Keiko Seki

Role of Fibronectin-Binding Proteins A and B in In Vitro Cellular Infections and In Vivo Septic Infections by Staphylococcus aureus

ABSTRACT Fibronectin-binding protein A (FnBPA) and FnBPB are important adhesins for Staphylococcus aureus infection. We constructed fnbA and/or fnbB mutant strains from S. aureus SH1000, which possesses intact rsbU, and studied the role of these adhesins in in vitro and in vivo infections. In intravenous infection, all fnb mutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice. fnbB mutant caused a more severe decrease in body weight than that caused by fnbA mutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced in fnbA or fnbA fnbB mutant infections; however, there was no significant reduction in fnbB mutant infections. In in vitro cellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important for in vitro and in vivo infections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.

Inhibition of Endothelial Interleukin-8 Production and Neutrophil Transmigration by Staphylococcus aureus Beta-Hemolysin

ABSTRACT Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-α). Migration of neutrophils across TNF-α-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.

Occurrence of Coagulase Serotype among Staphylococcus aureus Strains Isolated from Healthy Individuals -Special Reference to Correlation with Size of Protein-A Gene-

One‐hundred‐and‐nineteen strains of Staphylococcus aureus isolated from healthy individuals for 3 years between 1991 and 1993 were subjected to an investigation on the producibility of proteins including protein A, coagulase, enterotoxins and toxic‐shock syndrome toxin‐1. Especially, protein A was the center of our interest. Among these strains, 69, 43, 3 and 1 strains were found to have the protein‐A gene containing 5, 4, 3 and 2 IgG‐binding domains, respectively. On the other hand, only one strain was devoid of the protein‐A gene. There were some differences in the profile of the coagulase serotype between the group with 4 IgG‐binding domains and that with 5 IgG‐binding domains. Differences in the profile of toxin production were also observed between the two groups.

Conspicuous Ingestion of Staphylococcus aureus Organisms by Murine Fibroblasts In Vitro

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts‐adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophytics. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non‐professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.

Fibronectin Bound to the Surface of Staphylococcus aureus Induces Association of Very Late Antigen 5 and Intracellular Signaling Factors with Macrophage Cytoskeleton

ABSTRACT Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, α5 and β1 chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, β3 integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.

Intracellular Localization of Staphylococcus aureus within Primary Cultured Mouse Kidney Cells

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity‐tolerant (HOT) cells were obtained. The rate of cocci‐ingesting cells varied from 16.9% to 93.4% among those of the HOT cells at the end of 3‐hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK‐1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK‐1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast‐like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast‐like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30‐min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.

Altered Virulence of a Pleiotropic Staphylococcus aureus Mutant with a Low Producibility of Coagulase and Other Factors in Mice

The virulence of a pleiotropic Staphylococcus aureus mutant with an extremely low producibility of coagulase and other factors was investigated in mouse. A mutant strain, designated as CL‐1, showed the same LD50 and the same intrarenal proliferation as its parental strain, when the mutant organisms were inoculated in mice in high doses. The mutant organisms, however, showed a diminished intrarenal proliferation compared with its parental organisms in low doses. This mutant strain expressed a pleiotropic phenotype such as a concomitant reduction in the producibility of coagulase, α‐toxin, and Panton‐Valentine leucocidin. The total effect due to the reduction in producibility of various factors on the virulence of the mutant strain was investigated with studies on the bacterial resistance to the phagocytic activity of leucocytes. A possible role of coagulase and that of some other staphylococcal exoproteins in the pathogenesis of S. aureus were discussed.

Apoptosis Observed in BALB/3T3 Cells Having Ingested Staphylococcus aureus

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 μ/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I‐infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 ± 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron‐dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2‐ to 6‐hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.

Improved Methods for Detection and Serotyping of Coagulase from Staphylococcus aureus

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti‐coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6‐amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type‐specifically inhibited in the presence of type‐specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.

Epidemiologic Typing of Methicillin‐Resistant Staphylococcus aureus in Neonate Intensive Care Units Using Pulsed‐Field Gel Electrophoresis

To elucidate the mode of dissemination of methicillin‐resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed‐field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS‐polyacrylamide gel‐electrophoresis (SDS‐PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST‐1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A′, A″ and B′) were identified. The A type including A′ and A″ (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B′. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS‐PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic‐shock syndrome toxin (TSST)‐1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST‐1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.