Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida.

Stable infection of Bacillus anthracis laboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two such B. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines of Eisenia fetida earthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group of Bacillus species and related organisms, Wip1 and Wip4 were both infective only toward B. anthracis and certain B. cereus strains. The natural host of Wip4 remained constant for 3 years and was a B. cereus strain that expressed a B. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which such B. cereus- and B. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability of B. cereus sensu lato to form heat-resistant spores, the sensitivity of B. anthracis to the PlyG lysin, or the selective amplification of environmental phages cocultured with B. anthracis. Findings from this work indicate that B. cereus sensu lato and its phages are common inhabitants of earthworm intestines.

Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats.

Cpl-1, a pneumococcal phage lytic enzyme, was tested in rats with experimental endocarditis due to Streptococcus pneumoniae WB4. High-dose regimen Cpl-1 eliminated pneumococci from blood within 30 min and decreased bacterial titers in vegetations (>4 log10 CFU/g) within 2 h. Rapid bacterial lysis induced by Cpl-1 treatment increased cytokine secretion noticeably.

Selective response of dermal Th-1 cells to 20-50 kDa streptococcal cell-wall proteins in chronic plaque psoriasis.

We have recently described a dermal Th-1 subset in skin lesions of psoriasis which recognizes cell-wall extract isolated from group A streptococci (GAS). As a first step in the identification of the streptococcal proteins involved, dermal T-cell lines (TCL) cultured from the lesional skin of 12 human leucocyte antigen (HLA)-typed psoriasis patients were stimulated with GAS cell-wall extract and 14 fractions (MWt approximately 20-100 kDa) separated from the cell-wall extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution, stained for intracellular interferon-gamma(IFN-gamma) expression and analysed by flow cytometry. All the TCL responded to GAS cell-wall extract to varying extents (3.5-27.6% IFN-gamma+). This response was consistently directed against 20-50 kDa cell-wall fractions and inhibited by anti-HLA-DR antibody. TCL with higher responses to GAS cell-wall extract recognized a larger number of fractions within this range than the lower responder TCL. No difference between the level and pattern of response to the fractions was observed for TCL from HLA-DR7+ (n = 6) and HLA-DR7- (n = 6) individuals. This preliminary study has shown a selective response to lower MWt proteins expressed on GAS cell wall by skin Th-1 cells in psoriasis. Further studies are required to identify the proteins involved.

Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains.

Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination, against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan and thus may be more efficient for the prevention and elimination of pneumococcal colonization.

Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase.

Nasopharyngeal carriage is the major reservoir for Streptococcus pneumoniae in the community. Although eliminating this reservoir would greatly reduce disease occurrence, no suitable intervention has been available for this purpose. We show here that seconds after contact, a purified pneumococcal bacteriophage lytic enzyme (Pal) is able to kill 15 common serotypes of pneumococci, including highly penicillin-resistant strains. In vivo, previously colonized mice revealed undetectable pneumococcal titers 5 hours after a single enzyme treatment. Pal enzyme had little or no effect on microorganisms normally found in the human oropharynx, and Pal-resistant pneumococci could not be detected after extensive exposure to the enzyme.

Group A streptococci bind to mucin and human pharyngeal cells through sialic acid-containing receptors.

The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with (125)I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified alpha2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.

Skin T cell proliferative response to M protein and other cell wall and membrane proteins of group A streptococci in chronic plaque psoriasis.

To determine and compare the T cell response to M protein and other group A streptococcal (GAS) antigens, T cell lines (TCL) were cultured from the lesional skin of 33 psoriatic patients and 17 disease controls. GAS-reactive skin TCL were tested in proliferation assays with recombinant M6 protein, and extracts of cell wall and membrane from type M6 GAS and its corresponding M gene deletion mutant. Initially, GAS-reactive skin TCL were obtained from 16 of 25 (64%) psoriasis, and from seven of 17 (41%) control patients. Eleven psoriatic and four control GAS-reactive TCL proliferated to M6 cell wall extract, whereas all the TCL from both groups responded to the extract of M6 membrane proteins. This difference in response to the two extracts was significant for both groups of patients (psoriasis, P = 0.0335, controls, P = 0.0156). GAS-reactive TCL from a further eight psoriasis patients showed no difference in response to cell wall extract from M6 GAS (containing the M protein minus its C-terminus) compared to that of its corresponding M gene deletion mutant. Furthermore, GAS-reactive TCL did not proliferate to recombinant M6 protein. However, a small, but significant reduction in proliferation by the eight psoriatic GAS-reactive TCL to the M-negative (lacking the M protein C-terminus) compared to M6-positive membrane extract was observed (P = 0.01). These findings suggest that GAS-reactive T cells in skin lesions of chronic plaque psoriasis proliferate to streptococcal membrane and, to a lesser extent, cell wall proteins. However, psoriatic skin T cells do not recognize cell wall M protein.

Induction of autoimmune valvular heart disease by recombinant streptococcal m protein.

Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.

Non-M protein(s) on the cell wall and membrane of group A streptococci induce(s) IFN-gamma production by dermal CD4+ T cells in psoriasis.

Recently we have demonstrated that a disease-specific subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis produces interferon-gamma in response to group A streptococcal (GAS) antigens. To determine if these T cells recognize M or non-M protein, extracts from cell wall of type M6 GAS (M6W) and its isogenic M gene deletion mutant (M-W), M6 membrane extract (M6M) and recombinant M6 protein (rM6) were used to stimulate GAS-reactive T-cell lines from nine patients with chronic plaque psoriasis. T-cell lines were incubated with or without streptococcal extracts for 18 h in the presence of a transport inhibitor, stained for surface CD4 and intracellular cytokine expression, and analysed by flow cytometry. Variable numbers (0.2-34%) of CD4+ T cells produced interferon-gamma, in all but one of the T-cell lines tested, in response to M6W, M-W and M6M extracts. No significant difference between the response to M6W and M-W extracts was detected. In addition, rM6 protein failed to increase CD4+/interferon-gamma+ T-cell numbers in seven of nine T-cell lines compared to medium alone. For the group, there was a highly significant correlation between the responses to the three extracts (M6W vs M-W, P = 0.0005; M6W vs M6M, P = 0.0003; M-W vs M6M, P = 0.0001). Low or minimal numbers of interleukin-4- and interleukin-10-producing CD4+ T cells were occasionally induced. These findings suggest that a subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis patients produces interferon-gamma in response to non-M protein(s) present on the cell wall and membrane of GAS.

Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme.

Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C(1) termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of approximately 10(7) group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 10(7) live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease.

Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells.

We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of approximately 10(5) phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization.

Stronger proliferative response to membrane versus cell-wall Streptococcal proteins by peripheral blood T cells in chronic plaque psoriasis.

Proliferative responses of peripheral blood mononuclear cells (PBMC) to group A streptococcal (GAS) antigens have been studied in 24 patients with psoriasis and 15 disease controls. Extracts of cell wall (including M protein) from types M4 and M12 GAS, recombinant M6 protein, and both cell-wall and cell-membrane extracts from type M6 (M6+) GAS and its corresponding M gene deletion mutant (M6-) were tested. PBMC from psoriatic patients proliferated more strongly to cell-wall extracts from M12 versus M4 (P = 0.0348), and to M6+ versus M6- (P = 0.0019) GAS with, in most cases, moderate proliferation to recombinant M6 protein. The psoriatic response to M12 cell wall was significantly increased compared to the controls (P = 0.0032). In psoriatics, M6+ membrane extracts induced a markedly greater proliferation than those of cell wall (P = 0.0002); responses to M6+ (P = 0.0039) and M6- (P = 0.0114) membrane extracts were higher than those of the control PBMC. Both groups showed a decreased response to the M6- versus M6+ membrane extracts (P = 0.0030; P = 0.0181, respectively). This study has demonstrated that patients with psoriasis have a heightened circulating T-cell response to cell wall M protein and to non-M proteins present on the cell wall and membrane of GAS.

Antibodies to streptococcal surface enolase react with human alpha-enolase: implications in poststreptococcal sequelae.

The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.

Reactivity of rheumatic fever and scarlet fever patients' sera with group A streptococcal M protein, cardiac myosin, and cardiac tropomyosin: a retrospective study.

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.

A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion.

Nerve damage is the hallmark of Mycobacterium leprae infection, which results from M. leprae invasion of the Schwann cell of the peripheral nervous system. We have recently shown that the laminin-2 isoform, specially the G domain of laminin alpha2 chain, on the Schwann cell-axon unit serves as an initial neural target for M. leprae. However, M. leprae surface molecules that mediate bacterial invasion of peripheral nerves are entirely unknown. By using human alpha2 laminins as a probe, a major 28-kDa protein in the M. leprae cell wall fraction that binds alpha2 laminins was identified. After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. Deduced amino acid sequence of this M. leprae laminin-binding protein predicts a 21-kDa molecule (ML-LBP21), which is smaller than the observed molecular size in SDS/PAGE. Immunofluorescence and immunoelectron microscopy on intact M. leprae with mAbs against recombinant (r) ML-LBP21 revealed that the protein is surface exposed. rML-LBP21 avidly bound to alpha2 laminins, the rG domain of the laminin-alpha2 chain, and the native peripheral nerve laminin-2. The role of ML-LBP21 in Schwann cell adhesion and invasion was investigated by using fluorescent polystyrene beads coated with rML-LBP21. Although beads coated with rML-LBP21 alone specifically adhered to and were ingested by primary Schwann cells, these functions were significantly enhanced when beads were preincubated with exogenous alpha2 laminins. Taken together, the present data suggest that ML-LBP21 may function as a critical surface adhesin that facilitates the entry of M. leprae into Schwann cells.

Identification and characterization of a novel fibronectin-binding protein on the surface of group A streptococci.

Understanding the role surface proteins play in the interaction of group A streptococci with epithelial cells is an important step toward the development of new strategies to fight infections. Fibronectin-binding proteins in streptococci and staphylococci have been described as important mediators for adherence to eukaryotic cells. In the present study we describe a new Streptococcus pyogenes fibronectin-binding protein (PFBP). The gene encoding the PFBP protein (pfbp) was identified from an M12 strain genomic library. It encodes a protein of 127.4 kDa which contains the LPXTGX motif characteristic of cell wall-associated proteins in gram-positive organisms and is among the largest surface molecules described for group A streptococci. The pfbp gene is transcribed during cell growth and was present in several class I and II streptococcal strains tested. The deduced amino acid sequence of PFBP exhibits a variable N-terminal region and a conserved C-terminal region when compared to most fibronectin-binding proteins identified from other gram-positive bacteria. The N-terminal region presents a stretch of 105 amino acids with no homology with N-terminal regions of previously described fibronectin-binding molecules, while the C-terminal region contains three repeat domains that share significant similarity with the repeat regions of fibronectin-binding proteins from S. pyogenes, S. dysgalactiae, and S. equisimilis. The PFBP repeated region, when expressed on the surface of S. gordonii, a commensal organism, binds to soluble and immobilized fibronectin. This study also shows that, in addition to pfbp, a second gene homologous with that of protein F1 (which also codes for a fibronectin-binding protein) is transcribed during cell growth in the same S. pyogenes strain.

Immunological characterization of a protective antigen of Erysipelothrix rhusiopathiae: identification of the region responsible for protective immunity.

The gene encoding a protective protein antigen of the gram-positive bacterium Erysipelothrix rhusiopathiae, an important veterinary pathogen responsible for erysipelas in swine and a variety of diseases in animals, was cloned and sequenced. The gene encodes a polypeptide of 597 amino acids plus a putative signal sequence of 29 amino acids, resulting in a mature protein with a molecular mass of 69,017 Da. Sequence analysis of the gene product revealed a C-terminal region composed of nine tandem repeats of 20 amino acids and a total sequence that is nearly identical to that of the 64-kDa cell surface protein (SpaA) of the bacterium. Because of this similarity, the protein was designated SpaA.1. In this study, we examined whether the SpaA.1 protein could induce protective antibodies and whether we could identify the region involved in protective immunity. Both the mature SpaA.1 protein and its C-terminal repeat region, but not the N-terminal segment, were expressed in Escherichia coli and purified as a histidine-tagged fusion recombinant protein. Rabbit antiserum raised against the mature SpaA.1 protein passively protected mice from lethal challenge with a virulent homologous strain, Fujisawa-SmR, suggesting that protection is mediated by humoral antibodies. To determine which domain of the SpaA.1 protein is responsible for the observed protection, mice were actively immunized with either the mature SpaA. 1 protein or the C-terminal repeat region and then challenged with Fujisawa-SmR. The result showed that mice immunized with the mature SpaA.1 protein, but not the C-terminal repeat region, were protected, suggesting that the protection-eliciting epitope(s) is located within the N-terminal two-thirds of the SpaA.1 molecule. This was confirmed by passive immunization experiments in which the protective activity of rabbit antiserum, raised against mature SpaA. 1 protein, was not abolished by absorption with the purified recombinant C-terminal repeat region. In addition, antibodies specific for the C-terminal repeat region were unable to protect mice from lethal challenge. These results show that the N-terminal two-thirds of the SpaA.1 molecule may constitute a good vaccine candidate against erysipelas.

Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.

alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.