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 posttranslational 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 Nacyltransferase 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.

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

“…Although not directly tested in this work, based on similarities with Lnt, PE is likely the major source of palmitoyl for N-palmitoylation of CexE. In fact, Belmont-Monroy et al, 2020 suggested that PE is the major source of lipid due to an increase of PE in the aar mutant compared to the wild type. However, this seems counterintuitive as the deletion of aar results in an increase in aggR expression, which subsequently results in increased AatD production ( Morin et al, 2013 ; Santiago et al, 2014 ; Yasir et al, 2019 ); Aar is a negative regulator of aggR expression in EAEC 042 ( Santiago et al, 2014 ).…”

“…Thus, N-palmitoylation of glycine appears to be a rare or under-described event and to our knowledge this is the first report of enzyme-mediated N-palmitoylation of a protein at a glycine residue. The recent publication by Belmont-Monroy et al, 2020 identified AatD as an acyltransferase of Aap and CexE responsible for N-palmitoylation of a glycine residue. However, this study failed to recognise this modification as the first confirmed example of enzyme-mediated N-palmitoylation of a glycine or to demonstrate that the position and conservation of the N-terminal amino acids were crucial to acylation.…”

“…Previously, we described two outer membrane proteins (OMPs), CexE and Aap, associated with enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC), respectively ( Crossman et al, 2010 ; Sheikh et al, 2002 ). CexE has been implicated in prolonged bacterial shedding and increased severity of infection ( Rivas et al, 2020 ), whereas Aap influences biofilm formation and gut colonisation ( Belmont-Monroy et al, 2020 ; Sheikh et al, 2002 ). Despite Aap and CexE sharing only 18% amino acid identity, both proteins are secreted by the Aat system ( Belmont-Monroy et al, 2020 ; Nishi et al, 2003 ; Rivas et al, 2020 ).…”

“…CexE has been implicated in prolonged bacterial shedding and increased severity of infection ( Rivas et al, 2020 ), whereas Aap influences biofilm formation and gut colonisation ( Belmont-Monroy et al, 2020 ; Sheikh et al, 2002 ). Despite Aap and CexE sharing only 18% amino acid identity, both proteins are secreted by the Aat system ( Belmont-Monroy et al, 2020 ; Nishi et al, 2003 ; Rivas et al, 2020 ). The Aat system requires five proteins (AatPABCD) to facilitate protein secretion, two of which bear resemblance to components of the type I secretion system (T1SS): an OMP and a periplasmic adaptor protein (PAP).…”

“…First, Aap/CexE is translocated across the inner membrane into the periplasm by the Sec pathway ( Pilonieta et al, 2007 ). It then enters the Aat system to be secreted across the outer membrane ( Belmont-Monroy et al, 2020 ; Nishi et al, 2003 ; Rivas et al, 2020 ).…”