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Title: Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli}
Article:
Abstract:
In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores( enterobactin, salmochelins and yersiniabactin) and of a genotoxin( colibactin) are known to require a 4′-phosphopantetheinyl transferase( PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island( HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli( ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.
Introduction:
Escherichia coli is a normal resident of the lower-gut of humans and animals. Although usually a commensal, E. coli can be also a pathogen, associated with diarrheal disease and extra-intestinal infections[1],[2]. The majority of E. coli strains can be assigned to one of five main phylogenetic groups: A, B1, B2, D and E[3]. Strains of the distinct phylogenetic groups differ in their phenotypic and genotypic characteristics[4]–[6]. Extra-intestinal pathogenic E. coli( ExPEC), which display enhanced ability to cause infection outside the intestinal tract, carry specific genetic determinants or virulence factors that are clustered on different pathogenicity islands[7]. These virulence factors associated with extra-intestinal infections are nonrandomly distributed, and strains of the E. coli phylogenetic group B2 harbor the greatest frequency and diversity of virulence traits[8],[9]. As iron bioavailability is limited in the host, ExPEC are known to synthesize up to four types of siderophores involved in iron uptake: enterobactin, salmochelins, yersiniabactin and aerobactin[10],[11]. The biosynthesis of the first three requires a 4′-phosphopantetheinyl transferase( PPTase). These enzymes activate polyketide synthases( PKSs) and nonribosomal peptide synthetases( NRPSs) by catalyzing the transfer of a phosphopantetheinyl( P-pant) moiety from coenzyme A to conserved serine residues on PKSs and NRPSs[12],[13]. In organisms containing multiple P-pant-requiring pathways, each pathway generally involves a dedicated cognate PPTase[12]. In E. coli, the EntD PPTase is involved in the synthesis of enterobactin[14] and salmochelins, which are glycosylated forms of enterobactin[15]. The IroA locus responsible for salmochelins production is located either on a chromosomal pathogenicity island or on a transmissible plasmid[16]. Contrary to enterobactin, salmochelins are able to evade the mammalian innate immune response protein lipocalin 2( siderocalin) and are therefore more potent virulence factors[17]. The other siderophore necessitating a PPTase is yersiniabactin. This siderophore is encoded by the high-pathogenicity island( HPI) that was acquired through horizontal transfer[18]. The HPI core region was detected in more than 70% of ExPEC isolated from blood cultures, urine samples and cerebrospinal fluid[19]. While yersiniabactin production in Yersinia requires the YbtD PPTase encoded outside the HPI[20], no gene homologous to ybtD has been identified in the genome of E. coli strains producing yersiniabactin. The PPTase committed to the synthesis of yersiniabactin in E. coli remains unknown. We have shown that a number of E. coli strains from phylogenetic group B2 display also the pks island, which codes for the production of colibactin, a polyketide-non ribosomal peptide genotoxin[21]. Colibactin is known to induce DNA double-strand breaks, cell cycle arrest in G2-phase and megalocytosis in infected eukaryotic cells[21]. E. coli strains harboring the pks island can induce DNA damage in enterocytes in vivo and trigger genomic instability in mammalian cells[22]. In a rodent model of colon inflammation, colibactin was demonstrated to potentiate the development of colon cancer[23]. Surprisingly, colibactin is also required for the colonic anti-inflammatory properties of the probiotic E. coli strain Nissle 1917[24]. The synthesis of colibactin requires a PPTase encoded by the clbA gene located on the pks island[21]. Epidemiological studies revealed that the majority( 73. 1%) of the colibactin-positive E. coli strains was clinical ExPEC and that the pks island was significantly associated with a highly virulent subset of ExPEC isolates[25]. Strikingly, an analysis of the prevalence of the colibactin island among Enterobacteriaceae revealed that the pks island was constantly associated with the yersiniabactin gene cluster[26]. In this work we investigated a potential interplay between the biosynthetic pathways leading to the production of siderophores and of the colibactin genotoxin, through a possible functional interchangeability between PPTases in E. coli. We demonstrated that ClbA can contribute to the synthesis of siderophores both in vitro and in vivo. We proved in a mouse model of sepsis that the presence of either functional EntD or ClbA is required to maintain full virulence of ExPEC. This evidenced the interconnection between pathways leading to the synthesis of distinct secondary metabolites, via the PPTase ClbA. Therefore, the strict association of the pks island with HPI could have been selected in highly virulent E. coli isolates because ClbA can contribute to the synthesis of both the genotoxin and yersiniabactin.
Discussion:
Our work demonstrates the interplay between the biosynthetic pathways of a genotoxin and multiple siderophores. We have shown that ClbA, encoded by the pks island, is a promiscuous PPTase which promotes the synthesis of colibactin, yersiniabactin, enterobactin and consequently salmochelins. Although we demonstrated that ClbA could substitute for an entD mutation, the reciprocity was not observed. EntD seems to be specific for the synthesis of siderophores, which is consistent with other published reports[32]. In contrast, YbtD, the PPTase involved in yersiniabactin production in Yersinia was shown to substitute for a clbA mutation and allowed the production of colibactin. Attempts to relate conserved motifs of the group II subfamily of PPTases[12] with substrate specificity did not allow us to understand the functional promiscuity evidenced among certain PPTases, since type II PPTases usually have very remote primary sequences. Unfortunately, it is not possible to compare either the 3D structure of these PPTases because only the structure of Sfp is available[33]. Type II PPTases are predicted to have a similar folding and very similar secondary structures[33]. However it is difficult to draw conclusions on the folding of proteins and to correlate it with substrate specificity. Only the comparison of 3D high-resolution structures would provide information about the structure/function relationship of PPTases. Our work provides novel evidence that make PPTases promising targets for antibacterial development[34], because these enzymes are crucial for the biosynthesis of a multitude of a pathogen's collection of essential metabolites and virulence factors[35]. Iron is an essential element for survival of E. coli. Therefore, E. coli strains have evolved a strategy for iron acquisition which uses multiple siderophores with high-affinity for ferric iron. These include enterobactin, salmochelins, aerobactin and yersiniabactin[11]. Each siderophore has specific affinity for iron and may be differentially regulated to provide different advantages, potentially allowing extra-intestinal pathogenic E. coli( ExPEC) to adapt to different environmental conditions or to overcome host innate immunity[10],[36],[37]. In our model of sepsis, the ExPEC mutant that produced only aerobactin as a siderophore( strain SP15 entD clbA) was completely attenuated. This suggests that aerobactin plays a minor role in the iron uptake in this sepsis model; but the importance of each siderophore can be host and strain dependent[38]. Interestingly, either ClbA or EntD were able to restore the virulence of strain SP15 entD clbA. However, we have shown that colibactin synthesis cannot be sustained by EntD. This suggests that not colibactin, but the siderophore systems( alone or in combination) are critical during the first step of the infection in this mouse model of sepsis. Indeed, the bacterial loads in both spleen and blood were similar in animal infected with SP15 entD clbA mutant complemented either with ClbA or EntD. Analysis of bacteria present in the popliteal lymph node confirmed this analysis( data not shown). Since the carriage of the pks island is correlated with successful long-term gut colonization in humans[39], colibactin could be important for the commensal lifestyle of ExPEC. Moreover, our unpublished data suggest that the genotoxin colibactin could also play a role in natural sepsis since lymphocytes are susceptible to the genotoxin. Phylogroup B2, which includes the majority of ExPEC isolates, is considered to represent the evolutionary eldest lineage within the species[40]. Interestingly, the pks island found in B2 isolates is highly conserved, and is physically associated to a highly conserved High-Pathogenicity Island. This might even point towards a recent emergence of a distinct subgroup within phylogroup B2. In fact, epidemiological knowledge allows defining specific clonal lineages with high ExPEC virulence potential[41]. We believe that the most virulent and also the best colonizer of human gut resulted from a step-by-step acquisition and selection of different mobile elements. We propose here a scenario with the sequential integration of at least two pathogenicity islands and the cross talk via two PPTases( Fig. 8). At first, all E. coli strains produce at least one siderophore i. e. enterobactin. The entD gene and the other genes of the enterobactin system are part of the core genome and have been identified in all the E. coli strains isolated so far[42]. In contrast, the HPI encoding the yersiniabactin siderophore system devoid of any PPTase gene was acquired by horizontal gene transfer. Almost all E. coli HPIs appear to result from a single ancestor, which entered the E. coli species rather recently[43]. All strains of the phylogenetic group B2 and almost all of group D carry the HPI, whereas strains of groups A and B1 were found to be only occasionally HPI positive( Fig. 8,[19]). The spread of the HPI must have occurred in a dramatically fast fashion, which may indicate a strong selective pressure. We have shown in this study that EntD is actually the PPTase that mediates the synthesis of a functional yersiniabactin. E. coli strains that contain the HPI were demonstrated to be more virulent than isolates that lack the island[18]. Moreover, yersiniabactin is frequently associated with urinary tract infections[44],[45]. The pks island is known to be confined to the phylogenetic group B2. Besides, the pks island is highly represented within an especially highly virulent subset of B2 strains that exhibit extremely elevated virulence scores and an increased likelihood of causing bacteremia[25]. It has been previously demonstrated that all the E. coli strains that acquired the pks island encoding the colibactin through horizontal transfer, also displayed the HPI locus, with an integration site in tRNA asnW gene and asnT gene, respectively( Fig. 8;[26]). The pks island appears to be highly conserved( or even identical) in terms of nucleotide sequence in different E. coli isolates[26]. This may be a hint to a more recent acquisition of the pks island, compared to the HPI, which displays about 1–2% sequence divergence among the E. coli isolates( Schubert, unpublished data). We hypothesize that the association of the pks island with HPI has been selected in the highly virulent E. coli isolates because ClbA can contribute to the synthesis of both the genotoxin and yersiniabactin( and also enterobactin and consequently salmochelins). This deadly association is not confined in E. coli. Similar events also occurred in other pathogenic Enterobacteriacae since the pks island was also detected in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates where the island is also physically associated on the chromosome with the HPI locus[26].
The synthesis of numerous molecules involved in the virulence potential and fitness of pathogenic bacteria requires a particular enzyme family , i . e . phosphopantetheinyl transferases ( PPTases ) . To date , the synthesis of a given bioactive metabolite was thought to require a specific PPTase . As PPTases are being investigated as promising targets for antibacterial development , we addressed the question of a possible functional interchangeability between PPTases in Escherichia coli . PPTases are known to be involved in the synthesis of low-molecular weight iron chelators ( siderophores ) , and of a genotoxin named colibactin . Here we demonstrated interplay between the synthesis pathways leading to the production of siderophores and of colibactin . We showed that inactivation of both PPTases abolished the virulence of extra-intestinal pathogenic E . coli ( ExPEC ) in a mouse sepsis model . To our knowledge , this is the first demonstration of interplay between multiple PPTases-requiring pathways leading to the biosynthesis of functionally distinctive virulence factors , in a given microorganism . The extensive substrate specificity of PPTase ClbA could account for the co-selection and co-evolution of genomic islands encoding colibactin and yersiniabactin siderophore .