Certain methicillin-resistant Staphylococcus aureus strains contain a 230-kDa cell-wall protein which is not present on the surface of other staphylococci. The presence of this 230-kDa protein is associated with a negative test result in commercial assays designed to detect fibrinogen-binding proteins andlor protein A on the staphylococcal surface. We have purified and partially characterised the 230-kDa protein from a lysostaphin digest of a non-agglutinating methicillin-resistant S. aureus strain. Partial amino acid sequence data obtained from the purified protein did not reveal any significant similarities to known proteins which indicates that the protein is novel. The 230-kDa protein was very sensitive to proteolysis ; soluble plasmin, or plasmin formed on the bacterial-cell surface, rapidly degraded the 230-kDa protein to a 175-kDa form. The finding that the 230-kDa protein bound to lectins allowed its purification by affinity chromatography on immobilised wheat germ agglutinin. Furthermore, the degradation of the 230-kDa protein was associated with an increased adherence of non-agglutinating methicillin-resistant S. aureus cells to solid-phase fibronectin, fibrinogen or IgG.Keywords: Staphylococcus aureus ; bacterial adherence; surface proteins ; plasminogen ; plasmin.Staphylococcus aureus posesses several well-characterised surface molecules including protein A, fibrinogen-binding proteins and proteins that bind to the extracellular-matrix components fibronectin, laminin and collagen [I -61. These surface proteins have been suggested to mediate the adherence of S. aureus to eukaryotic cells and tissues which is an important initial event in bacterial infection. In addition, receptors for plasminogen (the precursor of the serine protease plasmin) have been reported on several bacteria such as p-hemolytic streptococci, S. aureus, Escherichia coli, Neisseria meningitidis and Neisseria gonorrhoeae [7 -121. Via these receptors the bacterial cells acquire a surface-associated plasmin activity which plays a role in microbial invasion and penetration of the extracellular matrix in vitro [9, 11 -1 31. Yersinia pestis pathogenicity has been shown to be linked to a plasmid-borne gene, pla, which encodes a plasminogen-activator protein [14]. Fimbria and flagella of E. coli, and certain M-proteins of group A streptococci, have been shown to be receptors for plasminogen [13,15, 161. Staphylococcal plasminogen receptors have not been characterised biochemically.The methicillin-resistant S. aureus strains are characterised by their resistance to a group of penicillins that was designed to avoid the effect of P-lactamase, an enzyme which destroys the p-lactam ring of the penicillin molecule [17]. The methicillin resistance is due to the production of an additional penicillinbinding protein, PBP2' or PBP2a, which has a greatly reduced affinity towards p-lactam antibiotics [IS]. PBP2' is encoded by the mecA gene but additional factors, such as the product of femA gene, are involved in the methicillin-resistant phenotype [1...

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15؇C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15؇C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nmdiameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.

Seventy-nine methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated during 1980 to 1990, were classified as MRSA Aggl-(14 strains) and MRSA Aggl+ (65 strains) strains on the basis of test results in slide agglutination assays designed to detect fibrinogen-binding protein (clumping factor) and protein A on the staphylococcal surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that lysostaphin digests of MRSA Agglstrains contained a high-molecular-weight protein which was not detected in digests of MRSA Aggl+ strains. Immunization of rabbits with an MRSAAggl strain produced an antiserum which agglutinated all MRSA Aggl strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68 coagulase-negative staphylococci showed agglutination in this assay. The anti-MRSA Aggl antiserum reacted mainly with a 230-kDa staphylococcal surface protein but also with a 175-kDa protein, probably formed by proteolysis of the former and a few slightly smaller proteins. These could not be immunologically detected in lysostaphin digests of MRSA Aggl+ strains. Purified antibodies reacting with the 230-kDa protein agglutinated all MRSA Agglstrains, indicating that the protein is located on the surfaces of staphylococci. The results suggest a tentative role for the 230-kDa protein or its fragments as a novel target to develop more efficient rapid identification methods for S. aureus, including MRSA.

We have studied the interactions of the G1 and G2 membrane glycoproteins of Uukuniemi virus, a bunyavirus, in virus particles and in Triton X-100-solubilized virus. The G1 glycoprotein in intact virus or in Triton solution could be oxidized into a covalent homodimer using Cu2+ ion as a catalyst. Immunoprecipitations of the glycoproteins from Triton-solubilized virus lysates showed that G1 and G2 do not form a stable heterodimeric or heterooligomeric complex. The oligomeric association of G1 and G2 was further analyzed using centrifugation in sucrose gradients in the presence of Triton X-100. The results indicate that G1 exists as a Triton-resistant pH-insensitive homodimer. This is in contrast to the behavior of G2, which exists as a homodimer and partially as a monomer at pH 6.4 or above and is dissociated completely into a monomer at pH 6.0 or below. The threshold for the dimer-monomer shift of G2 is between pH 6.2 and pH 6.0. Electron microscopy studies show that the surface structure of the virus particle undergoes a pH-dependent change. Studies on the kinetics of virus entry suggest that pH below 6.2 is necessary for the penetration of Uukuniemi virus.