NAD+Utilization inPasteurellaceae: Simplification of a Complex Pathway

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Abstract
For certain members of the Pasteurellaceae, such as H. influenzae, NAD+ is essential for growth due to the organism's apparent lack of NAD+ synthesis and recycling (18). The vestigial NAD+ pathway consists of an uptake system with minimal resynthesis activity (Fig. 1), and therefore, these organisms rely on extracellular NAD+ sources. The mucosa of the nasopharyngeal region and the upper respiratory and genital tracts are the niches which can be occupied by most Pasteurellaceae species in mammals, and when the commensal state inadvertently leads to disease, these species are also able to enter the bloodstream and the cerebrospinal fluid. It is suggested that such niches may provide microaerophilic conditions and, as deduced from the gene content of H. influenzae, the metabolism of these organisms seems to be more oriented toward reductive than oxidative conditions (69). The host compartments compensate for the bacterial NAD+ auxotrophy and contain NAD+ or related intermediates in concentrations appropriate for the survival and growth of bacteria. H. influenzae can grow well in either a complex (brain heart infusion) medium or a chemically defined medium as well as in a modified RPMI 1640-based tissue culture medium (12), if it is supplemented with 1 to 10 μM NAD+ (3, 34). Alternatively, other NAD+ sources, collectively termed “factor V,” such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), can also be supplied in the growth media (14, 26, 79). Although H. influenzae is isolated exclusively from humans, the organism can establish disease in animal models such as chinchilla (55), mouse (11), and rat (52). To our knowledge, the concentrations of NAD+, NMN, NR, and nicotinamide (NAm) in human body fluids are not well defined. It is known that human erythrocytes, for example, contain about 30 to 60 μM NAD+ (49), lymphocytes contain about 400 μM NAD+, and human serum contains about 50 to 60 nM NAD+ (91). Recently, NAD+ levels were determined for the body fluids of pigs and rats (57, 70). For example, in the pig model, NAD+ concentrations were determined for plasma, laryngeal-wash, tracheal-wash, and lung-wash samples and for cerebrospinal fluid and ranged from 0.18 to 1.52 μM. The sera of humans and rats are also known to harbor NAD+ pyrophosphatase and nucleotide phosphatase activities, indicating that the respective NAD+ intermediate products are available in the blood of these species (77). The kinetics of substrate uptake in H. parainfluenzae (apparent Km s of 0.55 μM NAD+ and 0.14 μM NR [14]) and the growth-limiting concentration of 1 μM NAD+ under laboratory conditions may indicate that a substrate concentration of 50 to 60 nM in human serum might be insufficient to support the growth of H. influenzae, although it has to be considered that free NR or NMN could also serve well as a factor V source, probably compensating for low NAD concentrations.

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