De novo histidine biosynthesis protects Mycobacterium tuberculosis from host IFN-γ mediated histidine starvation

Intracellular pathogens including Mycobacterium tuberculosis (Mtb) have evolved with strategies to uptake amino acids from host cells to fulfil their metabolic requirements. However, Mtb also possesses de novo biosynthesis pathways for all the amino acids. This raises a pertinent question- how does Mtb meet its histidine requirements within an in vivo infection setting? Here, we present a mechanism in which the host, by up-regulating its histidine catabolizing enzymes through interferon gamma (IFN-γ) mediated signalling, exerts an immune response directed at starving the bacillus of intracellular free histidine. However, the wild-type Mtb evades this host immune response by biosynthesizing histidine de novo, whereas a histidine auxotroph fails to multiply. Notably, in an IFN-γ−/− mouse model, the auxotroph exhibits a similar extent of virulence as that of the wild-type. The results augment the current understanding of host-Mtb interactions and highlight the essentiality of Mtb histidine biosynthesis for its pathogenesis.

Simulations Reveal Key Role of Arg15 for Promiscuous Activity in HisA Enzyme

The HisA enzyme catalyzes the first step of histidine biosynthesis via Amadori rearrangement of substrate ProFAR. Since it possesses the most conserved and ancient TIM-barrel fold, it provides an ideal...

Characterization of the coformycin biosynthetic gene cluster in Streptomyces kaniharaensis

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-5′-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

PA0335, a Gene Encoding Histidinol Phosphate Phosphatase, Mediates Histidine Auxotrophy in Pseudomonas aeruginosa

ABSTRACT The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2 (PA3165), hisH2 (PA3152), and hisF2 (PA3151) were found to be dispensable for histidine synthesis, whereas hisG (PA4449), hisE (PA5067), hisF1 (PA5140), hisB (PA5143), hisI (PA5066), hisC1 (PA4447), and hisA (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, hisH1 (PA5142) or hisD (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosa. IMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa. The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.

Salmonella Proteomic Profiling during Infection Distinguishes the Intracellular Environment of Host Cells

ABSTRACT Essential to bacterial pathogenesis, Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved the capacity to quickly sense and adapt to specific intracellular environment within distinct host cells. Here we examined S. Typhimurium proteomic remodeling within macrophages, allowing direct comparison with our previous studies in epithelial cells. In addition to many shared features, our data revealed proteomic signatures highly specific to one type of host cells. Notably, intracellular S. Typhimurium differentially regulates the two type III secretion systems (T3SSs) far more quickly in macrophages than in epithelial cells; bacterial flagellar and chemotaxis systems degenerate more quickly in macrophages than in HeLa cells as well. Importantly, our comparative analysis uncovered high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells. Targeted metabolomic measurements revealed markedly lower histidine levels within macrophages. Intriguingly, further functional studies established that histidine biosynthesis that is defective (due to a hisG mutation) renders the bacterium (strain SL1344) hypersensitive to intracellular shortage of this amino acid. Indeed, another S. Typhimurium strain, namely, strain 14028s, with a fully functional biosynthetic pathway exhibited only minor induction of the his operon within infected macrophages. Our work thus provided novel insights into S. Typhimurium adaptation mechanisms within distinct host cells and also provided an elegant paradigm where proteomic profiling of intracellular pathogens is utilized to discriminate specific host environments (e.g., on the basis of nutrient availability). IMPORTANCE Salmonella Typhimurium is one of the leading causes of foodborne bacterial infection. Nevertheless, how Salmonella adapts to distinct types of host cells during infection remains poorly understood. By contrasting intracellular Salmonella proteomes from both infected macrophages and epithelial cells, we found striking proteomic signatures specific to particular types of host cells. Notably, Salmonella proteomic remodeling exhibited quicker kinetics in macrophages than in epithelial cells with respect to bacterial virulence and flagellar and chemotaxis systems. Furthermore, we unveiled high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells, which is attributable to differing intracellular levels of this amino acid. Intriguingly, we found that a defective hisG gene renders a Salmonella strain hypersensitive to histidine shortage in macrophages. Overall, our work reveals specific Salmonella adaptation mechanisms in distinct host cells, which should aid in the development of novel anti-infection strategies.

Guarding the gateway to histidine biosynthesis in plants: Medicago truncatula ATP-phosphoribosyltransferase in relaxed and tense states

In the first committed step of histidine biosynthesis, adenosine 5′-triphosphate (ATP) and 5-phosphoribosyl-α1-pyrophosphate (PRPP), in the presence of ATP phosphoribosyltransferase (ATP-PRT, EC 2.4.2.17), yield phosphoribosyl-ATP. ATP-PRTs are subject to feedback inhibition by histidine that allosterically binds between the regulatory domains. Histidine biosynthetic pathways of bacteria, lower eukaryotes, and plants are considered promising targets for the design of antibiotics, antifungal agents, and herbicides because higher organisms are histidine heterotrophs. Plant ATP-PRTs are similar to one of the two types of their bacterial counterparts, the long-type ATP-PRTs. A biochemical and structural study of ATP-PRT from the model legume plant, Medicago truncatula (MedtrATP-PRT1) is reported herein. Two crystal structures, presenting homohexameric MedtrATP-PRT1 in its relaxed (R-) and histidine-bound, tense (T-) states allowed to observe key features of the enzyme and provided the first structural insights into an ATP-PRT from a eukaryotic organism. In particular, they show pronounced conformational reorganizations during R-state to T-state transition that involves substantial movements of domains. This rearrangement requires a trans- to cis- switch of a peptide backbone within the hinge region of MedtrATP-PRT1. A C-terminal α-helix, absent in bacteria, reinforces the hinge that is constituted by two peptide strands. As a result, conformations of the R- and T-states are significantly different from the corresponding states of prokaryotic enzymes with known 3-D structures. Finally, adenosine 5′-monophosphate (AMP) bound at the active site is consistent with a competitive (and synergistic with histidine) nature of AMP inhibition.