Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin.

Gram-positive bacteria deploy the type VII secretion system (T7SS) to facilitate interactions between eukaryotic and prokaryotic cells. In recent work, we identified the TelC protein from Streptococcus intermedius as a T7SS-exported lipid II phosphatase that mediates interbacterial competition. TelC exerts toxicity in the inner wall zone of Gram-positive bacteria; however, intercellular intoxication of sister cells does not occur because they express the TipC immunity protein. In the present study, we sought to characterize the molecular basis of self-protection by TipC. Using sub-cellular localization and protease protection assays, we show that TipC is a membrane protein with an N-terminal transmembrane segment and a C-terminal TelC-inhibitory domain that protrudes into the inner wall zone. The 1.9-Å X-ray crystal structure of a non-protective TipC paralogue reveals that the soluble domain of TipC proteins adopts a crescent-shaped fold that is composed of three α-helices and a seven-stranded ß-sheet. Subsequent homology-guided mutagenesis demonstrates that a concave surface formed by the predicted ß-sheet of TipC is required for both its interaction with TelC and its TelC-inhibitory activity. S. intermedius cells lacking the tipC gene are susceptible to growth inhibition by TelC delivered between cells; however, we find that the growth of this strain is unaffected by endogenous or overexpressed TelC, although the toxin accumulates in culture supernatants. Together, these data indicate that the TelC-inhibitory activity of TipC is only required for intercellularly transferred TelC and that the T7SS apparatus transports TelC across the cell envelope in a single step, bypassing the cellular compartment in which it exerts toxicity en route.

Polymicrobial subdural empyema: involvement of Streptococcus pneumoniae revealed by lytA PCR and antigen detection.

The authors report a case of a subdural empyema (SDE) caused by a coinfection with Streptococcus intermedius and Streptococcus pneumoniae, initially considered a S. intermedius infection only. An otherwise healthy 11-year-old female was admitted to the hospital after 5 days of illness. Symptoms were consistent with classical SDE symptoms and progressed rapidly with finally somnolence before the first neurosurgical procedure despite relevant antibiotic treatment. Primary MRI showed an interhemispheric SDE and a postoperative control CT scan showed progression of the empyema infratentorially. The empyema was evacuated twice, day 8 and 18, with good results. Primary samples showed growth of S. intermedius only. The severity of the clinical picture elicited supplementary samples, which were additionally positive for S. pneumoniae by an in-house specific lytA PCR and/or a commercial antigen test.

Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). We earlier reported that the bacterial lipoteichoic acid was detected at the sites of inflammation around damaged bile ducts in the livers of PBC, and PBC patients' sera showed high titers against streptococcal histone-like protein. Here, we investigated whether chronic bacterial exposure could trigger PBC-like epithelial cell damage in normal mouse. BALB/c mice were repeatedly inoculated with various bacteria for 8 weeks. At 1 week (Group 1) and 3, 4, or 20 months (long term; Group 2) after the final inoculation, mice were killed to obtain samples. In the livers of the Streptococcus intermedius (S.i.)-inoculated mice in Group 1, cellular infiltration was predominantly observed around the bile ducts over the hepatic parenchyma. In the S.i.-inoculated mice in Group 2, portal but not parenchymal inflammation was observed in the livers, and periductal cellular infiltrates were detected in the salivary glands. Both S.i.-inoculated Groups 1 and 2 BALB/c mice sera had antibodies against HuCCT1 biliary epithelial cells, anti-nuclear antibodies, and anti-gp210 antibodies, but not anti-mitochondrial antibodies. Immunoreactivity to histone-like DNA-binding protein of S.i. (S.i.-HLP) was detectable around the sites of chronic nonsuppurative destructive cholangitis in the portal area in the livers of both S.i.-inoculated Groups 1 and 2 BALB/c mice. Furthermore, anti-S.i.-HLP antibody bound to synthetic gp210 peptide, as well. Bacteria triggered PBC-like cholangitis, multifocal epithelial inflammation, and autoantibody production. Bacteria are likely involved in the pathogenesis of PBC and of associated multifocal epithelial inflammation.

Streptococcus cristatus attenuates Fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells may be invaded by a polymicrobial intracellular flora, including pathogens together with commensals. Various oral pathogens can induce the production of interleukin-8, a potent neutrophil chemotractant, in oral epithelial cells. Evidence from the gut suggests that commensal species may modulate inflammatory responses to pathogens. The aim of this study was to examine the interleukin-8 responses of oral epithelial cells to an oral pro-inflammatory species, Fusobacterium nucleatum, in combination with an oral commensal, Streptococcus cristatus. MATERIAL AND METHODS: KB, TERT-2, TR146 and SCC15 cells were cocultured with F. nucleatum and S. cristatus, either alone or in combination, at 37 degrees C in 5% CO2 under various conditions. The mRNA expression of interleukin-8 was analyzed by reverse transcription-polymerase chain reaction and protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: F. nucleatum alone evoked a potent interleukin-8 response, whereas S. cristatus alone did not induce significant interleukin-8 expression in oral epithelial cells. When present together, S. cristatus attenuated the F. nucleatum-induced interleukin-8 production in the four oral epithelial cell lines to varying degrees. The inhibitory effect of S. cristatus was independent of its viability and its co-aggregation with F. nucleatum, was not related to soluble bacterial products and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. CONCLUSION: Our data suggest that S. cristatus may exert immunomodulatory effects on the interleukin-8 response of oral epithelial cells to F. nucleatum challenge.

A possible role of histone-like DNA-binding protein of Streptococcus intermedius in the pathogenesis of bile duct damage in primary biliary cirrhosis.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). It was reported that anti-histone autoantibody was detected in PBC, suggesting that bacterial histone-like DNA-binding protein (HLP) may be involved in the pathogenesis of PBC. To identify bacterial species in PBC to confirm this possibility, serum reactivity to bacterial cells was studied by ELISA. The IgM class Streptococcus intermedius titers were significantly higher in PBC than chronic hepatitis due to hepatitis C virus (CH-C) and healthy subjects. Among the streptococci, S. intermedius was selected for further study. The antigenic peptide of S. intermedius of HLP was synthesized to examine the serum reactivity to Si-HLP. IgM class anti-Si-HLP peptide titers were significantly higher in PBC. Immunoreactivity to anti-Si-HLP was detected in the cytoplasm of biliary epithelial cells and inflammatory cells in the portal area in PBC patients' livers. Streptococci, especially S. intermedius, might play a key role in the pathogenesis of PBC, possibly involving HLP.

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways.

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.

Meningitis y absceso cerebral por Streptococcus intermedius en un paciente con infección por VIH-1 / Meningitis and brain abscess caused by streptpcoccus intermedius in a patient infected with HIV-1

Los estreptococos de grupo milleri se caracterizan por su tendencia a provocar infecciones piógenas invasoras en diferentes localizaciones. Las meningitis estreptocócicas no neumocócicas son poco frecuentes en pacientes adultos y pueden asociarse a la presencia de un absceso cerebral. Los abscesos cerebrales son colecciones localizadas dentro del parénquima cerebral que se originan como complicación de una infección, siendo los estreptococos microaerófilos y las bacterias anaerobiaslos microorganismos más frecuentemente aislados. Aunque no es inusual la presencia de colecciones intracraneales de etiología infecciosa en pacientes con infección por VIH-1, los abscesos cerebrales producidos por las bacterias piógenas habituales son muy infrecuentes y es T. gondiiel agente etiológico más frecuente. Aportamos un caso de meningitis y absceso cerebral por S. intermedius en un paciente con infección por VIH-1

Streptococcus milleri group have been recognized as an important pathogens for abscess formation in various organs. Streptococci other than Streptococcus pneumoniae are a rare cause of bacterial meningitis in adults and can be associated with the presence of an undiagnosed brain abscess. Brain abscess is a focal collection within the brain parenchyma wich can arise as a complication of a variety of infections. The most common etiologic organisms in clinical series have been microaerophilic streptococci and anaerobic bacterias. Although intracraneal mass lesions that occur as a result of infection have commonly been reported in patients infected with the human immunodeficiency virus, brain abscess due to the common bacterial pathogens are rarely described in HIV infected patients and Toxoplasma gondii is the organism most frecuently isolated from stereotactic brain biopsy in these patients. We report a patient with both HIV-1 infection and streptococcal meningitis secondary to brain abscess caused by S. intermedius

Isolation and some properties of fimbriae of oral Streptococcus intermedius.

Streptococcus intermedius 1208-1 carried linear fiber-like fimbriae that extended radially from the cell surface. The fimbriae were isolated by pipetting and sonication and were purified by ammonium sulfate precipitation followed by a column chromatography series. Heat treatment in the presence of sodium dodecyl sulfate resulted in the dissociation into smaller molecules. Rabbit antiserum raised against the purified protein reacted with fimbriae on the surface of bacteria under immunogold staining. Serotype g or g-related strains produced the fimbriae and aggregated in human saliva. The aggregation was inhibited by the anti-fimbriae immunoglobulin Fab fragment or the purified fimbriae.

Human salivary aggregation in Streptococcus intermedius type g strains: relationship with IgA.

Bacterial aggregation is an important step in elimination from the human body to protect against infection. Streptococcus intermedius K1K aggregates in human saliva. In this study, the salivary agglutinin was identified. The aggregation level was very strong in sonic-treated saliva and 1-microm filtrate. Preincubation of human saliva with anti-human alpha chain serum or anti-human whole saliva serum completely inhibited aggregation, but preincubation with anti-human micro chain serum or anti-Fc fragment of human IgG serum had no effect. Agglutinin of human saliva that could aggregate the strain K1K was purified using DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephacryl S200HR gel filtration. Purified salivary agglutinin was characterized with electrophoresis and immunological techniques, indicating that purified material was IgA. Bacterial aggregation was dependent on the presence of calcium. Saliva filtrate specimens from eight healthy men and eight women showed different aggregation activities. Three men and one woman had little activity. These data show that the present bacterial aggregation was an immunoreaction between IgA in saliva and the bacteria dependent on the levels of calcium. In addition, the IgA in human saliva related with possible calcium-dependent antigen(s) on the surface of strain K1K.

Platelet responses and anaphylaxis-like shock induced in mice by intravenous injection of whole cells of oral streptococci.

Intravenous injection of lyophilized whole cells of various oral streptococcal strains into muramyldipeptide (MDP)-primed C3H/HeN mice induces rapid anaphylactoid shock. Here we examined the mechanism underlying this shock. In non-primed mice, Streptococcus intermedius K-213K (SiK213) and Streptococcus constellatus T21 (ScT21) produced little or no sign of shock. In MDP-primed mice, SiK213 caused lethal shock, while ScT21 only had a weak effect. SiK213 induced decreases in blood platelets and 5-hydroxytryptamine (5HT) preceding the shock, while the effects of ScT21 were weak. The SiK213-induced 5HT decrease and shock were reduced by a complement-C5 inhibitor. These results suggest that (i). streptococcal bacterial cells can induce rapid platelet responses, (ii). complement-dependent degradation of platelets may be involved in streptococcus-induced shock, (iii). the streptococcus-induced platelet degradation or degranulation may occur largely in the systemic circulation, and (iv). platelets may play a role not only in infectious diseases caused by gram-negative bacteria, but also in diseases caused by gram-positive bacteria.