Pasteurella multocida is the causative agent of infectious diseases of economic importance such as fowl cholera, bovine hemorrhagic septicemia, and porcine atrophic rhinitis. However, knowledge of the molecular mechanisms and determinants that P. multocida requires for virulence and pathogenicity is still limited. To address this issue, we developed a genetic expression system, based on the in vivo expression technology approach first described by Mahan et al. (Science 259:686-688, 1993), to identify in vivo-expressed genes of P. multocida. Numerous genes, such as those encoding outer membrane lipoproteins, metabolic and biosynthetic enzymes, and a number of hypothetical proteins, were identified. These may prove to be useful targets for attenuating mutation and/or warrant further investigation for their roles in immunity and/or pathogenesis.Pasteurella multocida is an opportunistic veterinary and human pathogen with worldwide distribution. Certain serotypes are the etiologic agents of severe types of pasteurellosis, such as fowl cholera in avian species, hemorrhagic septicemia in cattle and buffalo, and atrophic rhinitis in swine. Despite considerable research into the mechanisms of immunity, virulence, and pathogenesis, safe and effective vaccines against pasteurellosis are still lacking and little is known of the molecular mechanisms of pathogenesis.Mahan et al. (35) first described a system to identify in vivo-expressed genes and termed this "in vivo expression technology" (IVET). Various IVET systems have since been designed and used in a number of different organisms (reviewed in references 9, 24, and 25). Information gained from these research efforts has identified a number of known virulence factors, metabolic and biosynthetic genes, and, interestingly, many genes with no known function. IVET systems provide an insight into the genes which are required for survival and multiplication in vivo, and the gene products identified may represent new targets for attenuating mutations, antimicrobial agents, or recombinant vaccines. The inactivation of genes identified by IVET systems has, in many cases, resulted in the attenuation of virulence, indicating an important role for these in vivo-expressed genes in pathogenesis (34,52). In addition, the in vivo promoters themselves could be utilized for heterologous antigen expression in vivo.Outer membrane protein preparations from in vivo-grown P. multocida cells protect birds from heterologous serotypes, whereas in vitro-grown bacteria provide protection only against the homologous somatic serotype (17,22,23). The in vivo-expressed antigens involved in providing heterologous protection have been termed the cross-protective factors. Much interest has been focused on identifying the cross-protective factors of P. multocida fowl cholera strains, yet none has been isolated and characterized to date. The IVET system provides a new approach for identifying such genes and overcomes the limitations of using in vitro media and conditions to mimic the host factors responsibl...

Pasteurella multocida is the causative agent of a range of diseases with economic importance in production animals. Many systems have been employed to identify virulence factors of P. multocida, including in vivo expression technology (IVET), signature-tagged mutagenesis, and whole-genome expression profiling. In a previous study in which IVET was used with P. multocida, nrfE was identified as a gene that is preferentially expressed in vivo. In Escherichia coli, nrfE is part of the formate-dependent nitrite reductase system involved in utilizing available nitrite as an electron accepter during growth under anaerobic conditions. In this study, we constructed an isogenic P. multocida strain that was unable to reduce nitrite under either aerobic or anaerobic conditions, thereby demonstrating that P. multocida nrfE is essential for nitrite reduction. However, the nrfE mutant was still virulent in mice. Real-time reverse transcription-PCR analysis indicated that nrfE was regulated independently of nrfABCD by an independent promoter that is likely to be upregulated in vivo.Pasteurella multocida is a gram-negative bacterial pathogen that is responsible for a number of diseases that are prevalent worldwide, including bovine hemorrhagic septicemia, avian fowl cholera, porcine atrophic rhinitis, and lapine snuffles. The worldwide economic cost of these diseases in production animals is significant, but despite considerable research, safe and effective vaccines against pasteurellosis are still lacking. The molecular mechanisms of P. multocida pathogenesis are still largely unknown, and only a few virulence factors have been identified. These factors include toxins (8), capsule (5, 9), iron acquisition proteins (4, 13, 25), and hemagglutinins (25). Therefore, it is likely that many virulence factors remain uncharacterized. Identification of novel virulence factors could be used to identify new candidate vaccine antigens or targets for antimicrobial compounds.Numerous methods have been utilized to identify genes expressed during Pasteurella infections, including in vivo expression technology (IVET) (21), signature-tagged mutagenesis (13, 16), and whole-genome expression profiling (6). In the P. multocida IVET study a number of genes that are upregulated in vivo in mice were identified (21). One of these genes, nrfE, was selected for further characterization.The formate-dependent nitrite reductase (Nrf) system is present in a number of enteric bacteria, including Escherichia coli and Salmonella spp., and in the species closely related to P. multocida, Haemophilus influenzae and Actinobacillus actinomycetemcomitans (28). The Nrf system in E. coli is encoded by a seven-gene operon (nrfABCDEFG) and uses nitrite as an alternate electron acceptor for oxygen during anaerobic growth. nrfA encodes a 50-kDa cytochrome that utilizes nitrite as an electron acceptor, while nrfBCD encode proteins that are essential for electron transfer to the catalytic subunit, NrfA (22). nrfEFG have been proposed to encode proteins that form a heme lyas...

No abstract

A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some serogroup Icterohaemorrhagiae agglutinating epitopes. However, LaiMut displayed an increased reactivity to antisera against related serogroups, suggesting that the disruption of some lipopolysaccharide epitopes resulted in greater exposure to cross-reactive epitopes, not accessible to antibodies in the wild type (LaiWT). Comparison of the nucleotide sequences of the lipopolysaccharide loci of LaiMut and Lai wild type (LaiWT) strains showed an inframe stop mutation in the gene encoding undecaprenyl-galactosyltransferase, a protein that provides a fundamental and nonredundant function essential for lipopolysaccharide biosynthesis. Despite this, the biosynthesis of lipopolysaccharide agglutinating antigens was not abolished by the mutation. Based on the phenotype of LaiMut and analysis of the domain structure of the undecaprenyl-galactosyltransferase in relation to the mutation, we propose that the mutation results in the expression of two functional proteins in place of the undecaprenyl-galactosyltransferase. We hypothesize that the loss of coordination of the coupled function afforded by the intact dual function protein present in the parent strain results in an inefficient production of lipopolysaccharide in LaiMut.