Alterations of the oxygen-evolving apparatus in a (448)Arg --> (448)S mutant in the CP47 protein of photosystem II under normal and low chloride conditions.

We have shown previously that a mutant which contained the alteration (448)R --> (448)S (R448S) in the CP47 protein of photosystem II exhibited a defect in its ability to grow and assemble functional photosystem II reaction centers under chloride-limiting conditions [Wu, J., Masri, N., Lee, W., Frankel, L. K., and Bricker, T. M. (1999) Plant Mol. Biol. 39, 381-386]. In this paper we have examined the function of the oxygen-evolving complex under chloride-sufficient (480 microM) and chloride-limiting (< 20 microM) conditions. When placed under chloride-limiting conditions, both the control strain K3 and R448S cells exhibit a loss of steady-state oxygen evolution, with t(1/2) of 16 and 17 min, respectively. Upon the addition of chloride, both recover their oxygen-evolving capacity relatively rapidly. However, R448S exhibits a much slower reactivation of oxygen evolution than does K3 (t(1/2) of 308 and 50 s, respectively). This may indicate a defect at the low-affinity, rapidly exchanging chloride-binding site [Lindberg, K., and Andréasson, L.-E. (1996) Biochemistry 35, 14259-14267]. Additionally, alterations in the distribution of S states and S-state lifetimes were observed. Under chloride-sufficient conditions, the R448S mutant exhibits a significant increase in the proportion of reaction centers in the S(3) state and a greatly increased lifetime of the S(3) state. Under chloride-limiting conditions, the proportion of reaction centers in both the S(2) and S(3) states increases significantly, and there is a marked increase in the lifetime of the S(2) state. These alterations are not observed in the control strain K3. Our observations support the hypothesis that (448)R of CP47 may participate in the formation of the binding domain for chloride in photosystem II and/or in the functional interaction with the 33 kDa protein with the photosystem.

Survival of Staphylococcus aureus inside neutrophils contributes to infection.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

Optical testing of condoms.

Condoms provide one of the most important means of preventing pregnancy and the spread of human immunodeficiency virus (HIV) and other sexually transmitted diseases. Pinhole defects may lead to the passage of sperm or viruses through the condom wall. Embedded particles, which may become dislodged in handling or use, may represent latent pinhole defects. Thin regions in the condom wall may lead to breakage in use. Testing for such defects in regulatory laboratories, or in the factories as part of production screening or quality assurance efforts, is a major tool for ensuring condom reliability. A new optical method for testing condoms is presented, sensitive to pinholes, thin regions, and embedded particles. The method could supplement or replace the existing, less powerful, electrical test techniques in either regulatory testing laboratories or in the factory. The optical techniques presented provide quantitative data on the condoms tested, rather than just accept/reject decisions. They thus support detailed monitoring of production or lot characteristics that is generally not possible with existing electrical techniques. The ability of the optical techniques to separately test for and identify not only pinholes, but also thin regions and embedded particles, which are not separately tested for with the electrical technologies, is an especially important new capability. Further, the new optical techniques allow the locations of defects found to be precisely marked, making possible a microscopic examination of defects useful in studying their structure and possible causes. The optical technique is also totally nondestructive, in that it does not alter the defects whose structures are of interest. Initial results are reported on the testing of latex condoms purchased in retail stores. Condoms identified optically as being potentially defective were subjected to industry standard leak and burst testing. Results of the various tests are summarized. The data suggest that use of the new optical test techniques could have prevented an unexpectedly large number of condoms with significant pinhole and thin region defects from reaching the public. Optical test systems could be used to support either regulatory, quality assurance, or clinical field testing of latex or nonlatex condoms. Due to their high rate capability and level of automation, they could also be applied to 100% screening testing in the factory. The new test techniques could also be used to detect defects in a wide range of other thin sheets and membranes.

PIP: Testing of latex condoms in factories and regulatory laboratories for pinhole defects, embedded particles, and thin regions represents a major strategy for ensuring condom reliability. A new optical method for testing condoms could supplement or even replace existing, less powerful electrical test techniques that require extensive manual handling. Optical techniques provide quantitative data on the condoms tested rather than simple accept/reject decisions, thereby enabling detailed monitoring of production or lot characteristics. This paper presents initial findings on the optical testing of 1813 nonlubricated latex condoms purchased in US retail stores. An extremely high level of correlation was found between the optical test results and the outcomes of standard leak-and-burst testing. Optical screening followed by leak testing produced 12 confirmed failures in this series. The majority of confirmed leaks resulted from holes substantially larger than the detection limits of both testing systems. The large pinhole and thin-region defects identified optically are of serious public health concern, even if the defects were incipient and only realized after handling.

Spontaneous elaboration of transforming growth factor beta suppresses host defense against bacterial infection in autoimmune MRL/lpr mice.

Infection with gram-negative and gram-positive bacteria remains a leading cause of death in patients with systemic lupus erythematosis (SLE), even in the absence of immunosuppressive therapy. To elucidate the mechanisms that underly the increased risk of infection observed in patients with systemic autoimmunity, we have investigated host defense against bacterial infection in a murine model of autoimmunity, the MRL/Mp-lpr/lpr (MRL/lpr) mouse. Our previous study implicated transforming growth factor beta (TGF-beta) in a novel acquired defect in neutrophil function in MRL/lpr but not congenic MRL/Mp-+/+ (MRL/n) mice (Gresham, H.D., C.J. Ray, and F.K. O'Sullivan. 1991. J. Immunol. 146:3911). We hypothesized from these observations that MRL/lpr mice would have defects in host defense against bacterial infection and that they would have constitutively higher local and systemic levels of active TGF-beta which would be responsible, at least in part, for the defect in host defense. We show in this paper that spontaneous elaboration of active TGF-beta adversely affects host defense against both gram-negative and gram-positive bacterial infection in MRL/lpr mice. Our data indicate that MRL/lpr mice, as compared with congenic MRL/n mice, exhibit decreased survival in response to bacterial infection, that polymorphonuclear leukocytes (PMN) from MRl/lpr mice fail to migrate to the site of infection during the initial stages of infection, that MRL/lpr mice have a significantly increased bacterial burden at the site of infection and at other tissue sites, and that this increased bacterial growth occurs at a time (> 20 h after infection) when PMN influx is greatly enhanced in MRL/lpr mice. Most intriguingly, the alteration in PMN extravasation during the initial stages of infection and failure to restrict bacterial growth in vivo could be duplicated in MRL/n mice with a parenteral injection of active TGF-beta 1 at the time of bacterial challenge. Moreover, these alterations in host defense, including survival in response to lethal infection, could be ameliorated in MRL/lpr mice by the parenteral administration of a monoclonal antibody that neutralizes the activity of TGF-beta. These data indicate that elaboration of TGF-beta as a result of autoimmune phenomenon suppresses host defense against bacterial infection and that such a mechanism could be responsible for the increased risk of bacterial infection observed in patients with autoimmune diseases.

Staphylococcus aureus microcapsule expression attenuates bacterial virulence in a rat model of experimental endocarditis.

The virulence of the Staphylococcus aureus strains that differed only in capsule expression was compared in a rat model of catheter-induced experimental endocarditis. The ID50 of all the strains was low (less than 3 x 10(3) cfu of S. aureus), suggesting that this model may be more sensitive than other animal models to differences in bacterial virulence. Compared with the wild-type strains that expressed type 5 or type 8 capsular polysaccharides, mutant strains devoid of capsule had significantly lower ID50 values. In contrast, a mutant that produced scant amounts of the type 5 polysaccharide had an ID50 similar to that of the parental type 5 isolate. As the bacterial inoculum was increased, each of the S. aureus strains reached final concentrations of 10(10)-10(11) cfu/g of vegetation; however, the nonencapsulated mutants colonized the left-sided vegetations at lower inocula than did the wild-type strains. This study indicates that microcapsule expression attenuates bacterial virulence in a rat model of catheter-induced endocarditis.

The role of fibronectin binding in the rat model of experimental endocarditis caused by Streptococcus sanguis.

Inactivation of fibronectin (Fn) binding by insertional mutagenesis of Streptococcus sanguis with Tn916 reduces virulence of this bacterium in the rat model of infective endocarditis (IE). Transconjugants were screened for Fn adherence using an ELISA adherence test. One transconjugant had a decreased adherence to immobilized Fn. Southern hybridization demonstrated that the insertion occurred only once in this mutant. The parent strain and mutant strain JL113 were used as challenge strains in a rat endocarditis model. These experiments demonstrated that the mutant had a reduced ability (P less than 0.05) to produce IE. Spontaneous excision of Tn916 from JL113 produced strains identical to both the parental and mutant phenotypes. One strain (JLR-19) that retained the mutant phenotype and one (JLR-15) that regained the parental phenotype for Fn binding were tested for their ability to produce IE. These strains demonstrated that the ability to bind Fn and to produce IE were correlated after Tn916 excision. The reduced virulence of the mutant suggested that adherence of S. sanguis to immobilized Fn plays an important role in the production of IE.

Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to beta-lactam antibiotics, and virulence.

The growth of Staphylococcus epidermidis sensu stricto and Staphylococcus saprophyticus on Memphis agar yielded up to 6 morphotypes with each strain. With S. epidermidis, one morphotype produced slime (rho) but became non-slime-producing (epsilon) at a high frequency. The slime-producing rho variants were methicillin-resistant and more virulent than methicillin-susceptible epsilon variants in an endocarditis model. With S. saprophyticus, phase variation was of higher frequency. Nitrosoguanidine mutagenesis produced a stable blue epsilon form that was more virulent than the parent in a mouse model of urinary tract infection. Mutants with the blue epsilon phenotype differed from gold epsilon parents in a variety of phenotypic properties, including increased resistance to oxacillin. These staphylococcal species have a high frequency of phase variation: Phase variants differ in antibiotic resistance and virulence, which is only partially correlated with suggested virulence factors such as slime production.

Molecular epidemiology of nosocomial, multiply aminoglycoside resistant Enterococcus faecalis.

The prevalence of high-level aminoglycoside resistance among Enterococcus faecalis at the Memphis VA Medical Center was 23.6% (59 of 250 isolates) from October to December 1986. Hybridization to a probe cloned from Ent. faecalis pIP1800 for 6' acetyltransferase-2" phosphotransferase (AAC6'-APH2") was observed in 55 (93.2%) of the resistant isolates and was associated with gentamicin resistance. Hybridization to a probe cloned from Ent. faecalis pJH1 for 3', 5" phosphotransferase type III (APH3', 5" III) was observed in 28 (47.4%) and was associated with streptomycin resistance. Twenty-five of the 32 isolates which were resistant to both gentamicin and streptomycin hybridized to both probes. Cell mating in conjunction with hybridization indicated that the AAC6'-APH2" gene is transferred separately from that for APH3', 5" (III), and the streptomycin resistant gene is cotransferred with the latter. The gentamicin-streptomycin resistant isolates therefore contain genes from two Ent. faecalis plasmids, and resistance to these two antibiotics appears to transfer separately. The genetic homogeneity of these isolates suggests nosocomial transmission of enterococci.

Pathogenesis of experimental endocarditis.

Numerous important observations concerning the pathogenesis of infective endocarditis have been made over the past 18 years. Many of these observations have been obtained with animal models of endocarditis in which an indwelling catheter has been used to produce predisposing endocardial lesions for subsequent infection. Unlike several previously used animal models, the catheter-induced endocarditis models have many characteristics similar to infective endocarditis in humans. Key findings regarding microbial adherence and persistence and the host response to endocardial infection have led to a better appreciation of the complex issues operative in the pathogenesis of endocarditis.

Adherence of Streptococcus sanguis to conformationally specific determinants in fibronectin.

The adherence of Streptococcus sanguis to specific receptors exposed or deposited at the site of endothelial damage may play an important role in the development of infective endocarditis. Adherence of the Challis strain of S. sanguis to gelatin (or collagen) and gelatin-binding components of plasma was examined with an enzyme-linked immunosorbent assay. S. sanguis adhered poorly to immobilized gelatin and to molecular or fibrillar collagen. However, in the presence of fresh human plasma, the adherence of S. sanguis to all three substrates increased as much as eightfold. Removal of gelatin-binding proteins eliminates the ability of plasma to enhance adherence of S. sanguis to the substrates. Addition of purified human plasma fibronectin (Fn) to the absorbed plasma restored the adherence-promoting ability in a dose-dependent manner. A similar dose-dependent increase in S. sanguis adherence was observed when increasing concentrations of Fn alone were added to the gelatin-coated assay wells. S. sanguis adherence to immobilized fibronectin could not be inhibited by preincubating either the bacteria or the gelatin-coated assay wells with Fn or by including excess soluble Fn in the assay mixture. Studies with peptides purified from trypsin digests of Fn indicated that the 160- to 180-kilodalton (kDa) fragments which retain both the gelatin-binding and the cell-binding regions of the intact molecule support adherence of S. sanguis to gelatin. The 160- to 180-kDa fragments inhibited the interaction of S. sanguis with immobilized Fn. In contrast, intact Fn and the 31-kDa amino-terminal fragment were unable to inhibit the adherence when used in equivalent or greater molar amounts. These in vitro results suggest that in the presence of whole plasma, S. sanguis binds to immobilized gelatin or collagen via Fn bound to the immobilized substrates. Our finding that adherence of S. sanguis to immobilized Fn can occur in the presence of large concentrations of Fn, whether in plasma or purified, indicates that a S. sanguis-binding domain is cryptic in the Fn molecule while in solution and is exposed by a conformational change when the Fn becomes bound to gelatin-coated plastic. The ability of peptide fragments of Fn to inhibit S. sanguis adherence is consistent with this hypothesis.