Is a NAD pyrophosphatase activity necessary for Haemophilus influenzae type b multiplication in the blood stream?

Haemophilus influenzae has an absolute requirement for factor V because it lacks all the biosynthetic enzymes necessary for the de novo synthesis of NAD. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mono-nucleotide (NMN) or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake for factor V in vivo. Recently, a hypothetical open reading frame (ORF), termed nadN, was identified to encode a gene product essential for H. influenzae growth on NAD. Here, we report its role in the virulent H. influenzae serotype b strain Eagan. Our results indicate that NadN of type b Eagan strains is involved in NAD uptake and in processing NAD to NR, which appears to be the substrate for an as yet unidentified cytoplasmic membrane NR transport system. Furthermore, we present data showing that H. influenzae type b nadN mutants are able to survive as well as Eagan, in vivo in the five-day-old infant rat model of human invasive disease. NAD pyrophosphatase and NMN 5'-nucleotidase activities were present in rat and human serum, implying that under infection conditions H. influenzae may obtain NR directly from its host.

NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

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.

Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation.

Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.

NADP and NAD utilization in Haemophilus influenzae.

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

In vivo transposon mutagenesis in Haemophilus influenzae.

In order to devise an in vivo insertion mutagenesis scheme for Haemophilus influenzae, a novel set of transposons has been constructed. These are Tn10-based minitransposons carried on pACYC184- and pACYC177-based replicons, which are suitable for in vivo transposition in H. influenzae. The transposon delivery system was designed to contain an H. influenzae-specific uptake signal sequence which facilitates DNA transformation into H. influenzae. The following mini-Tn10 elements have been made suitable for specific tasks in H. influenzae: (i) Tn10d-cat, which can be used to generate chloramphenicol-selectable insertion mutations; (ii) Tn10d-bla, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn10d-lacZcat, a chloramphenicol-selectable lacZ transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen H. influenzae.

[Proliferating ability of the lymphocytes of demodectic dogs during immune cell-stimulating therapy]. / Zur Proliferationsfähigkeit von Lymphozyten demodikosekranker Hunde bei immunzellstimulierender Therapie.

8 dogs with clinical manifest generalized demodicosis were treated with muramyldipeptide and the multipotent inducer PIND-ORF. Before treatment as well as 10 and 20 days after the beginning of therapy the lymphocyte stimulation indices of these animals were investigated and compared with the values of untreated healthy dogs. In these investigations it was determined that a PIND-ORF and muramyldipeptide therapy has the effect of rising the lymphocyte response to mitogen without, however, reaching the comparative values of healthy dogs.

[Sarcoptic mange of dogs: biology of the organism, epidemiology, pathogenesis, clinical aspect, diagnosis and treatment]. / Die Sarcoptes-Räude des Hundes: Erregerbiologie, Epidemiologie, Pathogenese, Klinik, Diagnose und Behandlung.

A review is presented on the biology of the causative agent, epidemiology, pathogenesis, clinical features, diagnosis and therapy of canine Sarcoptes scabiei infestation. This survey includes also clinical data of the period 1978-1986 in the Small Animal Hospital, Munich Veterinary Faculty. Several skin scrapings are usually necessary for diagnosis. For therapy application of acaricides once a week, altogether at least three times is sufficient. Simultaneously a decontamination of the dog's surroundings should be carried out.

The life cycle of Argas (Argas) africolumbae under constant abiotic and biotic conditions.

The developmental and reproduction pattern as well as the nondiapause behaviour of Argas (Argas) africolumbae, reared on domestic chickens and held at 27 degrees C and 90% RH, indicate that there may be 2-3 generations annually. The minimum incubation period of eggs requires 16.2 days (mean), irrespective of the day of oviposition. Larvae feed for 5-6 days and moult 10.2 days after detaching to N1. Generally, there are only 2 nymphal instars. All N1 develop to N2 13.7 days after feeding. After repletion, N2 moult to males and females within 28.3 days with a ratio of 1.12:1.0. Female determined N1 and N2 are always heavier (1.13 mg and 3.81 mg, respectively) and imbibe larger bloodmeals (4.19 mg and 17.45 mg, respectively) than male N1 and N2 (0.9 mg and 2.71 mg unfed weight and 3.04 mg and 12.33 mg bloodmeal size, respectively). Fed females begin to oviposit 9.5 days after engorgement and produce 102.9 eggs during a 14-day oviposition period.