The In Vitro Redundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium

Abstract

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that singlegene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ⌬purT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ⌬purN mutant was reduced approximately 50% in 20 h. The attenuation of the ⌬purT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C 1 units in vivo (CI ‫؍‬ 0.03 ؎ 0.03). In contrast, GlyA was the only important enzyme for the formation of C 1 units in vitro. The results with the ⌬gcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C 1 units and glycine by GlyA were not sufficient to ensure C 1 formation in S. Typhimurium in vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy in S. Typhimurium in vivo. Salmonella enterica is a common cause of foodborne disease worldwide. Annually, more than 93 million people have been estimated to suffer from nontyphoid salmonellosis, and more than 155,000 people succumb to the disease (1). Infection of mice with S. enterica serovar Typhimurium does not cause diarrhea but results in a systemic, life-threatening condition in which bacteria predominantly localize to cells of the immune system in the liver and spleen. For this reason, S. Typhimurium infection of mice is used as a model for systemic salmonellosis, including infection with the host-specific serovar S. Typhi (2).The diversity of intracellular and extracellular host niches occupied by S. Typhimurium is reflected in a high degree of metabolic flexibility (3). This flexibility is achieved through component and system-level redundancy in the metabolic network (4), and recent years have seen several studies of S. Typhimurium in order to understand the importance of metabolic redundancy for its pathogenic lifestyle (5-8). These studies have used genome-scale metabolic modeling to predict essential and combined lethal metabolic reactions; the latter group consists of two or more nonessential reactions which, when considered as one unit, are found to be essential. Such combinations are referred to as minimal cut sets in metabolic modeling (9).A list of 102 cut sets of metabolic reactions in S. Typhimurium was recently produced using a novel genome-scale metabolic model. Eac...

Methionine biosynthesis and transport are functionally redundant for the growth and virulence of Salmonella Typhimurium

“…Our description of this apparent redundancy between de novo synthesis and high-affinity transport of Met conflicted with previous studies that showed Met auxotrophs are defective for intracellular survival in macrophages and epithelial cells ( 22 , 23 ) and have reduced virulence in mice ( 24 , 28 ) and in 1-day-old chickens ( 34 ). This may be at least in part due to differences in the study design.…”

“…the spleen and liver) infections, which, in turn, may vary the bacterium's dependence on de novo biosynthesis and nutrient import pathways for growth in different tissue niches. The enzymes required for Met biosynthesis and the Met transporter have been implicated in the virulence of S. enterica and other bacteria ( 22 – 28 ). However, these pathways have not been systematically investigated for their role in the virulence of S. enterica .…”

Evaluation of pathogenicity of Salmonella Gallinarum strains harbouring deletions in genes whose orthologues are conserved pseudogenes in S. Pullorum

“…There is a growing body of evidence towards the linkage between the metabolism of a given pathogen and its virulence [ 17 , 18 , 19 , 20 , 21 , 22 ]. It was shown by Njoroge et al [ 18 ] that, in a glucose-dependent manner, the positive regulators Cra and KdpE bind to the promoter region of ler genes leading to expression of the locus of enterocyte effacement (LEE) genes of enterohemorragic Escherichia coli (EHEC).…”

Structural characterization of glycinamide-RNase-transformylase T from Mycobacterium tuberculosis

“…Total ten steps are involved in the purine salvage pathway to convert phosphoribosyl-pyrophosphate (PRPP) into inosine 5 ′ -monophosphate (IMP) [13,14]. In the third step, two enzymes PurT (glycinamide-RNase-transformylase T) and PurN (glycinamide-RNase-transformylase N), catalyze the formylation of 5 ′ -phosphoribosyl-glycinamide (GAR) to obtain formyl-phosphoribosyl-glycinamide (FGAR) using different formyl donors [11,[15][16][17] (Figure 1). PurT utilized ATP-dependent ligation of formate as a formyl donor instead of the cofactor N 10 -formyltetrahydrofolate (N 10 -formylTHF) by PurN, which is found in both prokaryotes and eukaryotes [13,18].…”

“…and α(8)(9)(10)(11)(12) and β(9)(10)(11)(12)(13)(14)(15)(16)(17). Each subunit can be divided into three domains, representing the A-, B-and C-domains.…”