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. Although the regulatory scheme of AggR is well understood, the biological relevance of half of AggR-regulated proteins remains unknown. In this study we provide experimental evidence that the AggR-regulated AatD is a novel N-acyltransferase restricted to pathogens of the order Enterobacterales, including EAEC, ETEC, Yersinia sp., and C. rodentium. AatD is structurally similar to Lnt. However, unlike Lnt which is essential for cellular functions, AatD is a dedicated N-acyltransferase required for post-translational modification of virulence factors. Aap was identified as a lipoprotein acylated by AatD. Lipid modification in Aap provides an important post-translational mechanism to regulate the trafficking, stability and subcellular localization of Aap. In the absence of AatD, Aap is retained in the periplasmic space and cannot be translocated to the bacterial surface, presumably, restricting the biological function of the protein. Our data suggest that AggR and Aar virulence regulators, not only regulate the expression of Aap virulence factor at the transcriptional level, but also regulate translocation of Aap to the bacterial surface, which is required for full virulence of EAEC, unveiling an important mechanism of virulence regulation.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco’s minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

Summary: The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I‐V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new Y eh D f imbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD , also present in most E. coli strains. Immuno‐labelling of EAEC strain 042 with anti‐AAF/II and anti‐YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro‐inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17‐2, 042, 55989, C1010, 278‐1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro‐inflammatory cytokines in supernatants of Caco‐2 cells infected with EAEC strains 042 and J7 and their isogenic Δ yehD mutants showed that these mutants were significantly defective in release of IL‐8 and TNF‐α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host’s ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.

The toxigenic Klebsiella oxytoca strains secrete tilymicin and tilivalline enterotoxins, which cause antibiotic-associated hemorrhagic colitis. Both enterotoxins are non-ribosomal peptides synthesized by enzymes encoded in two divergent operons clustered in a pathogenicity island. The transcriptional regulator Lrp ( l eucine-responsive r egulatory p rotein) controls the expression of several bacterial genes involved in virulence. In this work, we have uncovered novel findings that have significant implications. We determined the transcriptional expression of aroX and npsA , the first genes of each tilimycin (TM)/tilivalline (TV) biosynthetic operon in K. oxytoca MIT 09-7231 wild-type and its derivatives Δ lrp mutant and complemented strains. Our results suggest that Lrp directly activates the transcription of both aroX and npsA genes by binding to the intergenic regulatory region in a leucine-dependent manner. Furthermore, the lack of Lrp significantly diminished the cytotoxicity of K. oxytoca on HeLa cells due to reduced production of TM and TV. Altogether, our data present a new perspective on the role of Lrp as a regulator in cytotoxin-producing K. oxytoca strains and how it controls the expression of genes involved in the biosynthesis of their main virulence factors. IMPORTANCE: Tilimycin (TM) and tilivalline (TV) are enterotoxins that are a hallmark for the cytotoxin-producing Klebsiella oxytoca strains, which cause antibiotic-associated hemorrhagic colitis. The biosynthesis of TM and TV is driven by enzymes encoded by the aroX - and NRPS-operons. In this study, we discovered that the transcriptional regulator Lrp plays a crucial role in activating the expression of the aroX - and NRPS-operons, thereby initiating TM and TV biosynthesis. Our results underscore a molecular mechanism by which TM and TV production by toxigenic K. oxytoca strains is regulated and shed further light on developing strategies to prevent the intestinal illness caused by this enteric pathogen.

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco’s Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization.