Genetic analysis of the antivirulence loci involved in the biosynthesis of nicotinamide adenine dinucleotide in enteroinvasive Escherichia coli and Shigella

Abstract

Shigella species are the primary etiologic agents of bacillary dysentery or shigellosis, which remains a significant threat to public health, particularly in less developed countries where sanitation is poor. There are an estimated 160 million shigellosis episodes worldwide yearly, with 1.1 million deaths, predominantly in children younger than 5 years of age. The genus Shigella is divided into four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. However, Shigella has been known for a long time to be closely related to E. coli. There are very few characteristics that can distinguish Shigella strains from enteroinvasive E. coli (EIEC), which also cause dysentery. Furthermore, recent phylogenetic studies have suggested that Shigella and EIEC form a single pathotype of E. coli. My PhD work will be mainly focused on the study of evolution of the Shigella/EIEC pathotype from an ancestor commensal E. coli. The critical event in the transition toward a pathogenic life-style has probably relied on the acquisition, likely through horizontal transfer, of the virulence plasmid (pINV), which encodes all the genes required for invasion and for intra- and intercellular spreading, including their positive activators. Together with the acquisition of the pINV, the Shigella/EIEC pathotype had lost some traits that were important for survival in the environment, but redundant for the life inside the host. The loss of these specific functions has been hypothesized to be functional to the pathogenetic lifestyle of the Shigella/EIEC because the expression of these genes, defined antivirulence loci (AVL), might have been detrimental for the expression of the newly acquired virulence genes or because might have been redundant in the new niches. Unlike most E. coli strains, Shigella strains require nicotinic acid supplementation for growth on minimal medium. The nicotinic acid requirement is due to mutation in two unlinked loci, nadA and nadB, encoding the enzyme complex that converts L-aspartate to quinolinate (QUIN), a precursor of the pathway leading to de novo synthesis of NAD. Recently has been reported that QUIN is a strong and specific inhibitor of several virulence phenotypes of Shigella. In this study, has been used a combination of genetic, molecular, and genomic approaches to analyse if the requirement for nicotinic acid may be selected during the evolution toward a pathogenic lifestyle in both Shigella and EIEC strains. Genetic analysis of the antivirulence loci involved in the biosynthesis of NAD in enteroinvasive Escherichia coli and Shigella. The analysis of the evolutionary relationship among Shigella and E. coli indicates that Shigella strains have been derived repeatedly from different branches of the E. coli tree by convergent evolution involving both, gain and loss of genes. Shigella appears to have diverged from commensal E.coli more than enteroinvasive E. coli, a group of diarrhogenic E .coli which shares with Shigella the same pathogenicity process. A characteristic of Shigella is their strict requirement for an exogenous source of nicotinic acid to bypass defect in NAD synthesis. Nicotinamine adenine dinucleotide (NAD) is an essential cofactor in many cellular oxidation/reduction reactions and the maintenance of an optimal intracellular concentration of this nucleotide is of paramount importance. In Escherichia coli NAD derives from quinolate (QUIN) which is synthesized from L-aspartate in the so-called de novo pathway. QUIN is the result of the concerted action of two enzymes, the L-aspartate oxidase (nadB), and the quinolate synthase (nadA). The QUIN is converted by quinolinate phosforibosyl transferase (nadC) to nicotinic acid mononucleotide, which enters the pathway for NAD synthesis. In the absence of functional nadA and/or nadB, exogenous nicotinic acid can be used instead of QUIN to produce nicotinic acid mononucleotide. Loss of the function of any of the three genes nadB, nadA can render NAD auxotrophy. Requirement of nicotinic acid in enteroinvasive Escherichia coli. Enteroinvasive Escherichia coli (EIEC) represent a midpoint in the evolution between E. coli and Shigella species. The infection of EIEC is similar to that of Shigella, with an intracellular stage of replication and cell-to-cell spread. Based on phenotypic and biochemical evidence EIEC are more similar to E. coli than Shigella. Moreover EIEC are not a homogenous group respect to serotype, plasmid content, and biochemical features. Recent studies of the genetic relationships between pathogenic and commensal E. coli strains, by multilocus enzyme electrophoresis, ribosomal DNA restriction fragment length polymorphism or genome sequencing projects confirm the presence of EIEC among different clusters of E. coli species. Even though E. coli can be grouped in the same genus with Shigella because of genetic similarities, nicotinamide auxotrophy is observed less frequently in E.coli than in Shigella. Unlike Shigella, the EIEC strains serotype O135 isolated during a study on diarrheal diseases in children in Somalia, were able to grow as prototroph in minimal medium. To verify the nicotinic acid requirement in other EIEC we analyzed several strains isolated in different geographic areas, belong to different serotypes, and display different plasmid contents, but they are all positive in invasivity assay. Among the 15 EIEC strains studied, only four required nicotinic acid supplementation for grown in minimal medium. In these strains the nicotinic acid auxotrophy is due to alterations in nadB gene (L-aspartate oxidase), since the introduction of plasmid containing the nadB from E. coli K-12 restored the nicotinic independence. In addition nadA gene of these strains was able to complement E. coli nadA mutant. Sequence data of nadB from these four strains and site direct mutagenesis showed nadB disruption by insertion of an IS600 element after codon 52 (three strains) or inactivation of L-aspartate oxidase through a change of glycine74 in glutamate (one strain). Requirement of nicotinic acid in Shigella. There are several well known auxotrophic requirements of Shigella that are not found among most isolates of E. coli. Among these the nicotinic acid supplementation for growth on minimal medium is due to mutation in one or both unlinked loci nadA and nadB, encoding the enzyme complex that converts L-aspartate to quinolate. Recently as been reported that QUIN is a strong and specific inhibitor of Type three secretion system (TTSS) apparatus. An in silico analysis performed on genome sequences currently available on public databases highlights that nadB and/or nadA are always defective in Shigella and that its inactivation has been obtained by diverse strategies. We analyzed a collection of Shigella strains isolated over several years in different geographic areas to respect the acid nicotinic requirement. All Shigella strains examined required nicotinic acid or the intermediate quinolinic acid suggesting a deficiency in nadA or nadB locus. Than to analyze the molecular changes responsible of nadB or nadA silencing we cloned and sequenced the nadB or nadA loci of Shigella (12 S flexneri serotype 1-6, one S dysenteriae 1 and two S sonnei) and tested their ability to restore the nicotinic acid prototrophy in E. coli nadA or nadB mutant. All Shigella, except S. flexneri serotype 3a and S. dysenteriae 1, contained alterations in the nadB gene encoding for L-aspartate oxidase, the first enzyme steps in the pathway for de novo synthesis of NAD. This is an event that occurs through two strategies: point mutations leading to the replacement of an aminoacid by a stop codon, (S. flexneri serotype 1b, 2a, 4a and 5a) or a significant disruption by insertion of IS600 element (S. flexneri serotype 6 and S. sonnei). Inactivation of quinolate synthase (NadA) occurs through a change of cysteine 128 in tyrosine (S. flexneri serotype 1b, 2a and 4a), and change of prolyne 219 in leucine (S. dysenteriae 1) or by insertion of IS21 element (S. sonnei). S. flexneri serotype 3a, 5 and 6 contains a functional nadA gene able to complement E. coli nadA mutant. Since the introduction of a functional nadB restore the nicotinic acid independence only in S. flexneri serotype 5 the most likely explanation for the absence of quinolate synthase activity in S. flexneri serotype 3a and serotype 6 includes the possibility of mutations mapping outside the nadA gene that impair the functionality of quinolate synthase in these specific serotype. All together, our observations confirm and extend the concept that Shigella evolution proceeds through convergent evolution toward removal or inactivation of AVL by whatever alteration (IS or point mutation) leads to a more virulent phenotype. Although EIEC strains may be developing the full Shigella phenotype, they do not have the full set of characters that define Shigella strains. The majority of the EIEC analyzed in this study are nicotinic acid independent and since EIEC retained more characteristics of commensal E. coli than Shigella spp., these strains might reflect an earlier stage of the evolutionary process undergone by Shigella.