The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

Abstract

Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD ؉ salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2⌬pncA in this study) showed a similar growth rate but decreased NAD ؉ quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2⌬pncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2⌬pncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2⌬pncA mutant can be used as a novel live vaccine candidate. IMPORTANCERiemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD ؉ salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2⌬pncA led to a decrease in the NAD ؉ content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2⌬pncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2⌬pncA mutant can be used as a novel live vaccine candidate.

Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community

“…Other already described nicotinamidases are distributed in different clades throughout the phylogenetic tree, including a clade for yeasts ( Fig 2 , yellow) and nematodes ( Fig 2 , blue), where Saccharomyces cerevisiae and Caenorhabditis elegans nicotinamidases are included [ 18 , 24 , 26 , 28 , 29 , 56 ], respectively. The nicotinamidases from pathogenic microorganisms are distributed in three different clades, one including those of Borrelia burgdorferi , Leishmania infantum , Acinetobacter baumanii and the recently described nicotinamidase from Riemerella anatipestifer [ 4 – 6 , 18 , 20 ] ( Fig 2 , orange); and the others containing those of Mycobacterium tuberculosis [ 31 – 33 ] ( Fig 2 , red), and Streptococcus pneumoniae [ 18 , 34 ] ( Fig 2 , purple), respectively. Finally, it is worth mentioning that the clade described by Plasmodium falciparum nicotinamidase [ 18 ] and related members of the Plasmodium genus represented an special case in the phylogenetic tree ( Fig 2 , pink), since their sequences are longer than those of the rest of nicotinamidases, showing an N-ter extension of about 200 amino acids of unknown function.…”

“…However, human and mammalian genomes do not encode nicotinamidases and convert NAM directly into nicotinamide mononucleotide (NMN), which is then adenylated back to NAD + in a step catalyzed by NMN adenylyltransferase (EC 2.7.7.1). This absence of nicotinamidase activity in humans and its crucial role in the NAD + salvage pathways of not only the human pathogens involved in Lyme disease [ 4 ] and infantile visceral leishmaniasis [ 5 ], but also in an epizootic disease of domestic ducks caused by Riemerella anatipestifer [ 6 ], makes this enzyme a promising drug target.…”

“…The first nicotinamidase activity was reported in 1952 [ 10 ], since when the activity of 24 more of these enzymes has been measured [ 6 , 11 – 37 ]. However, their catalytic efficiency toward their respective natural substrate, nicotinamide, has only been reported in 12 of them [ 11 , 12 , 14 , 16 – 18 , 29 , 31 ], and only in 6 of these also toward other nicotinamide analogs [ 11 , 16 , 17 , 20 , 29 , 31 ].…”

Bergeyella cardium: Clinical Characteristics and Draft Genome of an Emerging Pathogen in Native and Prosthetic Valve Endocarditis

“…Virulence determinants of Bergeyella species are incompletely understood and are an emerging area of research. Recently, pncA , which encodes a nicotinamidase in the related species R. anatipestifer, was identified as an important virulence factor and determinant of invasive cardiac disease in animal models [11]. We identified a related nicotinamidase gene with 71% nucleotide similarity; it is possible that this gene also serves as a key virulence determinant in B. cardium .…”

Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence

“…Several virulence factors of R . anatipestifer have been identified, including VapD 5 , CAMP cohemolysin 6 , outer membrane protein A 7 , putative genes associated with lipopolysaccharide (LPS) synthesis 8 – 10 and nicotinamidase 11 . In our previous study, 49 virulence-associated genes were identified by transponson mutagenesis.…”