The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level.

Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of alpha- and beta-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for alpha- and beta-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.

Field trials of a vaccine against bovine mastitis. 1. Evaluation in heifers.

A vaccine was developed against bovine mastitis based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated, unencapsulated Staph, aureus and Streptococcus spp. cells. This vaccine was tested on 30 heifers during a 7-mo period. The 30 heifers were randomly assigned to three groups of 10 heifers each. The prepartum group received two injections of the vaccine at 8 and 4 wk before calving, and the postpartum group received two injections at 1 and 5 wk after calving. The control group received two injections of a placebo at 8 and 4 wk before calving. The vaccine or the placebo was administered subcutaneously in the brachiocephalicus muscle of the neck. The frequencies of intramammary infections caused by Staph. aureus were reduced from 18.8% for heifers in the control group to 6.7 and 6.0% for heifers in the prepartum and postpartum groups, respectively. This protective effect was maintained for at least 6 mo. The relative risk of mastitis caused by Staph. aureus was 0.31 and 0.28 for heifers in the prepartum and postpartum groups, respectively, compared with that for heifers in the control group. The results of the trial indicated the effectiveness of the vaccine in decreasing the incidence of intrammammary infections caused by Staph. aureus. A slight but nonsignificant increase occurred in fat production in the milk of vaccinated cows. The vaccine had no observable effect on somatic cell count or streptococcal infections.

Field trials of a vaccine against bovine mastitis. 2. Evaluation in two commercial dairy herds.

A vaccine against bovine mastitis was developed. The vaccine was based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated unencapsulated Staph. aureus and Streptococcus spp. cells. In this study, the vaccine was evaluated in 164 cows from two commercial dairies (A and B) during a 4-mo period. Two doses of the vaccine were administered subcutaneously to 82 cows in the brachiocephalicus muscle of the neck within a 4-wk interval. The results of this trial revealed significantly fewer intramammary infections caused by Staph. aureus at various levels of severity (clinical, subclinical, and latent) in cows that were vaccinated. The odds ratios of all types of intrammammary infections caused by Staph. aureus for dairies A and B, which were determined by a logistic model, were 1.84 and 1.89, respectively, for quarters of vaccinated cows and quarters of control cows. The colony counts for Staph. aureus in milk from infected quarters of vaccinated cows were significantly lower than those in milk from infected quarters of control cows. Also, the somatic cell counts per milliliter in milk from vaccinated cows were significantly decreased when the initial somatic cell count was < 500,000 cells/ml at the start of the trial. The vaccine had no observable effect on fat production in milk or on streptococcal infections.

In vivo expression of exoprotein synthesis with a Sae mutant of Staphylococcus aureus.

The expression of exoprotein synthesis of Staphylococcus aureus Sae mutant RC121 and its parental strain was studied under in vivo growth conditions. Cultures of both strains were inoculated into dialysis sacs implanted in sheep peritoneum. Results indicated that similar to in vitro grown mutant cells, Sae mutant RC121 shows diminished synthesis of alpha- and beta-hemolysin, coagulase, DNase and protein A. However, in vitro and in vivo grown mutant cultures showed different exoprotein profiles in SDS-PAGE; some bands from in vivo mutant cultures were diminished or missing and others appeared as more concentrated, when compared with the pattern of the in vivo grown parental strain, while all the exoprotein bands from the in vitro cultures of the mutant were diminished or missing as compared to the in vitro grown parental strain. The virulence of the Sae mutant, assayed by intraperitoneal injection in mice, was lower than that of the parental strain after both in vivo and in vitro growth conditions.

Phenotypic characterization and virulence of a sae- agr- mutant of Staphylococcus aureus.

A sae::Tn551 agr::tetM double mutant was constructed and characterized. The production of several exoproteins (e.g., beta-hemolysin, DNase, and proteases) by this mutant was determined and found to be lower than the already diminished production of either isogenic single mutant sae- or agr-. The double mutant also showed, like the agr- mutant, null production of alpha- and delta-hemolysins and diminished levels of lipase. The reduced levels of many exoproteins in the double mutant as compared with their already diminished levels in either single mutant suggest that there is an additive or synergistic interaction between the two mutations involved, sae- and agr-. However, inactivation of both loci, sae and agr, had a different effect on the two exoproteins that are up regulated in the agr- mutant; thus, coagulase dropped to levels close to the null levels of the sae- parental strain, while extracellular protein A displayed the high levels characteristic of the agr- single mutant. The virulence of the sae- agr- double mutant, determined by intraperitoneal injection in mice, was found to be significantly diminished as compared with that of the sae+ agr+ parental strain or the sae- agr+ single mutant.

Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins.

A Tn551 insertional pleiotropic mutant defective in the production of several exoproteins was isolated from Staphylococcus aureus 196E and characterized. The pleiotropism of the mutant was due to a single insertion of the transposon as evidenced by Southern blot hybridization and by the transfer of its phenotype by transduction to S. aureus ISP479. The mutants showed diminished or null levels of alpha- and beta-hemolysis, DNase, coagulase, and protein A in the supernatants of broth cultures. Production of proteases, lipase, staphylokinase, or enterotoxin A was not modified. The mutants did synthesize the cell-bound form of protein A and also the extracellular form of this protein coded by pRIT11, which lacks the COOH-terminal segment of the molecule. These observations suggest that the sae locus does not involve a positive regulatory gene acting at the transcriptional level. The phenotype of the mutant was different from that of other insertional mutants affecting exoprotein synthesis, such as agr, xpr, or sar. This new mutation has been designated sae (for S. aureus exoprotein expression).

[Characterization of a transpositional mutant of Staphylococcus aureus underproducing exoproteins]. / Caracterización de una mutante transposicional de Staphylococcus aureus hipoproductora de exoproteínas.

A pleiotropic transpositional mutant derived from Staphylococcus aureus strain RC46, from bovine origin, was isolated and characterized. This mutant showed decreased production of several exoproteins such as alpha- and beta-hemolysins, DNase, coagulase and extracellular protein A. The production of cell-bound protein A, proteases and delta-hemolysin was not affected. The pleiotrophy of this mutant, designated sae (for S. aureus exoprotein expression), is due to a single insertion of transposon Tn917, as indicated by Southern blot hybridization. The sae mutant showed decreased virulence since its LD50 determined by intraperitoneal injection in mice was 32 times higher than that of the parental strain RC46.

Characterization of a Tn925-induced mutant of Staphylococcus aureus altered in exoprotein production.

An insertional mutant was isolated from a bovine Staphylococcus aureus strain after membrane-mating with a Streptococcus faecalis strain carrying conjugative plasmid pCF10::Tn925. This mutant, designated RC128, showed enhanced production of alpha-hemolysin and proteases and decreased production of coagulase, extracellular protein A, DNase, lipase and delta-hemolysin. No difference was found in the production of beta-hemolysin. Both, Southern blot analysis and transfer of the pleiotropic mutant phenotype by transduction, indicated that the mutation was originated from a single insertion of transposon Tn925. The LD50 determined by intraperitoneal administration in mice showed that mutant RC128 was slightly less virulent than its parental strain.

Identification of a nucleotide deletion in parts of polypeptide 3A in two independent attenuated aphthovirus strains.

A set of antisera specific for each viral polypeptide of foot-and-mouth disease virus was used to provide a full comparison of polypeptides of two strains attenuated for cattle with respect to their parental virulent strains. Both attenuated strains, belonging to serotypes O1 Campos and C3 Resende, were obtained through serial passages of the corresponding virulent strains in chicken embryos. Although mutations were scattered throughout the genome, both attenuated strains showed an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral polypeptide 3A faster than that of their respective wild-type strains. To determine the nature of this alteration, the nucleotide sequences of the genomic region encoding this polypeptide were determined. Comparative sequence analysis of wild-type and attenuated strains revealed 57 and 60 nucleotide deletions in the attenuated strains O1 Campos and C3 Resende, respectively. These studies, in conjunction with our previous analysis of recombinant viruses between wild-type and attenuated strains, which concluded that the major determinants of attenuation are located in the 3' half of the viral genome, strongly suggest that the alteration in polypeptide 3A of the attenuated strains is important for their reduced virulence in cattle.

Behavior of intertypic recombinants between virulent and attenuated aphthovirus strains in tissue culture and cattle.

Two aphthovirus intertypic recombinants between the virulent strain A Venceslau and guanidine-resistant attenuated mutants of either strain C3 Resende or O1 Campos were obtained in an attempt to establish the region(s) of the viral genome responsible for attenuation in cattle. Recombinants that inherited the 3' half of the genome from either attenuated parent and the 5' half from the virulent strain were selected and analyzed with respect to their ability to grow in cells of bovine origin and for their virulence in cattle. The results obtained support our previous conclusion, derived from studies with homotypic recombinants between attenuated aphthovirus type O1 and its original virulent strain, that the host range restriction phenotype for fetal bovine kidney cells of the attenuated strain is inherited from the 3' half of the genome. For the intertypic recombinants, however, this restriction is enhanced, presumably by the presence of a heterologous 5' half of the genomic region. In addition, we demonstrate that the results in vitro correlate with those of virulence tests in cattle.

Biochemical characterization of an aphthovirus type C3 strain Resende attenuated for cattle by serial passages in chicken embryos.

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Isolation and characterization of recombinants between attenuated and virulent aphthovirus strains.

A guanidine-resistant mutant of the attenuated strain of aphthovirus type 01 strain Campos and the original wild-type strain were crossed to generate recombinant viruses. Two independently derived recombinant viruses were isolated. One isolate (RI) contained the P1 (structural proteins) gene region of attenuated strain and P3 (polymerase precursor) gene region of the wild-type strain. The other isolate (RII) had a genomic structure complementary to that of RI, this is, P1 of the wild-type strain and P3 of the attenuated virus. Recombinant RII inherited some in vitro phenotypic markers that were characteristic of the attenuated strain, whereas the RI recombinant had in vitro behavior that was similar to that of the wild-type strain. The data obtained suggest that the polymerase precursor (P3) of the attenuated strain (01 Campos) could be involved in the determination of the attenuated phenotype for fetal bovine kidney cells and, eventually, for cattle.