Hypersensitivity pneumonitis and antigen identification--An alternate approach.

OBJECTIVES: Identification of the causal antigen for patients with hypersensitivity pneumonitis (HP) is challenging in a standard clinical setting. The purpose of this pilot study was to determine whether it was possible to evaluate the home/workplace of patients, and identify the causal antigen. METHODS: Using a case-control study design we compared the presence of antibody to antigen collected in the environment of individuals with HP and controls consisting of family members/co-workers. Based on patient interviews, homes/workplaces were evaluated and suspected sources of antigen collected for use in immunoassays. RESULTS: Nineteen individuals with HP participated with 15 classified as having fibrotic disease. Up to 54 bulk samples were collected from each patient's environment, with multiple isolates (antigens) cultured from each. Of the seven individuals who tested positive to one or more environmental samples, three had a positive response to more than 1 antigen from the environmental sample (range 1-9). Twelve individuals tested positive to antigen(s) on a standard panel, with only one overlapping with the antigen from the home/workplace sample. A significant association existed between results of interviews/site evaluations, and ability to collect antigen eliciting a positive response (p < 0.001). CONCLUSION: Antigen identification was successful for patients with 'active' disease. Antigens for which patients test positive on standard panels may not be present in their environment. One benefit to patient-centered testing is the ability to develop recommendations specific to their environment. As most individuals tested positive for >1 antigen, further investigation is warranted to determine the actual antigen responsible for disease.

Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence.

Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.

Identification of an Actinobacillus pleuropneumoniae consensus promoter structure.

Actinobacillus pleuropneumoniae promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless luxAB genes of Vibrio harveyi. The degree of expression was quantifiable, and only high-expression or "hot" promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for A. pleuropneumoniae was identified. The consensus promoter sequence for A. pleuropneumoniae was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the A. pleuropneumoniae consensus with other prokaryotic consensus promoters showed that the A. pleuropneumoniae consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical -10 element and a similar but truncated -35 element. However, the A. pleuropneumoniae consensus promoter is unique in the spacing between the -10 and -35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an A. pleuropneumoniae promoter is shorter than the spacing identified for Escherichia coli and Bacillus subtilis promoters.

A genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine.

Actinobacillus pleuropneumoniae is a gram negative pleiomorphic rod that is the causative agent of a severe, highly infectious and often fatal pleuropneumonia in swine. We have previously reported the construction of genetically-defined stable riboflavin auxotrophs by replacement of a portion of the APP riboflavin biosynthetic operon (ribGBAH) with an antibiotic cassette encoding resistance to kanamycin, and have demonstrated that such riboflavin auxotrophs are avirulent. In this study, we evaluated riboflavin auxotrophs of A. pleuropneumoniae for their ability to stimulate protective immunity against pleuropneumonia. An initial challenge experiment demonstrated that intramuscular vaccination with a live attenuated serotype 1A rib mutant, in a vaccine formulation that included a limiting amount of exogenous riboflavin, provided better protection against challenge with virulent A. pleuropneumoniae than either intratracheal immunization or intramuscular immunization with live bacteria in the absence of exogenous riboflavin. Subsequent studies in which the vaccine inoculating dose, concentration of exogenous riboflavin, and serotype of the vaccine strain were varied demonstrated that immunization with live avirulent riboflavin auxotrophs could elicit significant protection against experimental challenge with both homologous and heterologous virulent serotypes of A. pleuropneumoniae.

Identification of in vivo induced genes in Actinobacillus pleuropneumoniae.

We have developed an in vivo expression technology (IVET) system to identify Actinobacillus pleuropneumoniae gene promoters that are specifically induced in vivo during infection. This system is based upon an avirulent riboflavin-requiring A. pleuropneumoniae mutant and a promoter-trap vector (pTF86) that contains, in sequence, the T4 terminator, a unique Bam HI site, a promoterless copy of the V. harveyi luxAB genes, and a promoterless copy of the B. subtilis ribBAH genes in the E. coli - A. pleuropneumoniae shuttle vector pGZRS19. Sau 3A fragments of A. pleuropneumoniae genomic DNA were cloned into the Bam HI site in pTF86 and transformed into the A. pleuropneumoniae Rib- mutant. Pigs were infected with pools of 300-600 transformants by endobronchial inoculation and surviving bacteria were isolated from the pigs' lungs at 12-16 h post-infection. Infection strongly selected for transformants containing cloned promoters which drove expression of the vector ribBAH genes and allowed survival of the Rib- mutant in vivo. Strains that survived in vivo, but which minimally expressed luciferase activity in vitro, should contain cloned promoters that are specifically induced in vivo. Ten clones, designated iviA-J, were isolated which contain promoters that are induced in vivo during infection. These ivi clones were shown to be induced in the animal by luminescence of infected tissue and by direct assay of bacteria recovered from bronchoalveolar lavage. Four of these clones were putatively identified by amino acid sequence similarity as ilvI, the ilvDA operon, the secE-nusG operon, and the mrp gene. This is the first report of an IVET system for use in the family Pasteurellaceae, as well as the first report of an IVET system utilizing an infection model of pneumonia in the natural host.

Cloning and characterization of a gene encoding an antigenic membrane protein from Actinobacillus pleuropneumoniae with homology to ABC transporters.

Actinobacillus pleuropneumoniae is a pathogenic bacterium responsible for a highly contagious and often fatal form of bronchopneumonia in swine. Survival from a natural infection generally results in immunity from further infection by all 12 common serotypes, suggesting the presence of common protective antigens. We have identified one of the antigenic membrane proteins from A. pleuropneumoniae serotype 5, and cloned the gene which encodes it. This gene is found in all 12 serotypes, and encodes a protein with a predicted molecular mass of 30 kDa. Sequence analysis revealed that this antigen has a typical signal sequence characteristic of lipoproteins, and is likely to be secreted and inserted into the periplasmic side of the inner membrane. The gene shows high homology to the surface antigen CjaA of Campylobacter jejuni and to solute binding proteins of the ABC transporter family. The probable role of this protein in substrate binding and transport was supported by the presence of an upstream gene with significant homology to ATP binding proteins of the same family. In Escherichia coli, the cloned gene produced a protein which reacted strongly with convalescent sera from swine infected with A. pleuropneumoniae serotype 5, and weakly with sera from swine infected with serotype 1A or from swine vaccinated with a killed bacterin of serotype 1A or 5. It thus appears that this antigen displays some crossreactivity between serotypes, and may be less exposed in bacterins than in live cells. This protein, designated ApaA, may have an important role in nutrient acquisition and in the pathogenesis of infections caused by A. pleuropneumoniae.

The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells.

Infection of human epithelial cells by Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) increases the rate of degradation of LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. Several lines of evidence indicate that the neisserial IgA1 protease is directly responsible for this LAMP1 degradation. LAMP1 contains an IgA1-like hinge region with potential cleavage sites for the neisserial type 1 and type 2 IgA1 proteases. Neisserial type 2 IgA1 protease cleaves purified LAMP1 in vitro. Unlike its wild-type isogenic parent, an iga mutant of N. gonorrhoeae cannot affect LAMP1 turnover and its growth in epithelial cells is dramatically reduced. Thus, IgA1 protease cleavage of LAMP1 promotes intracellular survival of pathogenic Neisseria spp.

A riboflavin auxotroph of Actinobacillus pleuropneumoniae is attenuated in swine.

Actinobacillus pleuropneumoniae is the etiological agent of a highly contagious and often fatal pleuropneumonia in swine. A riboflavin-requiring mutant of A. pleuropneumoniae serotype 1, designated AP233, was constructed by deleting a portion of the riboflavin biosynthetic operon (ribGBAH) and replacing it with a gene cassette encoding kanamycin resistance. The genes affected included both the alpha- and beta-subunits of riboflavin synthase as well as a bifunctional enzyme containing GTP cyclohydrase and 3,4-dihydroxy-2-butanone-4-phosphate synthase activities. AP233 was unable to grow in the absence of exogenous riboflavin but otherwise was phenotypically identical to the parent wild-type strain. Experimental infection studies with pigs demonstrated that the riboflavin-requiring mutant was unable to cause disease, on the basis of mortality, lung pathology, and clinical signs, at dosages as high as 500 times the normal 50% lethal dose for the wild-type parent. This is the first demonstration of the attenuation of A. pleuropneumoniae by introduction of a defined mutation in a metabolic gene and the first demonstration that mutations in the genes required for riboflavin biosynthesis can lead to attenuation in a bacterial pathogen.

Molecular characterization of a common 48-kilodalton outer membrane protein of Actinobacillus pleuropneumoniae.

Previous studies have shown that a vaccine prepared from outer membranes of Actinobacillus pleuropneumoniae serotype 5 can elicit protective immunity in swine against challenge with either serotype 5 or serotype 1. These results suggest the presence of common subcapsular surface antigens, such as outer membrane proteins, that contribute to cross-protective immunity. We have identified a 48-kDa outer membrane protein that is common to all 12 capsular serotypes of A. pleuropneumoniae but is not present in the outer membranes of related species of gram-negative swine pathogens. This protein is immunogenic in swine infected with either A. pleuropneumoniae serotype 5 or 1A, as well as in swine vaccinated with A. pleuropneumoniae serotype 5 outer membranes. This 48-kDa protein is readily detected in outer membranes produced by sucrose density gradient centrifugation, but it is sarcosyl soluble and therefore not found in outer membranes prepared by detergent treatment. The gene encoding the 48-kDa outer membrane protein has been cloned from A. pleuropneumoniae serotype 5 and and has been designated aopA, for Actinobacillus outer membrane protein A. The gene is 1,347 bp in length and encodes a protein, designated AopA, of 449 amino acids with a predicted molecular weight of 48,603. Southern blot analysis under high-stringency conditions showed that strains of all 12 serotypes of A. pleuropneumoniae contain DNA homologous to this gene, as do strains of two closely related species, Actinobacillus suis and Pasteurella multocida. Whether antibodies against the AopA antigen contribute to cross-protective immunity against A. pleuropneumoniae infection remains to be determined.

Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.

A targeted mutagenesis system for Actinobacillus pleuropneumoniae.

We describe methods for the mutagenesis of cloned Actinobacillus pleuropneumoniae (Ap) genes and for the construction of Ap mutants by allelic exchange. We used these methods to construct isogenic mutants of Ap which no longer synthesized a 48-kDa outer membrane protein (AopA). The native aopA locus was replaced with a mutated locus that had been inactivated by insertion of a gene (KmR) encoding kanamycin resistance from Tn903. The inactivated aopA locus was cloned into a conjugative, R6K-derived, lambda pir-dependent suicide vector and introduced into Ap using a filtermating technique. Southern and Western blot analyses indicated that the wild-type locus was replaced by the mutated locus through either single- or double-crossover events, and that AopA was no longer produced by either type of mutant. These methods were used successfully to construct AopA- mutants in Ap serotypes 1 and 5. These methods should be generally useful in constructing mutant loci which can be used to analyze the roles of various Ap genes in the pathogenesis of contagious pleuropneumonia in swine.

Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B.

Cross-protection experiments were conducted to determine whether antigenic differences located within the lipopolysaccharides (LPS) of Actinobacillus pleuropneumoniae subtypes 1A and 1B were important with respect to the efficacy of whole cell, formalin-inactivated bacterins. Based on clinical signs, lung lesions scores and mortality rates, pigs immunized with A. pleuropneumoniae subtype 1A were partially protected against severe challenge with both subtypes 1A and 1B. In contrast, 1B vaccinated pigs were not protected against severe challenge with subtype 1A but were partially protected against 1B challenge. Cross-reactive serum antibody levels were measured with an ELISA using outer membranes of subtype 1A or 1B as the coating antigen. Serum antibodies were detected against both subtypes within 2 weeks after the first immunization. Antibody levels increased with time and were generally higher against the homologous subtype coating antigen. We conclude that antigenic variation within a capsular serotype, due to antigenic variation within LPS, can result in the failure of whole cell bacterins to provide protection against challenge with the same capsular serotype. This lack of cross-protection within a capsular serotype provides partial explanation for vaccination failures observed under field conditions.

Antigenic differences within Actinobacillus pleuropneumoniae serotype 1.

Two distinct antigenic subtypes of Actinobacillus pleuropneumoniae serotype 1 were identified via coagglutination (Co-A) and designated as 1A and 1B. The reference strains, ATCC 27088 (1A) and ISU 158 (1B), were used to prepare hyperimmune rabbit sera for Co-A reagents. Of 75 serotype 1 field isolates tested by Co-A, 35 isolates typed as 1A, 12 as 1B and 28 as 1A/1B. Significant cross-reactivity between the 2 subtypes was found in the Co-A and was eliminated in 20/28 1A/1B strains by using Co-A reagents prepared with rabbit sera absorbed with the heterologous reference strain. However, twelve isolates (5 1A and 7 1A/1B; 16%) showed no reaction with Co-A reagents prepared with absorbed sera. Immunoblots of outer membranes (OM) prepared from APP 1A or 1B reference strains and field isolates indicated that antigenic differences between subtypes 1A and 1B were located within the high molecular weight (MW) region of the gels (40-100 kDa). Hyperimmune rabbit sera against 1A or 1B and sera from pigs vaccinated with whole-cell, formalin inactivated 1A or 1B bacterins reacted with the high MW region only in strains of the homologous subtype. In contrast, 4 of 5 sera from 1B infected pigs and 2 of 5 sera from 1A infected pigs reacted with all APP serotype 1 strains regardless of subtype. Apparently, infection exposed cross-reactive antigenic determinants that were not exposed by immunization with killed bacteria preparations. SDS-PAGE gels with LPS purified from APP 1A, 1B, 9 and 11 showed that 1A, 9 and 11 LPS O-antigens had an identical smooth ladder pattern, while 1B LPS was distinctly different. In immunoblots with OM or LPS and in dot-immunobinding assays with LPS, rabbit antiserum against APP 1A reacted with 1A, 9 and 11. In contrast, rabbit antiserum against APP 1B only reacted with APP 1B and weakly with APP 9 in the OM immunoblot and with LPS from APP 1B, 9 and 11 in the LPS immunoblot and dot-immunobinding assay. We conclude that 2 subtypes of APP serotype 1 can be distinguished based on their antigenic differences. These differences are located, at least in part, within the LPS O-antigens. LPS O-antigens from APP 1B appear more antigenically similar to APP 9 LPS than to either APP 1A or APP 11 LPS. There may also be antigenic differences in the capsular polysaccharides of APP 1A and 1B strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence analysis and complementation studies of the argJ gene encoding ornithine acetyltransferase from Neisseria gonorrhoeae.

Clinical isolates of Neisseria gonorrhoeae frequently are deficient in arginine biosynthesis. These auxotrophs often have defects in the fifth step of the arginine biosynthetic pathway, the conversion of acetylornithine to ornithine. This reaction is catalyzed by the enzyme ornithine acetyltransferase, which is a product of the argJ gene. We have cloned and sequenced the gonococcal argJ gene and found that it contains an open reading frame of 1,218 nucleotides and encodes a peptide with a deduced Mr of 42,879. This predicted size was supported by minicell analysis. This gene was capable of complementing both Escherichia coli argE and argA mutations and of transforming an ArgJ- strain of N. gonorrhoeae to Arg+. Southern blots were able to detect bands that specifically hybridized to the gonococcal argJ gene in genomic DNA from Pseudomonas aeruginosa but not E. coli, a result that reflects the divergent nature of the arginine biosynthetic pathway in these organisms.

Molecular characterization of the argJ mutation in Neisseria gonorrhoeae strains with requirements for arginine, hypoxanthine, and uracil.

Arginine auxotrophs are commonly encountered among clinical isolates of Neisseria gonorrhoeae. Arginine auxotrophs which also require hypoxanthine and uracil (AHU strains) compose a unique set of strains that are highly homogeneous and are believed to be clonally derived. The Arg- phenotype of these strains is due to a lesion in the argJ gene encoding ornithine acetyltransferase. We have cloned the mutant argJ gene from an AHU strain and compared the sequence of this gene to the wild-type argJ gene. The mutant gene contained a 3-bp deletion within a repetitive region of the argJ gene. This mutation was restored to the wild-type sequence in a naturally occurring Arg+ revertant of the AHU strain. This deletion was detected in a wide variety of other AHU strains but not in other ArgJ- strains or in ArgJ+ strains, supporting the theory that AHU strains are clonally derived.

Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease.

Sequence of the argF gene encoding ornithine transcarbamoylase from Neisseria gonorrhoeae.

The gonococcal argF gene encoding ornithine transcarbamoylase (OTCase) contains an open reading frame of 993 nucleotides which starts with a GUG codon and encodes a peptide with a deduced Mr of 36,731. We compared the predicted amino acid (aa) sequence to OTCase sequences previously determined for Escherichia coli and Pseudomonas aeruginosa and found that highly conserved regions in the genes from these organisms were also conserved in Neisseria gonorrhoeae, including those aa known to be important for carbamoyl phosphate and ornithine binding. In the flanking regions of the gene were found 15-bp inverted repeats that may serve as transcriptional termination signals, and which contain the neisserial DNA-uptake sequence.

In vitro antibacterial activity of faeriefungin, a new broad-spectrum polyene macrolide antibiotic.

The in vitro activity of faeriefungin, a new pentaene macrolide lactone antibiotic produced by Streptomyces griseus var. autotrophicus, against 263 clinical bacterial isolates was examined. In contrast to the related fungicidal antibiotics nystatin and amphotericin B, which show no activity against bacteria, faeriefungin showed bactericidal activity against all species of gram-positive bacteria examined. MICs for these species ranged from 8 to 64 micrograms/ml, and the MIC for 90% of the isolates tested was 32 micrograms/ml. Isolates of some fastidious gram-negative species, including Neisseria gonorrhoeae, N. meningitidis, and Haemophilus influenzae, were slightly susceptible to faeriefungin, with MICs ranging from 16 to 128 micrograms/ml, but all members of the families Enterobacteriaceae and Pseudomonadaceae, with the exception of Pseudomonas cepacia, were completely resistant to faeriefungin at the concentrations tested. Faeriefungin is also active against fungi, nematodes, and mosquito larvae. The mode of action of faeriefungin against both bacteria and fungi is under investigation.

Effect of Pasteurella haemolytica-derived endotoxin on pulmonary structure and function in calves.

The role of endotoxin in the pathogenesis of acute pneumonic pasteurellosis is uncertain. Recently, we reported that Escherichia coli-derived endotoxin given by airway inoculation fails to induce lung injury in calves. Because Pasteurella haemolytica-derived endotoxin may differ substantially from E coli in its pathogenicity, we repeated these studies with Pasteurella endotoxin. Intratracheal inoculation of P haemolytica endotoxin caused hypoxemia and increased the alveolar-arterial oxygen differences without causing hypercarbia or changes in lung mechanical properties and volumes. In contrast, IV inoculation of endotoxin caused systemic hypotension, leukopenia, gas exchange impairment, increased total pulmonary resistance, and decreased dynamic compliance. Both routes of inoculation increased serum endotoxin concentrations and were associated with areas of pulmonary hemorrhage, edema, and acute inflammation. We concluded that P haemolytica-derived endotoxin is pathogenic by IV and airway routes of inoculation, and therefore differs from E coli endotoxin in its ability to induce lung lesions in calves.