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Journal of Bacteriology, October 2006, p. 6719-6727, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00432-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

MINI-REVIEW

NAD⁺ Utilization in Pasteurellaceae: Simplification of a Complex Pathway

Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef Schneider Str. 2, E1, 97080 Würzburg, Germany

Bacteria of the family Pasteurellaceae are well known to exist in close relationship with mammalian hosts, mostly as constituents of commensal bacterial flora. However, at the same time, some species also have the ability to cause serious diseases. For example, Mannheimia haemolytica and Haemophilus somnus cause pneumonia in cattle (15); Pasteurella multocida is a common animal pathogen but occasionally causes meningitis in humans (40, 84); and Actinobacillus actinomycetemcomitans, although a commensal inhabitant of the oral cavity in humans, is also the leading agent of juvenile periodontitis (60), and in progressive dental disease, it can cause endocarditis (63). Other Actinobacillus spp., such as A. pleuropneumoniae, A. lignieresii, A. suis, and A. hominis, cause disease in animals. In humans, Haemophilus parainfluenzae and Haemophilus influenzae account for about 10% of the constant flora of the healthy upper respiratory tract, with H. parainfluenzae being more frequent but less pathogenic than H. influenzae. In particular, H. influenzae is capable of causing serious respiratory infections, and until the introduction of conjugative vaccines against the capsule, it was the leading cause of bacterial meningitis in young children (53). Finally, Haemophilus ducreyi can be isolated from the genital mucosa of patients suffering from chancroid disease, which is prevalent predominantly in developing countries (7, 78, 81).

MAKING IT SMALL: THE NAD⁺ PATHWAY OF THE PASTEURELLACEAE

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 K_ms 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.

View larger version (20K): [in this window] [in a new window]   FIG. 1. NAD+ utilization model. The current model of the NR, NMN, and NAD+ utilization pathway in H. influenzae is shown. As with other Pasteurellaceae family members, the nadV gene encodes a nicotinamide phosphoribosyltransferase, which is highlighted in light gray. The gene products involved are as follows: OmpP2, a general porin allowing facilitated diffusion of NMN and NAD; e (P4), a lipoprotein of the outer membrane with an acid phosphatase activity required to dephosphorylate NMN; NadN, an NAD pyrophosphatase that releases NMN and AMP from NAD and harbors nucleotide phosphatase activity; PnuC, which acts as an NR-specific permease; NadR, which is endowed with two enzymatic functions, namely, ribonucleotide kinase and NMN adenylyltransferase activity. In addition, the kinase activity of NadR is feedback regulated and is essential for NR transport. IM, inner membrane. PRPP, phosphoribosyl phosphate.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Feedback control and NR uptake model. Experimental data demonstrate that no accumulation of NR is detectable in a nadR NRK (NR-kinase deficient) background (48), indicating that NR phosphorylation is required for NR uptake. NRK is the subject for feedback inhibition by the end product NAD+; hence, NR uptake and NAD+ synthesis are feedback regulated by NAD+ (48). Proposed "open" and "closed" channel forms for PnuC which might respond and interact with NadR are indicated.

THE ENTRY OF NICOTINAMIDE RIBOSIDE IS A REGULATED PROCESS

In H. influenzae, a permease-encoding gene which possesses some distant relationship (20% amino acid identity) to PnuC from Escherichia coli (PnuC_Ec) was identified (32). This protein is located in the cytosolic membrane, and H. influenzae mutants with mutations in pnuC (pnuC_Hi) are deficient in NR uptake (32, 75). When pnuC_Ec was expressed in an H. influenzae pnuC mutant, only NR was taken up, not other pyridine substrates (75). A recent analysis of Salmonella enterica serovar Typhimurium also confirmed the NR substrate specificity for PnuC from serovar Typhimurium (PnuC_ST) (30). Therefore, it can be assumed that homologues of PnuC in general represent NR-specific transporters (30, 75). An investigation of characteristics and kinetics of NR transport (14) showed that NR uptake is carrier dependent and saturable and that ATP hydrolysis, but not proton motive force, is the relevant energy source. However, whether PnuC represents the primary active carrier system needs further analysis (75). Evidence for the involvement of a second protein function necessary for NR uptake in H. influenzae came from the observation that mutants with mutations in nadR were defective in NR transport (48) and that pnuC mutants could be created, implying a secondary uptake route (35). In Salmonella, the NadR protein acts as a repressor able to control the transcription of the nadB, nadA-pnuC, and pncB genes, depending on cellular NAD⁺ levels (64, 65). NadR directly influences the transport activity of PnuC (19, 88), thereby stimulating substrate uptake if cellular NAD⁺ levels are low (89). The first evidence for an association between enzymatic activity and NadR in E. coli came from Raffaelli and coworkers after the identification a signature nucleotidyltransferase (H/T)GHI motif found in archaeal species (68). They demonstrated that NadR is endowed with NMNAT activity. The crystal structure of NadR from H. influenzae, determined by Singh et al., revealed a detailed domain architecture and exposed a second functional feature, namely, the NRK domain (80). According to the protein structure, the NMNAT activity is located at the N-terminal part of the protein, and the NRK domain is located at the C-terminal part of NadR from H. influenzae (NadR_Hi) (Fig. 3) (42, 80). Results derived from site-directed mutagenesis studies of the Walker A, B, and LID motifs finally revealed that NR transport depends on the NRK activity of NadR_Hi (48). Based on NadR structural analysis, "nonactive" NAD⁺ binding sites were identified (Fig. 3), and it was postulated that feedback regulation mechanisms may act on NadR (80). The end product inhibition was experimentally confirmed, and it was shown that the kinase activity and, hence, NR uptake were targeted by feedback inhibition (48). The feedback inhibition could be abrogated by altering amino acid residue W256, which is part of the "nonactive" NAD⁺ binding site (48). Taken together, these findings indicate that NR is predominantly taken up by PnuC (Fig. 2), a protein that most likely fulfills the requirements of an type channel, according to a current transporter classification scheme (10, 74). Further, NR uptake relies on the NRK activity of NadR, most likely because phosphorylation to NMN prevents substrate efflux and NMN subsequently serves for NAD⁺ synthesis (Fig. 2). It is noteworthy that NadR can be found as soluble cytoplasmic and cytoplasmic membrane-associated proteins in both H. influenzae and S. enterica serovar Typhimurium (19, 48). However, no direct interaction with PnuC has been demonstrated so far.

View larger version (42K): [in this window] [in a new window]   FIG. 3. Ribbon diagram representing a NadR monomer of H. influenzae (adapted from reference 80 with permission of the publisher). The secondary-structure elements are indicated for strands and helices. The ß-strands are labeled from a to j, and the -helices are labeled from A to J. For H. influenzae, structural and biochemical analyses revealed the presence of an NMNAT and a NRK motif (80).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Comparison of the NAD reductive pathways of Enterobacteriaceae and Pasteurellaceae. (Left) Enzyme functions of the Enterobacteriaceae are presented. Three pathways are shown: (i) the de novo biosynthetic pathway, (ii) the pyridine nucleotide salvage pathway, and (iii) the uptake pathway. The de novo biosynthesis pathway consists of nadB (L-Asp oxidase), nadA (quinolinic acid synthase), nadC (quinolinic acid phosphoribosyltransferase), nadD (deamido-NAD pyrophosphorylase), nadE (NAD synthase), and nadF (NAD kinase). The pyridine nucleotide salvage pathway consists of pncA (NAm deamidase), pncB (NaMN pyrophosphorylase), pncC (NMN deamidase), and pnuE (NAD pyrophosphatase). The uptake/resynthesis system consists of pnuC (NR permease), pnuB and pnuD encoding an as yet undefined NMN uptake system (64), and nadR (NR kinase and NMN adenylyltransferase). (Right) Summary of the characterized protein factors of the family Pasteurellaceae: nadV (phosphoribosyl pyrophosphate transferase), pnuC (NR permease), and nadR (NR kinase and NMN adenylyltransferase). For more details, see the text.

Although NadR is completely dispensable in Enterobacteriaceae, it is the essential key component for NAD⁺ biosynthesis in H. influenzae. In the Pasteurellaceae, the function of NadR is to synthesize NAD⁺ and to control NAD⁺ synthesis and NR uptake rate (48) (Fig. 1 and 2), although the latter is not achieved at the level of transcriptional regulation that has been demonstrated for NadR in Enterobacteriaceae (64). NadR in S. enterica serovar Typhimurium (NadR_ST) was first described as a transcriptional regulatory protein able to bind NAD⁺ and ATP and to act as the repressor for several genes required for NAD⁺ de novo biosynthesis and pyridine nucleotide cycling (PNC) (19, 20, 64, 65, 88). It was postulated that the interaction between NadR and PnuC interferes with the transcriptional activity of NadR under low NAD⁺ concentrations, and recently it was shown in Salmonella that NadR directly binds NAD⁺ and uses it as a corepressor to repress genes involved in NAD⁺ biosynthesis. In vitro, in the absence of NAD⁺, NadR possesses only weak DNA binding activity, which is impaired if nucleotides such as ATP are present (65). In contrast to NadR_ST, no N-terminally located helix-turn-helix DNA binding motif is present in NadR_Hi. Therefore, it was postulated that NadR_Hi has no function as a transcriptional regulator protein (42). A genome survey and reconstruction revealed that this helix-turn-helix motif is also lacking in the NadR sequence of bacteria outside of the family Pasteurellaceae, yet it is present in NadR derived from H. ducreyi, A. actinomycetemcomitans, and P. multocida (42). However, NadR-dependent gene regulation has not so far been investigated in these organisms. A comparison of the enzymology of NadR derived from H. influenzae with that from S. enterica serovar Typhimurium has revealed some distinct differences. For example, NadR_Hi has a significantly better NMNAT activity level, which is about 22-fold higher than the NMNAT activity of NadR_ST (42). In contrast, NadR_ST has a significantly higher NRK catalytic activity than that of NadR_Hi. These results may indicate that in Enterobacteriaceae, NadR supports substrate flow into the PNC salvage pathway rather than being required to synthesize NAD⁺ directly (42). Whether this is a distinctive feature of NadR in PNC is unknown because the proper regulation of the substrate flow within these pathways is also unknown. In summary, NadR in general represents an amazing multifunctional regulator/enzyme complex able to integrate enzymatic, transport, and transcriptional/regulatory characteristics. It is likely that this multifaceted enzyme is the result of intramolecular dissection and specification which have occurred during evolution in order to differentially optimize NadR for the commensal environments of the respective species.

PATHWAY REDUCTION: GAPS AND CLOSURES ON THE NAD⁺ PATHWAY IN PASTEURELLACEAE

For pathogenic and commensal bacteria, little is known about their specific nutrient requirements or to what extent their nutrient requirements dictate host dependency. For endosymbionts, such as Buchnera, "Candidatus Blochmannia," and Wigglesworthia spp. within insects, some molecular aspects of interdependence and symbiotic interactions are known and have been recently reviewed (90). However, a common theme among many highly adapted commensal, pathogenic, and obligate intracellular bacteria is a minimized genome with a reduced set of metabolic pathways, determined mostly by reduced mobile genetic elements and reduced genome contents (8, 25, 27, 51). As deduced from genomics, the physiological repertoire in bacteria of the family Pasteurellaceae is restricted compared to that in free-living planktonic bacteria. For example, Fig. 4 shows an overview of the characterized enzymes involved in NAD synthesis in Enterobacteriaceae and Pasteurellaceae. As NAD and NADP are two of the most important coenzymes in redox reactions, the bacteria must maintain such cofactors at concentrations around 1 mM (64). In particular, as studies in S. enterica serovar Typhimurium have shown, NAD⁺ is synthesized via two major pathways: (i) the de novo pathway and (ii) the pyridine salvage pathway (Fig. 4). Like most prokaryotes, S. enterica serovar Typhimurium utilizes L-aspartate and dihydroxyacetone phosphate to form the intermediate product quinolinate via an NadAB-dependent reaction (Fig. 4). Quinolinate is subsequently converted to nicotinic acid mononucleotide (NaMN) by means of the phosphoribosyl phosphate-dependent enzyme NadC. The NaMN adenylyltransferase activity of NadD is further responsible for the conversion of NaMN to NaAD, followed by the amidation of NaAD via NAD synthase (NadE). The NAD⁺ salvage pathway is used for two purposes: (i) to recycle degradative products of NAD⁺ back to NAD⁺, and (ii) for the assimilation of exogenous NAD⁺. The basic function of the pyridine salvage pathway shown for Enterobacteriaceae is to recover NAD⁺ degradation products, e.g., NAm (delivered via yet poorly defined routes, e.g., NAD and NMN glycosylhydrolases, NAD⁺-dependent ligases, or NAD⁺-dependent ADP-ribosylation). The pyridine salvage starts by a conversion of NAm to nicotinic acid, catalyzed by the enzymatic activity of NAm aminohydrolase (PncA). The next step is the PncB-catalyzed synthesis of NaMN, which represents an intermediate in the de novo pathway to NAD (Fig. 4) (summarized in reference 64). Although most of the NAD⁺ (80 to 90%) is recovered by pyridine nucleotide cycles, an additional transport system accounts for NR/NMN uptake (64).

Assuming that all three branches of the NAD⁺ pathway are used by free-living bacteria, one might speculate that long-term exposure to an environment enriched for pyridines may favor the maintenance of the uptake system over the other two branches. By comparing the numbers of genes necessary for synthesis and maintenance of cellular NAD⁺ levels in Enterobacteriaceae, for example, with a corresponding number in Pasteurellaceae, it becomes obvious that genome and pathway reductions are interconnected and that evolution has selected and simplified complex pathways (Fig. 4).

As shown by functional and homology analyses, nadR and pnuC in H. influenzae represent the only significant remnants of the complex NAD⁺ pathway present in Enterobacteriaceae (Fig. 4) (18). In spite of the description of the protein components involved in NAD⁺ metabolism of H. influenzae presented here (Table 1), there are some gaps in our knowledge that need to be closed. For example, it was observed that [¹⁴C]NAm is released into the supernatant of H. parainfluenzae cells which had been incubated in buffer and [¹⁴C]NAD (14). It was speculated that this might have been the result of a cytoplasmically located NAD⁺/NMN glycohydrolase which produces and secretes NAm into the extracellular medium in order to eliminate elevated NAD⁺ levels. NAD⁺ glycohydrolase activity was also detected in Enterobacteriaceae (64), but besides secretory toxins with ADP-ribosylating activity or secreted NADase (44), no other gene products could be annotated as NAD⁺ glycohydrolase. Nevertheless, additional gene candidates were recognized in H. influenzae that are genetically associated with the components of the NAD⁺ pathway. For example, immediately downstream from and overlapping the 3' end of nadR is a hypothetical open reading frame (HI0762) which is most likely organized into a single operon with nadR. In silico analysis indicates that nadR-HI0762 is highly conserved within the genomes of the Pasteurellaceae (Table 1), and protein domain searches revealed that HI0762 is related to the metallophosphoesterases of the pfam00149.11 family (46). However, knockout mutations in HI0762 do not lead to any detectable NAD⁺ growth or utilization phenotype (unpublished results). In addition, a second gene (HI0205) located upstream of nadN encodes a gene product that is located in the periplasmic space (unpublished results). With the exception of H. influenzae and A. actinomycetemcomitans, no other sequences homologous to HI0205 and/or NadN seem to be present in the other Pasteurellaceae genomes analyzed so far (Table 1). On the other hand, mutants with knockout mutations in HI0205 lack any NAD⁺ utilization phenotype (unpublished results). A transposon insertion in HI0432 was recently identified in the infant rat model as a cause of attenuation (33). The significant homology of the HI0432 product with NADH pyrophosphatase of E. coli (21) may indicate that this enzyme regulates the level of reducing power by the hydrolysis of NADH to AMP and NMN. Homology searches further revealed that this putative NADH pyrophosphatase is conserved within the genomes of Mannheimia succiniciproducens, P. multocida, A. actinomycetemcomitans, A. pleuropneumoniae, and A. succinogenes but is absent in the genomes of H. ducreyi and H. somnus (Table 1). Further investigations are necessary to address these gene functions in H. influenzae.

NAD⁺: CONSERVATIONS AND DIFFERENCES BETWEEN PROKARYOTES AND EUKARYOTES

In both prokaryotes and eukaryotes, NAD⁺ is usually synthesized via two major pathways: the de novo pathway and the salvage pathway. Whereas in prokaryotes, for example, Enterobacteriaceae, the nicotinate moiety of NAD⁺ is synthesized from L-aspartate (Fig. 4), in eukaryotes the de novo pathway takes place via the kynurenine pathway using L-tryptophan (41). The essential role for tryptophan as a precursor for eukaryotic NAD⁺ biosynthesis in dogs is revealed by the fact that nicotinic acid and nicotinamide-deficient diets lead to potentially life-threatening pellagra-like black tongue symptoms (17). For NAD⁺ salvage, eukaryotes and prokaryotes can use niacin (nicotinic acid and nicotinamide substrates) that is phosphoribosylated to generate NaMN. NaMN is further adenylated to nicotinic acid adenine dinucleotide and subsequently aminated to yield NAD⁺ (Fig. 4) (67). Besides the Preiss-Handler salvage pathway, alternative NAD⁺ biosynthesis routes which also might have derived from prokaryotic ancestors exist in eukaryotes. For example, one conserved eukaryotic enzyme in humans, the pre-B-cell colony-enhancing factor, is a homologue of the herein described NadV (73). Instead of a nadV homologue, which is lacking in Saccharomyces cerevisiae, an equivalent of pncA converts nicotinamide into nicotinic acid and delivers this substrate to the Preiss-Handler pathway (24). In eukaryotes, NMN or NaMN can be further metabolized by several highly conserved pyridine nucleotide adenylyltransferases (PNAT1 to PNAT3) (45). Furthermore, nucleotide kinases are also presumed to occur naturally in human eukaryotic cells. For instance, a tiazofurin kinase seems to be responsible for the activation of a nucleotide-based anticancer prodrug in order to generate tiazofurin adenine dinucleotide (13). Subsequently, as deduced from the NadR pathway of H. influenzae, NR was investigated as a substrate for eukaryotic kinases, resulting in the identification of NR kinases in yeast and humans (6). NR kinases are in general very interesting, since, first, NR may now be considered an important nutrient factor, and, second, NR kinases are involved in the activation of prodrugs for antitumor therapies, such as benzamide riboside, tiazofurin, and selenazofurin (13, 76).

Beyond the well-established role of NAD⁺ as an important coenzyme in redox reactions in vertebrates, NAD⁺ also regulates the intracellular free Ca²⁺ concentrations in human monocytes (23) and modulates the innate immune response by acting as a substrate for the ectoenzyme CD38 (5, 61, 62, 66). CD38 was described as an integral membrane glycoprotein of inflammatory cells that, besides functioning as a signal transduction and adhesin molecule, also catalyzes the hydrolysis of NAD⁺ (NAD⁺ glycohydrolase) and the formation of cyclic ADP-ribose (31). It was speculated that NAD⁺ is not normally found in high concentrations in the extracellular environment (such as the serum [see above]) but that its concentration might increase in sites of inflammation, either passively due to cell lysis or actively via the NAD⁺ transporter (66). A direct link between bacterial infection and CD38-mediated NAD⁺ signaling has been demonstrated, since CD38-deficient neutrophils were unable to migrate toward a gradient of the bacterially derived chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (61). Therefore, in the host, extracellular NAD⁺ ensures bacterial growth, e.g., of H. influenzae, but also serves as a substrate for CD38 signaling. Based on these observations, it would be interesting to see whether competition for the signal and substrate molecule NAD⁺ occurs and whether bacterial metabolism interferes with innate immune responses, thereby favoring bacterial infection. It is not known whether NAD⁺, besides being a cofactor for NadR activity, serves as a signaling molecule in bacteria. However, in A. pleuropneumoniae, growth on limiting concentrations of NAD⁺ causes a change in the OM protein profile, an increased adhesiveness, an alteration in capsule expression, and elevated fimbria production (58, 82).

NAD⁺ METABOLISM: AN ANTIMICROBIAL TARGET?

Very recently, an interesting study was published that identified relevant in vivo metabolic pathways of S. enterica serovar Typhimurium which could be targeted for the development of new broad-spectrum chemotherapy (4). Unfortunately, most of the identified essential enzymes and pathways were either absent in other pathogens or are already the targets of antimicrobial agents. Another strategy, based on genetic footprinting and comparative genomics, has revealed the cofactor biosynthetic pathways as potential broad-spectrum antimicrobial drug targets (22). For example, in E. coli, the NAD⁺ biosynthetic nadDE genes (Fig. 4) are essential for cell viability (22). However, such a conclusion may not be true for all bacteria. For H. influenzae, for instance, this would suggest that the enzymatic activities of NadR could not compensate for the missing NadD and NadE functions, and yet they clearly do so in the host environment. Given the complexity and diversity of NAD⁺ biosynthesis in different bacterial species, it seems unlikely that a broad-range antimicrobial drug could be developed based on the inhibition of a single enzyme function, perhaps with the exception of NAD⁺ kinases. However, human NAD⁺ kinases are highly homologous to their bacterial counterparts (43), and therefore targeted drug design might not be a reasonable approach. Considering human NAD⁺ biosynthesis and its essentiality among the nad genes in bacteria, Gerdes and coworkers have suggested that the NadD gene product possessing NaMN adenylyltransferase activity should be prioritized as a potential antimicrobial target (22). By focusing on the NAD⁺ pathway within the Pasteurellaceae, only the PnuC-NadR or NadV-NadR pathways could serve as a narrow-spectrum target. With H. influenzae, pnuC mutants are nonviable in an infant rat model (32), and the construction of knockout mutations for the entire nadR gene failed (42, 48). By analyzing the two enzymatic domains of NadR, it was possible to delete the NRK domain only if nadV was expressed in H. influenzae (48), indicating the essential nature of the N-terminally located NMNAT domain. These data suggest that NMNAT activity seems to be a valid antimicrobial target for the Pasteurellaceae.

Substrates of nicotinamide analogs, such as 3'-aminopyridine (3'-AmP)-based inhibitors, have been investigated as inhibitors of bacterial growth, e.g., in H. influenzae and Staphylococcus aureus (28, 50). In H. influenzae, 3'-AmP and 3'-aminopyridine adenine dinucleotide (3'-AAD) uptake uses the NR and NAD⁺ routes (75). The selection for 3'-AmP-resistant isolates in a genetically engineered nadV⁺ background revealed the accumulation of point mutations in nadR. Some mutations were found to be located in the respective kinase domain of NadR, i.e., in the Walker B motif. This indicates that a deficient kinase activity is responsible for the reduced entry of 3'-AmP and subsequent synthesis to 3'-AAD (75). The target for the toxic activity of 3'-AAD is most likely located downstream of the PnuC-NadR pathway, in which 3'-AAD displaces NAD⁺ from NAD⁺-dependent redox enzymes. It has also been shown that 3'-AAD is a growth inhibitor under anaerobic conditions (75) and, therefore, that 3'-AAD might target anabolic rather than metabolic pathways.

CONCLUSIONS

The survival of bacteria with a reduced metabolic capacity would seem possible only if the bacteria committed themselves to a long-term association with hosts that could deliver relevant essential substrates. The NAD⁺ auxotrophy as such may help to explain why many of the Pasteurellaceae have developed into mammal-associated bacterial species. However, further specificity of some Pasteurellaceae species for particular hosts, such as humans, needs further detailed trait analysis. A valuable and powerful tool which supports our understanding of how certain niches bias the adaptation of specialized lifestyles is provided by comparative genomics. The main focus, however, should not concentrate entirely on the identification of new gene functions and pathways constituted by unknown hypothetical genes. Instead, much can be learned by the comparative analysis of well-established pathways, e.g., as shown here for the NAD⁺ pathway. Such investigations not only provide hints about the substrate availability within the respective host environments but also expose how evolution has optimized and enhanced such pathways in order to allow bacteria to successfully conquer particular niches. As shown here, comparative genomics in combination with detailed pathway analysis has exposed bottlenecks in enzyme functions which were otherwise buried or hidden within redundant pathways. To counteract global antibiotic resistance, it is necessary to develop novel broad-range inhibitors, although limitations to this strategy have been exposed (4). Therefore, novel strategies might include the development of second-line inhibitors which diminish essential pathway activities and thereby cause the attenuation of bacterial colonization. For example, H. influenzae and H. ducreyi strains are responsible for a considerable burden of disease, and it therefore seems reasonable to argue that the NAD⁺ pathway should be targeted in the future to develop narrow-spectrum antimicrobial agents, especially as antimicrobial resistance among H. influenzae may increase further.

ACKNOWLEDGMENTS

This work was funded by DFG grant Re 1561-1.2.

We thank H. Zhang for contributing NadR structural data and Mark Anthony and Christoph Schoen for critically reading the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef Schneider Str. 2, E1, 97080 Würzburg, Germany. Phone: 49 931 20146159. Fax: 49 931 20146445. E-mail: joachim.reidl{at}mail.uni-wuerzburg.de.

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