Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica.

Pasteurella haemolytica biotype A serotype 1 is the principal etiologic agent of bovine pneumonic pasteurellosis. A clear understanding of the pathogenesis of this disease and the mechanisms of resistance to it has been limited by a lack of information on the important antigens of the organisms. Using recombinant DNA techniques we have cloned a segment of DNA from P. haemolytica A1 that encodes three proteins of 28, 30, and 32 kDa. Two of these proteins, 30 and 28 kDa, react strongly on a Western blot with a bovine serum raised against live cells of P. haemolytica A1. The gene for the 30 kDa protein was localized to a 3.1 kbp EcoRI fragment, and expression of the 30 kDa protein was found to be independent of an E. coli promoter. The 30 kDa protein comigrated with a 30 kDa P. haemolytica protein that was susceptible to radioiodination and presumably exposed on the bacterial cell surface. The other principal radiolabeled P. haemolytica proteins were 100, 45, and 15 kDa. Antibodies against the 30 kDa protein, isolated from E. coli carrying the recombinant plasmid, recognized 30 kDa and 15 kDa proteins in P. haemolytica serotypes 1-15 and caused agglutination of whole P. haemolytica A1 cells. Cattle vaccinated with live P. haemolytica, P. haemolytica outer membrane proteins, or the cloned 30 kDa protein developed antibodies to the cloned 30 kDa protein as detected by Western blotting and densitometry. Sera were obtained from cattle vaccinated with live or killed P. haemolytica or saline and challenged with P. haemolytica. Those sera were evaluated for antibody responses to the cloned 30 kDa protein. High antibody responses to the 30 kDa protein significantly correlated (P less than 0.01) with resistance to challenge. From these studies it is concluded that the 30 kDa protein represents a surface antigen of P. haemolytica A1 that may be important in inducing immunity to P. haemolytica.

Identification of a potentially important antigen of Pasteurella haemolytica.

To identify antigens which may be important for stimulating immunity to pneumonic pasteurellosis, a bovine antiserum to whole P. haemolytica was used to screen a recombinant lambda gt11/P. haemolytica expression library. One of the recombinant bacteriophage clones identified with the bovine antiserum, SW20C, expressed a fusion protein which was also recognized by rabbit antiserum to partially purified P. haemolytica culture supernatant and was found to be immunogenic in guinea pigs. The guinea pig antibody recognized a 100 kDa protein in P. haemolytica cell lysates. Sequence analysis of the cloned DNA from SW20C identified a fragment of 1446 bp with a small open reading frame that was contiguous with the lacZ sequence. The 153 bp P. haemolytica-specific open reading frame encoded a polypeptide of approximately 6 kDa. Homology searches of Genbank and the EMBL data bases revealed no homology of this open reading frame with any other bacterial sequences including P. haemolytica leukotoxin and Ssa1. Evaluation of sera from calves that were scored either susceptible or resistant to experimental pneumonic pasteurellosis demonstrated a significant (P < 0.001) correlation between the intensity of the antibody response to the SW20C antigen and resistance to disease.

Relationship between the care index and mean dmft/DMFT.

OBJECTIVE: To assess the relationship between the care index and the mean dmft/DMFT in different populations of 5-, 12- and 14-year old children. DESIGN: Information from recent studies of 5-, 12- and 14-year old children, co-ordinated by the British Association for the Study of Community Dentistry (BASCD) was used. The correlation between the mean dmft/DMFT and the care index for each health district was calculated for each age group, along with the linear regression equation of the care index in terms of dmft/DMFT. RESULTS: All the correlation coefficients were negative indicating that the care index would be expected to increase as the disease levels decrease. For the 5-year-old children in 1995/96 three other factors for the same time period; dentist:population ratio, percent of children registered and standardised mortality ratio (SMR), were also included in the model. This did not change the negative relationship between dmft and the care index. CONCLUSIONS: The strong negative relationship between the care index and mean dmft/DMFT levels indicated that in a situation where disease levels are reducing over time, the care index would be expected to increase. Conversely in a situation where disease levels were increasing the care index would be expected to fall.

A campaign encouraging dental attendance among adolescents in Scotland: the barriers to behaviour change.

Qualitative consumer research was used to develop a health promotion campaign for school pupils aged 15-17 years to encourage them to attend a dentist for examination. The campaign used a combination of conventional health education about the benefits of dental care together with incentives for attending. The emphasis throughout was to establish an association with young style and group norms of social attractiveness. This study was part of the evaluation of the campaign. The aim was to identify the characteristics of those who responded positively to the campaign and to identify barriers to behaviour change. Those who responded were mainly female, intended to stay on at school beyond the age of 16 years and were more likely to be frequent attenders. Apathy and a lack of felt need were the main barriers to responding. Easier access to care and targeting a younger age group might enhance the success of similar interventions.

A survey of job stress and job satisfaction among DSAs in the north-west of England.

This study was a survey of job stress and job satisfaction among DSAs in general practice in the north-west of England during 1993. The results suggest that severe overall job stress and dissatisfaction were not prevalent but do present an important problem for a minority. The chief sources of stress are ranked. Those which caused moderate to severe stress were: running behind time, feeling under-valued by the dentist and handling difficult patients. Those experiencing greater stress outside work were more likely to report stress within it. Having a regular staff meeting, an annual salary review and a clear job description were associated with significantly less job stress.

Diagnosis and management of toothwear in two children in the same family: a case report.

The incidence of toothwear would appear to be on the increase in children. The main aetiological factor seems to be the increasing consumption of acidic drinks. This paper reports on a sister and brother with erosion whose parents were reluctant to accept that drinking fruit juice through a straw could have resulted in the damage their children's teeth sustained.

Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants.

The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.

Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly.

The retroviruses of the avian sarcoma-leukosis virus group synthesize their viral protease (PR) in two precursor forms--as a carboxy-terminal domain of the Gag precursor and as an embedded domain within the Gag-Pol precursor. We have shown previously that the Gag-derived PR is fully capable of processing the Gag precursor in the absence of the embedded PR (R.P. Bennett, S. Rhee, R.C. Craven, E. Hunter, and J.W. Wills, J. Virol. 65:272-280, 1991). In this study, we examined the question of whether or not the PR domain of Gag-Pol has an essential role in the maturation of the Pol proteins. The Gag-Pol precursor was expressed in the absence of Gag by use of a simian virus 40-based vector in which the gag and pol reading frames were fused. The fusion protein accumulated to high levels in transfected cells without being released into the medium but could be rescued into particles by coexpression of the Gag protein from a second vector. The resulting particles contained mature Gag and Pol proteins and active reverse transcriptase (RT). Using this complementation system, the effects of PR defects in the Gag and/or Gag-Pol proteins on the activation of RT were examined. The results showed that the presence of a functional PR on the Gag precursor, but not on Gag-Pol, was required for full activation of RT. The embedded PR of Gag-Pol was unable to carry out any detectable processing of the Gag precursor and was able to activate RT to only a low level in the absence of a functional Gag PR domain. Finally, some point mutations in the Gag-Pol PR domain inhibited activation of RT in trans by a wild-type PR, suggesting that the correct conformation of the PR domain in Gag-Pol is prerequisite for activation of RT.

Effect of repeated in vitro transfer of Pasteurella haemolytica A1 on encapsulation, leukotoxin production, and virulence.

Pasteurella haemolytica serotype 1 was transferred daily for 128 serial passages on both unsupplemented brain heart infusion agar and the same basal medium supplemented with bovine blood, horse serum, and yeast extract. Repeatedly transferred cultures were shown to retain the ability to produce both capsular material and leukotoxin. Furthermore, intact organisms were found to be as toxic in vitro for bovine leukocytes and as virulent for mice as unpassaged cultures. These results indicate that the precaution of using only freshly isolated cultures in the study of this organism may not be necessary.

Flagellar preparations from Pseudomonas aeruginosa: isolation and characterization.

Flagella from various strains of Pseudomonas aeruginosa were isolated by shearing the flagella followed by differential centrifugation to obtain typical filaments as viewed through an electron microscope. Electrophoretic analysis showed a major protein band corresponding to a flagellin with molecular weight of 53,000. Among the strains tested, flagellar antigen (FAg) preparations isolated from strains 1244 and 1210 routinely gave the highest percentage of flagellin, with the least amount of protein impurities, when grown on succinate-mineral salts medium. All FAg preparations contained 3 to 10 micrograms of 2-keto-3-deoxyoctonate-positive material per mg of protein. Strain PA-103 lacked flagella and exhibited no flagellin band, and preparations from PA-103 had a relatively higher content of 2-keto-3-deoxyoctonate. The isolation of highly purified, single-banded flagellin could be accomplished by elution of the 53,000-molecular weight gel band. Amino acid analysis showed 16 amino acids, but no proline. Antisera to FAg preparations were used to demonstrate inhibition of motility of strains RM-46 and M-2. Heated RM-46 FAg antisera and PA-103 antisera did not inhibit motility.

Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells.

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.

Second-site suppressors of Rous sarcoma virus Ca mutations: evidence for interdomain interactions.

The capsid (CA) protein, the major structural component of retroviruses, forms a shell that encases the ribonucleoprotein complex in the virion core. The most conserved region of CA, approximately 20 amino acids of the major homology region (MHR), lies within the carboxy-terminal domain of the protein. Structural and sequence similarities among CA proteins of retroviruses and the CA-like proteins of hepatitis B virus and various retrotransposons suggest that the MHR is involved in an aspect of replication common to these reverse-transcribing elements. Conservative substitutions in this region of the Rous sarcoma virus protein were lethal due to a severe deficiency in reverse transcription, in spite of the presence of an intact genome and active reverse transcriptase in the particles. This finding suggests that the mutations interfered with normal interactions among these constituents. A total of four genetic suppressors of three lethal MHR mutations have now been identified. All four map to the sequence encoding the CA-spacer peptide (SP) region of Gag. The F167Y mutation in the MHR was fully suppressed by a single amino acid change in the alpha helix immediately downstream of the MHR, a region that forms the major dimer interface in human immunodeficiency virus CA. This finding suggests that the F167Y mutation indirectly interfered with dimerization. The F167Y defect could also be repaired by a second, independent suppressor in the C-terminal SP that was removed from CA during maturation. This single residue change, which increased the rate of SP cleavage, apparently corrected the F167Y defect by modifying the maturation pathway. More surprising was the isolation of suppressors of the R170Q and L171V MHR mutations, which mapped to the N-terminal domain of the CA protein. This finding suggests that the two domains, which in the monomeric protein are separated by a flexible linker, must communicate with each other at some unidentified point in the viral replication cycle.

Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein.

The mature cores of all retroviruses contain a major structural protein known as the CA (capsid) protein. Although it appears to form a shell around the ribonucleoprotein complex that contains the viral RNA, its function in viral replication is largely unknown. Little sequence similarity exists between the CA proteins of different retroviruses, except for a region of about 20 amino acids termed the major homology region (MHR). To examine the role of the CA protein in particle assembly and release, mutants of Rous sarcoma virus were created in which segments of CA were deleted or single conserved residues in the MHR were altered. The ability of the deletion mutants to release particles at rates similar to the wild-type protein demonstrated that the CA domain of Gag is not an essential component of the minimal budding machinery. Certain point mutations in the MHR region did block assembly and release in certain cell types, presumably by perturbing the global structure of the Gag precursor. Another group of MHR substitutions produced noninfectious or poorly infectious particles that were normal in their content of gag and pol gene products and viral RNA. The mutants were capable of initiating reverse transcription in vitro; however, the association of CA protein with the core was compromised, as indicated by its sensitivity to extraction with nonionic detergent. Prominent blebs on the virion envelope also indicated a disturbance at the membrane. Finally, an anti-peptide serum directed against MHR was found to react with the uncleaved Gag protein but not with mature CA, suggesting that MHR undergoes a dynamic rearrangement upon liberation from the polyprotein. We conclude that the MHR is involved in the very late steps in maturation of the virion (i.e., ones that occur after budding is initiated) and is essential for proper function of the core upon entry into a new host cell.

Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly.

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the active protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

Rous sarcoma virus direct repeat cis elements exert effects at several points in the virus life cycle.

Two approximately 135-nucleotide (nt) direct repeats flank the Rous sarcoma virus (RSV) oncogene src and are composed of two smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt). These sequences have been reported to contain cis-acting signals necessary for RNA packaging and elements that allow cytoplasmic accumulation of unspliced RNA (cytoplasmic transport elements). In this report, we show that avian fibroblasts infected with the Prague A strain of RSV with precise deletions of both dr1 elements express src and are transformed by this mutant virus but production of virus particles is very low and virus spread throughout the culture requires several weeks. We show that the replication defect is due to complex effects on viral RNA transport, viral RNA half-life, and virus particle assembly. The dr1 elements may contain binding sites for a permissive cell-specific factor(s) that facilitates efficient nuclear-cytoplasmic transport, RNA stability, and cytoplasmic utilization of unspliced viral RNA. The implications of these results for understanding the defects of nonpermissive virus infections in mammalian cells are discussed.

Loss of virulence associated with absence of flagellum in an isogenic mutant of Pseudomonas aeruginosa in the burned-mouse model.

To test the importance of flagella as a virulence factor, virulent strain Pseudomonas aeruginosa M-2 was mutagenized, and a Fla mutant was isolated. An M-2 Fla spontaneous motile revertant was isolated from M-2 Fla. The three strains were biochemically and morphologically identical. Assay of Fla in the burned-mouse model showed a severe loss of virulence. The Fla revertant was fully virulent. The importance of P. aeruginosa motility (flagella) for virulence in burns infections is indicated.