. UMA raw data were acquired and aligned by using the Makerlynx software (version 4.1) based on the m/z value and the retention time of ion signals. Metabolites were identified using the iMass Bank database.

Fig 2. Phylogenetic analysis of AatD / Lnt family.

Phylogenetic analysis of the aminoacid sequence of Lnt-AatD family using the MAFFT algorithm, subdivided the family into two well-conserved N-acyltransferase lineages; those close to AatD or to Lnt (Panel A). The AatD was only found in pathogens of the order Enterobacterales (annotated in blue). A hypothetical structural model for AatD was generated based on its closest structural homolog Lnt from P. aeruginosa (5N6M) by using Phyre2 algorithm, with 99.2% of confidence (Panels B and C).

Fig 3. Aap accumulates in the periplasm of 042aatD.

Periplasmic fractions of 042 derivatives containing individual deletions of aafA, aap, and aatD or double deletion (042aatD/aap and 042aap/aatD); created by transposon (T) or lambda red (λ) mutagenesis procedures, were analyzed by Coomassie blue in 20% SDS-PAGE and Western blot using a polyclonal antibody against Aap (Panel A). 042aatD aap and 042aap aatD strains have deletions in aap and aatD but the Km marker is found in the aap or aatD locus, respectively. The 042aatD strain was complemented in trans with pAatD_EAEC plasmid and analyzed as in Panel B. Anti-MBP and anti-DnaK antibodies were used as markers of periplasmic and cytoplasmic fractions, respectively.

Fig 4. Compartmentalization of Aap is affected in absence of AatD.

Aap was analyzed in whole bacterial fractions (WBF) (Panels A and B), periplasmic fractions (PF) (Panels C and D) and membrane fractions (MF) (Panel E) of 042 derivatives by Coomassie blue SDS-PAGE and Western blot. Relative amounts of Aap were quantified in WBF and PF by ELISA using a polyclonal antibody against Aap (Panels B and D). Data are representative of three independent experiments run in triplicates. Anti-MBP and Anti-DnaK antibodies were used as markers of periplasmic and cytoplasmic fractions, respectively. Anti-AafA antibody was used as a marker for MF. Asterisks indicate significant difference by one-way ANOVA Bonferroni’s post-test (**, P < 0.001).

Fig 5. AatD is required for efficient translocation of Aap to the bacterial surface.

042 derivatives were grown statically in DMEM overnight at 37°C. Bacterial cells were harvested, washed with PBS and incubated with anti-Aap polyclonal antibody for 1 h, followed by incubation with Alexa-488-conjugated secondary antibody (green, for Aap staining) and Hoechst 33342 stain (blue, for DNA staining) (Panel A-E). As a control for surface localization, Wt 042 was incubated with anti-AafA polyclonal antibody and Alexa-488-conjugated secondary antibody (green, for fimbriae staining) (Panel F). Bacterial cells were analyzed using a LSM-710 laser-scanning confocal microscope (Zeiss, Germany). Representative confocal images taken with the 64X oil objective are shown (Panel A-F). Polar localization of Aap at the bacterial surface is shown in magnified sections of the images (Panel A, C, E and F). Percentage of bacteria expressing Aap on the surface from the total bacteria population of 5–6 microscopic fields, of at least three independent experiments, is shown in panel G. Asterisks indicate significant difference by one-way ANOVA Bonferroni’s post-test (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

Fig 6. Aap-mCherry accumulates at the poles in the periplasm of 042aatD.

042 and 042 derivatives (042aap, 042aatD aap and 042aatD aatC) transformed with pAap_59-cherry were grown statically in DMEM overnight at 37°C. Bacterial cells were harvested, washed with PBS and incubated with CellBrite stain (green, for membrane staining) and Hoechst 33342 stain (blue, for DNA staining) for 1 h. Bacterial cells were analyzed using a LSM-710 laser-scanning confocal microscope (Zeiss, Germany). Magnified sections of representative confocal images taken with the 64X oil objective are shown (Panel A-T). The localization of Aap_59-cherry overlapping with bacterial membranes (Panel G, H), at the periplasmic poles (Panel K, L) and homogenously diffused in the periplasm (Panel O, P) are indicted with arrows. 042aatD aap (pLpp_23-cherry) was included as a control (S, T). Original images are shown in S3 Fig

Fig 7. Mutation of E207 residue in the AatD catalytic triad causes accumulation of Aap in the periplasm.

Residues E207 and C316 in the putative catalytic triad of AatD (illustrated in Panel A) were changed to A (Ala) by site-directed mutagenesis. The grow rates of the 042aatD strain transformed with plasmids encoding AatD variants (pAatD_E207A, pAatD_C316A and pAatD_EAEC), Wt 042 and 042aatD were assessed in triplicate at 37°C over 6 h period (Panel B). Periplasmic fractions of these strains were analyzed by western blot using anti-Aap, anti-MBP and anti-DnaK antibodies (Panel C).

Fig 8. Aap does not accumulate in the periplasm of 042aatD complemented with Lnt.

042aatD strain was transformed with pLnt_EAEC and control pAatD_EAEC plasmid. Periplasmic fractions of 042aatD derivatives were analyzed by coomassie blue stain SDS-PAGE and western blot using anti-Aap polyclonal antibody. Anti-MBP and anti-DnaK antibodies were used as markers of periplasmic and cytoplasmic fractions, respectively.

Fig 9. AatD and Lnt, but not AatD_E207A can reestablish surface-expression of Aap in 042aatD.

042 derivatives were grown statically in DMEM overnight at 37°C. Bacterial cells were harvested, washed with PBS and incubated with anti-Aap polyclonal antibody for 1 h, followed by incubation with Alexa-594-conjugated secondary antibody (red, for Aap staining), CellBrite (green, for bacterial membrane staining) and Hoechst 33342 (blue, for DNA staining). Bacterial cells were analyzed using a LSM-710 laser-scanning confocal microscope (Zeiss, Germany). Representative confocal images taken with the 64X oil objective are shown (Panel A-T). Polar localization of Aap overlapping with membranes at the bacterial surface is shown in magnified sections of the images (Panel D, L, T). Percentage of bacteria expressing Aap on the surface from the total bacteria population of 5–6 microscopic fields, of at least three independent experiments, is shown in panel U. Asterisks indicate significant difference by one-way ANOVA Bonferroni’s post-test (**, P < 0.001; ***, P < 0.0001).

Fig 10. Aap is an acylated lipoprotein.

Aap_042-H6 was purified from 042aap(pAap_042-H6) and 042aatD aap(pAap_042-H6) by nickel affinity columns (Panel A). The samples were analyzed by LC-MS (Panels B and D). A cleavage site for Signal peptidase I was predicted in Aap using the LipoP algorithm (Panel C). Acylation of Aap was confirmed by LC-MS analysis (Panel D). Posttranslational modification of Aap is illustrated in Panel E.

Fig 11. Genomic rearrangements of the aat operon and conservation of AatD function.

Illustration of the genomic rearrangements of the aat operon in the order Enterobacterales (Panel A). The Aap and CexE proteins share 23.7% identity and 61.3% homology in 93 aa overlap (Panel B). Periplasmic fractions of 042aatD complemented in trans with encoding aatD genes of EAEC (aatD_EAEC), ETEC (aatD_ETEC) and C. rodentium (aatD_Cr) were analyzed by coomassie blue SDS-PAGE and western blot using a polyclonal antibody against Aap (Panel C). Periplasmic fractions of 042aatD aap transformed with plasmids encoding the homologous Aap of ETEC (CexE) and EAEC strain 042 (Aap₀₄₂) were analyzed as in Panel D. Of note that despite the 61.3% of similarity between of both Aap and CexE proteins, anti-Aap antibodies did not recognize CexE protein (Panel D, WB with anti-Aap).

Fig 12. Evaluation of 04aatD mutant in the streptomycin-treated mouse model.

The streptomycin-treated mouse model was used to evaluate the role of AatD in vivo. Groups of 10 C57BL/6 mice were coinfected with 10⁷ cfu/ml of 042 / 042aatD (Panels A and C) and 042aatD / 042aatD(pAatD_EAEC) (Panels B and D). Bacterial shedding was determined every day for 7 days (Panel A and B). Mice were euthanized on day 7 post-inoculation to determine bacterial colonization of ileum, cecum and colon (Panels C and D). The competitive index (CI) was determined by the ratio of Wt 042 to 042-derivative recovered from mice compared to the ratio of bacteria in the inoculum. Mutants with a CI of < -0.5 or > +0.5 are considered less-colonizer or hyper-colonizer, respectively. Data are representative of two independent experiments. Asterisks indicate significant difference by T- test (*, P < 0.01; **, P < 0.001).

Fig 13. Model of Acylation of Aap in EAEC.

The hypothetical model was constructed based on previously published Aat and Lnt translocation models. The model depicts the synthesis of AatPABCD operon in the bacterium cytoplasm following the activation of AggR. Aat proteins are translocated into the periplasm by the Sec-apparatus, where AatA, AatB and AatP assemble into the Aat channel, while AatC provides the energy to activate the Aat translocon. Aap is acylated at the inner membrane by AatD. Presumably, proper acylation of Aap ensure its translocation through the Aat channel toward the bacterial surface.