NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

Abstract

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5-nucleotidase activity. The e (P4) protein is also shown to have NMN 5-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.Haemophilus influenzae, a gram-negative facultative anaerobic bacterium, is responsible for significant morbidity and mortality in young children (9, 35). In order to cultivate H. influenzae, complex medium is required, and if it is not blood based, it must contain two growth factors: nicotinamide adenine dinucleotide (NAD) and hemin (6). Early biochemical investigations established that nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (10, 20, 31). The NAD dependency of H. influenzae was confirmed by the absence of the genes encoding the enzymes necessary for the de novo biosynthesis of NAD (8). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (4, 11). For H. parainfluenzae the K m for transport is about 0.55 M for NAD and 0.14 M for NR, while the V max for NR is about four times that of NAD (4). This implies that NR is the substrate for an as-yet-unidentified inner membrane transporter, a proposal that is supported by the observation that NAD cannot be taken up into the cytosolic compartment as an intact molecule. Limited NAD salvage capacity resides within the H. influenzae cytosol, which can be demonstrated if cell extracts are incubated with NR or NMN, indicating the presence of an NMN adenylyl transferase or an NAD pyrophosphorylase activity (5, 16).

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis

“…These findings indicate that while e (P4) is essential for utilization of NAD (10 μg/ml; 15 μM), it is not necessary for the utilization of NMN (10 μg/ml; 30 μM). These data confirm the report of Kemmer et al that an e (P4) mutant did not grow on media containing NAD below 35 μM but grew on 15 μM NMN [7]. Therefore, media in subsequent growth studies was supplemented with NMN at 10 μg/ml.…”

“…To address the question of the role of e (P4) in heme utilization, we eliminated the complicating factor of NAD utilization by performing growth studies with NMN to satisfy the factor V requirement. Although e (P4) has also been implicated in utilization of NMN there is no discernible impact of a hel insertion mutation on utilization of high levels of NMN in either a prior report or in our laboratory [7]. At high levels of free heme (20 μg/ml) no difference in growth was seen between the wild type and hel mutant strains.…”

“…The growth phenotype cannot be attributed to a defect in NMN utilization since one would expect to see the reduced growth at all heme concentrations if this were the case. Since an e (P4) mutation has no impact on utilization of nicotinamide riboside (NR) [7], in some growth studies NMN was replaced with NR generated from NMN as previously described [15]. Growth studies with NR gave similar results to those utilizing NMN (data not shown).…”

“…In addition, a fusion protein retaining the coding sequence for the first 30 amino acids of the mature e (P4), thus lacking the heme-binding site, was unable to complement the hemA mutation in E. coli [3]. Point mutations in either the conserved aspartate residues or the proposed heme binding site have not been analyzed in H. influenzae for an impact on heme acquisition; however, point mutations of D64 and D66 in H. influenzae reportedly impacted utilization of NAD [7]. Other members of the bacterial class C NSAP family that have been partially characterized include HppA of Helicobacter pylori, OlpA of Chyseobacterium meningosepticum, LppC of Streptococcus equisimilis, and SapS of Staphylococcus aureus [19][20][21][22][23].…”

“…Reidl et al concluded that the report with respect to heme utilization was erroneous, stating "it seems unlikely that e (P4) is involved in haem uptake" [6]. The fact that growth studies in the original report were performed on media containing NAD [3] supports this view, since e (P4) is necessary for utilization of NAD [3,7]; H. influenzae lacks the enzymes for de novo synthesis of NAD, thus having an absolute requirement for this nutrient [8]. However, this conclusion does not adequately address the observation that e (P4) complements an E. coli hemA mutant for growth on heme (the E. coli hemA mutant is unable to synthesis heme de novo and the E. coli outer membrane is impermeable to heme) [3].…”