Oral streptococci show diversity in resistance to complement immunity.

PURPOSE: Mechanisms underlying systemic infections by oral species of Mitis (Streptococcus mitis, Streptococcus oralis) and Sanguinis (Streptococcus gordonii, Streptococcus sanguinis) commensal streptococci are poorly understood. This study investigates profiles of susceptibility to complement-mediated host immunity in representative strains of these four species, which were isolated from oral sites or from the bloodstream. METHODOLOGY: Deposition of complement opsonins (C3b/iC3b), and surface binding to C-reactive protein (CRP) and to IgG antibodies were quantified by flow cytometry in 34 strains treated with human serum (HS), and compared to rates of opsonophagocytosis by human PMN mediated by complement (CR1/3) and/or IgG Fc (FcγRII/III) receptors. RESULTS: S. sanguinis strains showed reduced susceptibility to complement opsonization and low binding to CRP and to IgG compared to other species. Surface levels of C3b/iC3b in S. sanguinis strains were 4.5- and 7.8-fold lower than that observed in S. gordonii and Mitis strains, respectively. Diversity in C3b/iC3b deposition was evident among Mitis species, in which C3b/iC3b deposition was significantly associated with CR/FcγR-dependent opsonophagocytosis by PMN (P<0.05). Importantly, S. gordonii and Mitis group strains isolated from systemic infections showed resistance to complement opsonization when compared to oral isolates of the respective species (P<0.05). CONCLUSIONS: This study establishes species-specific profiles of susceptibility to complement immunity in Mitis and Sanguinis streptococci, and indicates that strains associated with systemic infections have increased capacity to evade complement immunity. These findings highlight the need for studies identifying molecular functions involved in complement evasion in oral streptococci.

A digenic human immunodeficiency characterized by IFNAR1 and IFNGR2 mutations.

Primary immunodeficiencies are often monogenic disorders characterized by vulnerability to specific infectious pathogens. Here, we performed whole-exome sequencing of a patient with disseminated Mycobacterium abscessus, Streptococcus viridians bacteremia, and cytomegalovirus (CMV) viremia and identified mutations in 2 genes that regulate distinct IFN pathways. The patient had a homozygous frameshift deletion in IFNGR2, which encodes the signal transducing chain of the IFN-γ receptor, that resulted in minimal protein expression and abolished downstream signaling. The patient also harbored a homozygous deletion in IFNAR1 (IFNAR1*557Gluext*46), which encodes the IFN-α receptor signaling subunit. The IFNAR1*557Gluext*46 resulted in replacement of the stop codon with 46 additional codons at the C-terminus. The level of IFNAR1*557Gluext*46 mutant protein expressed in patient fibroblasts was comparable to levels of WT IFNAR1 in control fibroblasts. IFN-α-induced signaling was impaired in the patient fibroblasts, as evidenced by decreased STAT1/STAT2 phosphorylation, nuclear translocation of STAT1, and expression of IFN-α-stimulated genes critical for CMV immunity. Pretreatment with IFN-α failed to suppress CMV protein expression in patient fibroblasts, whereas expression of WT IFNAR1 restored IFN-α-mediated suppression of CMV. This study identifies a human IFNAR1 mutation and describes a digenic immunodeficiency specific to type I and type II IFNs.

Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production.

Oral viridans group streptococci are the major commensal bacteria of the supragingival oral biofilm and have been detected in human atheromatous plaque. Atherosclerosis involves an ongoing inflammatory response, reportedly involving chronic infection caused by multiple pathogens. The aim of this study was to examine the invasion of human aortic endothelial cells (HAECs) by oral viridans group streptococci and the subsequent cytokine production by viable invaded HAECs. The invasion of HAECs by bacteria was examined using antibiotic protection assays and was visualized by confocal scanning laser microscopy. The inhibitory effects of catalase and cytochalasin D on the invasion of HAECs were also examined. The production of cytokines by invaded or infected HAECs was determined using enzyme-linked immunosorbent assays, and a real-time polymerase chain reaction method was used to evaluate the expression of cytokine messenger RNA. The oral streptococci tested were capable of invading HAECs. The number of invasive bacteria increased with the length of the co-culture period. After a certain co-culture period, some organisms were cytotoxic to the HAECs. Catalase and cytochalasin D inhibited the invasion of HAECs by the organism. HAECs invaded by Streptococcus mutans Xc, Streptococcus gordonii DL1 (Challis), Streptococcus gordonii ATCC 10558 and Streptococcus salivarius ATCC 13419 produced more cytokine(s) (interleukin-6, interleukin-8, monocyte chemoattractant protein-1) than non-invaded HAECs. The HAECs invaded by S. mutans Xc produced the largest amounts of cytokines, and the messenger RNA expression of cytokines by invaded HAECs increased markedly compared with that by non-invaded HAECs. These results suggest that oral streptococci may participate in the pathogenesis of atherosclerosis.

Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3')-III in commensal viridans group streptococci

High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3’)-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3’)-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3’’)-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci (AU)

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Failure of viridans group streptococci causing bacteremia in pediatric oncology patients to express superantigens.

Group A Streptococcus pyogenes causes a distinctive clinical disorder, streptococcal toxic shock syndrome, mediated by superantigenic bacterial exotoxins. Oncology patients with viridans group streptococcal sepsis frequently present with a streptococcal toxic shocklike syndrome of unclear pathogenesis. Viridans group streptococci isolated from pediatric oncology patients with streptococcal toxic shocklike illnesses do not possess homologs of known superantigen genes. Supernatants from cultures of these bacteria also fail to stimulate T-cell proliferation, suggesting these bacteria do not commonly elaborate superantigens. Adjunctive treatment with intravenous immunoglobulin, which is advantageous in streptococcal toxic shock syndrome, may not benefit these patients.

Secretory IgA, salivary peroxidase, and catalase-mediated microbicidal activity during hydrogen peroxide catabolism in viridans streptococci: pathogen coaggregation.

Viridans streptococci can kill methicillin-resistant Staphylococcus aureus (MRSA) through the production of hydrogen peroxide (H2O2). However, several hundred viridans streptococci cells are necessary to kill 1 cfu of MRSA. We analyzed the potency of bactericidal and fungicidal effector molecules induced by catabolism of H2O2 in the oral cavity. Secretory IgA (SIgA) and an unidentified salivary component bound Streptococcus sanguinis, a viridans streprococcus, and MRSA into coaggregates. In these coaggregates, salivary peroxidase and the MRSA catalase produced singlet molecular oxygen (1O2) from H2O2 produced by viridans streptococci. SIgA converted 1O2 into ozone, which has potent bactericidal and fungicidal activity. We calculated that <10 cfu of Streptococcus sanguinis were necessary to kill 1 cfu of MRSA in the coaggregate. SIgA, Aspergillus niger catalase, and H2O2 in saliva killed Candida albicans, which is highly resistant to reagent H2O2. Together with indigenous bacteria and innate immunity, SIgA potentially constitutes a novel system that may sustain oral homeostasis.

Human T-cell responses to oral streptococci in human PBMC-NOD/SCID mice.

We investigated cellular and humoral immune responses to oral biofilm bacteria, including Streptococcus mutans, Streptococcus anginosus, Streptococcus sobrinus, and Streptococcus sanguinis, in NOD/SCID mice immunized with human peripheral blood mononuclear cells (hu-PBMC-NOD/SCID mice) to explore the pathogenicity of each of those organisms in dental and oral inflammatory diseases. hu-PBMC-NOD/SCID mice were immunized by intraperitoneal injections with the whole cells of the streptococci once a week for 3 weeks. FACS analyses were used to determine the percentages of various hu-T cell types, as well as intracellular cytokine production of interleukin-4 and interferon-gamma. Serum IgG and IgM antibody levels in response to the streptococci were also determined by enzyme-linked immunosorbent assay. S. anginosus induced a significant amount of the proinflammatory cytokine interferon-gamma in CD4(+) and CD8(+) T cells in comparison with the other streptococci. However, there was no significant differences between the streptococci in interleukin-4 production by CD4(+) and CD8(+) T cells after inoculation. Further, S. mutans significantly induced human anti-S. mutans IgG, IgG(1), IgG(2), and IgM antibodies in comparison with the other organisms. In conclusion, S. anginosus up-regulated Th1 and Tc1 cells, and S. mutans led to increasing levels of their antibodies, which was associated with the induction of Th2 cells. These results may contribute to a better understanding of human lymphocyte interactions to biofilm bacteria, along with their impact on dental and mucosal inflammatory diseases, as well as endocarditis.

Glucosyltransferases of viridans group streptococci modulate interleukin-6 and adhesion molecule expression in endothelial cells and augment monocytic cell adherence.

Recruitment of monocytes plays important roles during vegetation formation and endocardial inflammation in the pathogenesis of infective endocarditis (IE). Bacterial antigens or modulins can activate endothelial cells through the expression of cytokines or adhesion molecules and modulate the recruitment of leukocytes. We hypothesized that glucosyltransferases (GTFs), modulins of viridans group streptococci, may act directly to up-regulate the expression of adhesion molecules and also interleukin-6 (IL-6) to augment monocyte attachment to endothelial cells. Using primary cultured human umbilical vein endothelial cells (HUVECs) as an in vitro model, we demonstrated that GTFs (in the cell-bound or free form) could specifically modulate the expression of IL-6, and also adhesion molecules, in a dose- and time-dependent manner. Results of inhibition assays suggested that enhanced expression of adhesion molecules was dependent on the activation of nuclear factor kappaB (NF-kappaB) and extracellular signal-regulated kinase and that p38 mitogen-activated protein kinase pathways also contributed to the release of IL-6. Streptococcus-infected HUVECs or treatment with purified IL-6 plus soluble IL-6 receptor alpha enhanced the expression of ICAM-1 and the adherence of the monocytic cell line U937. These results suggest that streptococcal GTFs might play an important role in recruiting monocytic cells during inflammation in IE through induction of adhesion molecules and IL-6, a cytokine involved in transition from neutrophil to monocyte recruitment.

Induction of immune response to Streptococcus pneumoniae by administration of oral viridans streptococci via phosphorylcholine determinant.

Expression of the phosphorylcholine (PC) epitope was examined in 48 viridans streptococcal strains, including Streptococcus pneumoniae R36a as the positive control, and their immunogenicity to induce an S. pneumoniae-cross-reactive response was evaluated in mice. Thirteen strains were found to express the PC epitope, while no obvious association was found between the taxonomic categories and PC expression. Serum antibody responses to S. pneumoniae cells were induced in mice by intraperitoneal injection of the PC-positive, but not PC-negative, strains. The cross-reactive antibodies induced by non-pneumococcal oral streptococci were readily inhibited by free hapten PC. IgM was the sole isotype of the anti-pneumococcal and anti-PC antibodies, and the phenomenon of immunological memory was not observed. Since the anti-PC antibody is critically important for resistance against pneumococcal infection in mice, the present results indicate the possibility that PC-expressing oral commensal bacteria have a significant influence on the hosts' responsiveness to S. pneumoniae.

Streptococcus sinensis may react with Lancefield group F antiserum.

Lancefield group F streptococci have been found almost exclusively as members of the 'Streptococcus milleri' group, although they have been reported very occasionally in some other streptococcal species. Among 302 patients with bacteraemia caused by viridans streptococci over a 6-year period, three cases were caused by Streptococcus sinensis (type strain HKU4T, HKU5 and HKU6). All three patients had infective endocarditis complicating their underlying chronic rheumatic heart diseases. Gene sequencing showed no base differences between the 16S rRNA gene sequences of HKU5 and HKU6 and that of HKU4T. All three strains were Gram-positive, non-spore-forming cocci arranged in chains. All grew on sheep blood agar as alpha-haemolytic, grey colonies of 0.5-1 mm in diameter after 24 h incubation at 37 degrees C in ambient air. Lancefield grouping revealed that HKU5 and HKU6 were Lancefield group F, but HKU4T was non-groupable with Lancefield groups A, B, C, D, F or G antisera. HKU4T was identified by the Vitek system (GPI), API system (20 STREP) and ATB system (ID32 STREP) as 99 % Streptococcus intermedius, 51.3 % S. intermedius and 99.9 % Streptococcus anginosus, respectively. Using the same tests, HKU5 was identified as 87 % Streptococcus sanguinis/Streptococcus gordonii, 59 % Streptococcus salivarius and 99.6 % S. anginosus, respectively, and HKU6 as 87 % S. sanguinis/S. gordonii, 77 % Streptococcus pneumoniae and 98.3 % S. anginosus, respectively. The present data revealed that a proportion of Lancefield group F streptococci could be S. sinensis. Lancefield group F streptococci should not be automatically reported as 'S. milleri'.