Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

Mannose Induces the Release of Cytopathic Factors from Acanthamoeba castellanii

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.Acanthamoeba spp. are protozoal parasites capable of infecting the skin, brain, and eye (10, 15, 17, 31, 32, 37). Corneal inflammation produced by Acanthamoeba was first recognized in 1973 and has since been intimately associated with contact lens wear (15, 31). Often the disease displays a ring-like neutrophilic stromal infiltrate with an overlying epithelial ulcer. The epithelium often undergoes a recurrent cycle of healing and breakdown during the progression of the disease. Topical or systemic treatment with antibiotics, antifungals, corticosteroids, and antivirals is often ineffectual (2). Typical treatment consists of around-the-clock hourly topical treatments with propamidine isothionate, polyhexamethylene biguanide, neomycin, or chlorhexidine, alone or in combination. This therapeutic regimen may continue for weeks. Many patients receive therapeutic corneal transplants, which can be reinfected by quiescent parasites residing in the periphery of the cornea.Parasite binding to the corneal epithelium is believed to be an important first step in the infectious cascade of Acanthamoeba keratitis. We have shown that adherence of Acanthamoeba to corneal buttons in vitro varies among mammalian species and correlates with susceptibility to experimental Acanthamoeba keratitis (14, 19, 35). Parasitic infections, such as Acanthamoeba keratitis, often occur in a sequential manner and are initiated by the pathogen’s adherence to host cells. Bacteria, fungi, and amoebae have been shown to bind to epithelial cells via lectin-glycoprotein interactions (5, 6, 11, 18, 20–22, 26, 27, 40). The cell surface of Pseudomonas aeruginosa is decorated with lectins which bind surface glycoproteins of the epithelium to be invaded (30, 39). Entamoeba histolytica also utilizes glycoproteins as receptor ligands for adherence to the gastrointestinal epithelium (6, 16, 25–29). Binding of Acanthamoeba polyphaga and A. castellanii to corneal epithelial cells in culture is inhibited by mannose (18, 40). Subsequent studies have indicated that the binding of A. castellanii to corneal epithelial cells is mediated by a 136-kDa mannose-binding protein on the trophozoite cell membrane (40).The pathophysiology of Acanthamoeba keratitis is poorly understood. Several studies have demonstrated that Acanthamoeba trophozoites can induce either cytolysis or apoptosis of target cells in vitro (1, 7, 24, 33, 34). Pathogenic Acanthamoeba trophozoites produce a variety of proteases which are believed to facilitate parasite penetration into the corneal stroma (9). Once in the stroma, Acanthamoeba trophozoites secrete collagenolytic enzymes which contribute to the dissolution of the stromal matrix (13).This study was undertaken to examine the cytopathic mechanisms employed by Acanthamoeba during the initial phase of ocular infection. We tested the hypothesis that blocking parasite binding to corneal epithelial cells with mannose would prevent parasite-mediated cytolysis and invasion of the corneal stroma. The results, however, indicate that although mannose blocks parasite binding, it also facilitates the release of cytolytic factors which kill corneal epithelial cells.     

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (10⁶ L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-γ limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-γ in combination with other cytokines including TNF-α facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-α secretion and IFN-γ-mediated MPC activation (46).IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-γ, TNF-α, and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10–12). Using this murine model of Legionnaires’ disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

Isotype Switching Increases Efficacy of Antibody Protection against Cryptococcus neoformans Infection in Mice

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.Cryptococcus neoformans is a fungus which is a frequent cause of life-threatening meningoencephalitis in patients with impaired immunity (22, 25). Cryptococcosis has been reported to occur in 6 to 8% of patients with AIDS (7). In immunocompromised individuals, C. neoformans infections are often incurable with conventional antifungal agents, and these patients frequently require lifelong therapy (45). The difficulties involved in the management of cryptococcosis in immunocompromised individuals have led to a reexamination of the potential of antibody-mediated immunity for prevention and therapy of cryptococcal infections. A polysaccharide-tetanus toxoid (TT) conjugate vaccine which is highly immunogenic and can elicit protective antibodies in mice has been made (3, 8, 9). In addition, several monoclonal antibodies (MAbs) have been shown to modify the course of infection in mice, and these may be useful in therapy of human infection (12, 14, 28, 42, 43).Cell-mediated immunity is generally acknowledged to provide important host defense against C. neoformans infection (4, 20, 26, 31, 42). In contrast, the role of antibody-mediated immunity in host resistance is less certain (2), but there is considerable evidence that administration of some MAbs can modify the course of infection in mice (8, 12, 14, 16, 28, 33). C. neoformans is unusual among fungal pathogens in that it has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM) (6), which is important for virulence (5). The capsular polysaccharide has been shown to produce a variety of deleterious effects including inhibition of phagocytosis (21), interference with antigen presentation (39), shedding of adhesion molecules (11), inhibition of leukocyte migration (10), and alterations in cytokine production by host effector cells (24, 40, 41). Antibodies to the C. neoformans capsular polysaccharide may contribute to host defense through multiple effects including enhanced opsonization (13, 18, 23, 30, 44), clearance of polysaccharide antigen (15), promotion of granuloma formation (14), and release of oxygen- and nitrogen-derived oxidants (27, 38).In previous studies, we demonstrated that immunoglobulin G3 (IgG3) MAbs are not protective in various mouse models of cryptococcal infection (32, 42). When one of these nonprotective IgG3 MAbs was switched to IgG1, the IgG1 significantly prolonged animal survival (32, 42). In the present study, we analyzed two families of IgG switch variants generated in vitro from two nonprotective IgG3 MAbs with different epitope specificities. We found that MAbs with different isotypes have different protective efficacies and that switching of nonprotective IgG3 MAbs to IgG1, IgG2b, and IgG2a significantly increased antibody protective efficacy. These studies demonstrate a complex relationship among efficacy of antibody protection, epitope specificity, and isotype.     

Immunological Relationship between the Class I Epitope of Streptococcal M Protein and Myosin

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the α-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.Acute rheumatic fever (ARF) is an inflammatory disease that can follow group A streptococcal pharyngitis. The most serious clinical manifestation is rheumatic carditis; however, arthritis, chorea, erythema marginatum, or subcutaneous nodules may be present (40, 41). The pathogenesis of ARF is thought to be mediated by autoimmune mechanisms activated during a streptococcal infection (40). The autoimmune hypothesis is supported by a number of previous observations, including a time interval of at least 3 weeks between the initial streptococcal throat infection and the onset of ARF (40, 41), the identification of heart-reactive immunoglobulin (Ig) and complement deposits in the myocardium of patients with fatal rheumatic carditis (25–27, 30), and the elevation of heart-reactive antibodies in the sera of patients with ARF (46). Cardiac myosin has been identified as one of the cardiac antigens recognized by these heart-reactive antistreptococcal autoantibodies (13, 29).Streptococcal M protein, an α-helical coiled-coil protein, structurally and immunologically mimics host tissue antigens, particularly the rod region of myosin (12, 14, 15, 17, 34, 35). Sequence analysis has revealed that streptococcal M proteins contain blocks of internally repeated amino acid sequences referred to as A, B, and C repeat regions (19). The NH2-terminal nonrepeat and A repeat regions contain determinants of type specificity, while epitopes found in the B and more highly conserved C repeat regions may be common to different M serotypes (19). While there are nearly 100 different serological types of group A streptococcal M protein, epidemiological studies indicate that only a limited number of M protein serotypes are associated with ARF outbreaks (6). This finding suggests that certain M protein serotypes may be more rheumatogenic than others. In a previous attempt to classify streptococcal serotypes according to their rheumatogenic capacity, Widdowson identified human antisera directed to a non-type-specific protein moiety of M protein known as M-associated protein (44, 45). However, a more recent classification scheme has been proposed by Bessen and colleagues, in which streptococcal serotypes were grouped based on the expression of a conserved surface-exposed M protein epitope (4). It was demonstrated that the M serotypes associated with the majority of ARF outbreaks possessed an epitope (class I) defined by monoclonal antibody (MAb) probes 10B6 and 10F5. The sequence of the 10B6 and 10F5 epitope was localized to a 15-amino-acid fragment within the C repeat region of the type 6 M protein (23). The remaining serotypes (class II) lack this epitope or the determinant is structurally inaccessible in those strains. There was a close parallel between serotypes designated class I and those serotypes previously classified as M-associated protein I by Widdowson (44, 45). The fact that only certain serotypes within class I streptococci are rheumatogenic implies that these organisms are of a phenotype that is capable of inducing ARF (4). This implication is supported in part by a recent publication in which it was shown that sera of ARF patients contained high levels of antibodies to the class I epitope, suggesting that their disease was the result of an infection by a class I streptococcus (5).Elevated titers of antibodies to many streptococcal antigens (2), including M protein and the self-antigen myosin (12–15, 17, 29), are associated with ARF. While antibodies to M protein are crucial for the opsonization of streptococci, they have also been implicated in the immunological cross-reactions between streptococci and host tissue antigens such as cardiac myosin (12–15, 17, 29). In earlier studies, many of these cross-reactive epitopes have been localized to the N-terminal, hypervariable A and B repeat regions of the M molecule (12, 15, 17). Myosin-reactive antibodies, found to be elevated in almost all cases of ARF (13), have been shown to bind to human heart tissue and to cross-react with streptococcal M protein (12). Previous studies have demonstrated that immunization of animals with the cell walls of certain strains of group A streptococci resulted in the production of heart-reactive antibodies which could be adsorbed with streptococcal extracts containing streptococcal M protein (16, 24, 28). Human MAbs or myosin affinity-purified antibodies produced from patients with ARF cross-reacted with streptococcal M protein and human cardiac myosin and contributed to the presence of heart-cross-reactive antistreptococcal antibodies in ARF (12, 13, 39). More recent studies have identified cytotoxic antistreptococcal/antimyosin MAbs from rheumatic carditis patients (1). Antimyosin antibody has been shown to deposit in the heart tissues of susceptible mice (31), and a cytotoxic mouse antistreptococcal/antimyosin antibody which binds to the surface of heart cells and to the α-helical coiled coil molecule laminin has been described (10).Identification of myosin cross-reactive epitopes of M protein recognized in ARF has been reported for the amino-terminal half of the molecule (12, 15, 17), and a study by Vashishtha and Fischetti demonstrated antimyosin antibody responses to the C repeat region. However, the reactivity was directed only to denatured myosin (43). More recently, studies of the C repeat or carboxy-terminal region of M protein have shown T-cell cross-reactions with myosin (38). The goal of the present study was to investigate the possibility that the class I epitope in the C repeat region of M protein cross-reacts immunologically with myosin. In this study we show that MAb 10B6, which recognizes the class I epitope of M protein, reacts with cardiac and skeletal myosin. This study also demonstrates that ARF and UNC sera react with a site in the conserved C repeat region of M protein within the class I epitope of rheumatogenic M protein serotypes. The new data show that in addition to previously described N-terminal epitopes, the class I epitope of streptococcal M protein is immunologically cross-reactive with myosin.(Portions of this work were presented at the XIII International Lancefield Society Meeting on Streptococci and Streptococcal Diseases at the Pasteur Institute in Paris, France, in September 1996.)     

d-Lactate Production and [14C]Succinic Acid Uptake by Adherent and Nonadherent Escherichia coli

Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [¹⁴C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.     

Streptococcal Histone-Like Protein: Primary Structure of hlpA and Protein Binding to Lipoteichoic Acid and Epithelial Cells

In addition to its role in the nucleoid, the histone-like protein (HlpA) of Streptococcus pyogenes is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the hlpA genes were cloned from S. pyogenes, S. gordonii, S. mutans, and S. sobrinus, using PCR amplification, and sequenced. The encoded HlpA protein of S. pyogenes has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of Bacillus species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below. S. pyogenes did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (P < 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.

Prokaryotes contain several small, basic, heat-stable proteins in association with the nucleoid. These proteins bind to single- and double-stranded DNA without obvious sequence specificity and are termed histone-like proteins; however, they do not have sequence homology with eukaryotic histones (for reviews, see references 13, 19, 33, and 37). The best-studied histone-like proteins are HU of Escherichia coli (4, 15, 29, 35, 38) and HB of Bacillus species (10, 23, 24, 31, 44). HU is a heterodimer of HU1 and HU2 proteins, which contain 90 amino acid residues each and have 70% sequence identity. HB is a protein highly homologous to HU but existing as a homodimer of a 92-amino-acid subunit (10, 23, 24, 31). Although the biological functions of histone-like proteins are not fully understood, they are known to wrap DNA and restrain negative supercoiling (4, 35). The resulting alterations in DNA structure and topology affect several cellular processes, including initiation of DNA replication (11, 51), DNA partitioning and cell division (12, 50), binding of repressors (3, 17, 30, 34), and transposition of bacteriophage Mu (43).In addition to the physiological functions of bacterial histone-like proteins, HlpA (previously called GAG-BP and HBP) of Streptococcus species may contribute fortuitously to the virulence of these bacteria when the protein is released into the tissues during infection. Purified HlpA binds selectively in vitro to heparan sulfate in proteoglycans of heart and kidney basement membranes (1, 5, 6, 49). The accumulation of intravenously administered HlpA on renal basement membranes of mice and rabbits and the ensuing in situ immune complex formation (7, 20) indicate that it might be an important virulence factor in acute poststreptococcal glomerulonephritis and the glomerulonephritis that is often associated with streptococcal endocarditis in humans (21, 47). Tissue-bound HlpA may serve as a nidus for in situ immune complex formation leading to the inflammation and immunopathology that typify these diseases. The HlpAs of Streptococcus pyogenes, S. mutans, S. gordonii, and S. mitis are immunologically cross-reactive and exhibit identical binding activities for basement membranes in animal tissues (5, 6, 49).This study was undertaken to clone and sequence hlpA from group A and viridans group streptococci, to compare the primary structure of HlpAs, and to evaluate the ability of these bacteria to release HlpA protein into the culture medium during growth. The hlpA genes of four Streptococcus species encode proteins of 91 amino acids that have at least 90% sequence identities. Members of the viridans group streptococci released more HlpA during stationary phase of growth than did the group A streptococci, and extracellular HlpA was complexed with soluble lipoteichoic acid (LTA). These antigen complexes bind to the surfaces of human epithelial cells in vitro and can lead to immune complex formation in situ.     

Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers

Yersinia enterocolitica is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of Y. enterocolitica biotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb Yersinia virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes: ail (7.2%), myfA (11.7%), ystA (0.9%), and ystB (85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (P ≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (P ≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of Y. enterocolitica may invade cells by a novel mechanism. These results indicate that some clinical isolates of Y. enterocolitica which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic Yersinia species.Yersinia enterocolitica is an important human pathogen which causes a variety of disorders, ranging from nonspecific diarrhea to invasive disease such as mesenteric lymphadenitis, hepatosplenic abscesses, and septicemia (5, 10, 37). The heterogenous nature of Y. enterocolitica, including differences in virulence, has led to the division of the species into subgroups based upon biochemical behavior and lipopolysaccharide O antigens (5, 37). At present, six biotypes are recognized, of which biotype 1B and biotypes 2 through 5 are regarded as including primary pathogens (5, 22, 26, 37, 40). The primary pathogenic strains of Y. enterocolitica are recognized in part by their ability to invade tissue culture cells in large numbers (7, 27, 29, 35). Genes which contribute this ability include the inv and ail genes on the bacterial chromosome and yadA, which is borne on a ca. 70-kb virulence plasmid known as pYV (4, 7, 27). Interestingly, however, other pYV-borne genes impede bacterial penetration of epithelial cells and macrophages, with the result that Y. enterocolitica is located extracellularly in infected animals (9, 15). Chromosomal genes other than inv and ail which may also contribute to virulence include yst (also known as ystA), which encodes a heat-stable enterotoxin (YST-a), myf, which encodes the production of fibrillae (Myf), and the urease gene complex (11, 12, 21).Biotype-1A strains of Y. enterocolitica, which generally are considered to be avirulent, are highly heterogenous, and include a large number of O serogroups (5, 37). They occur throughout the world in a wide range of environments and generally lack the genotypic and phenotypic markers associated with virulence of classical invasive strains of Y. enterocolitica, such as pYV, ail, myf, ystA, or a functional inv gene (12, 21, 28, 35, 40, 41, 45). Moreover, biotype-1A strains of environmental origin do not colonize the gastrointestinal tracts of experimentally inoculated animals (33, 42, 48). Despite these observations, some biotype-1A strains have been implicated as a cause of gastrointestinal disease. For example, a nosocomial outbreak of gastroenteritis in Canada involving nine patients was attributed to a strain of Y. enterocolitica biotype 1A, serogroup O:5 (36). In several countries, moreover, including Australia, Canada, The Netherlands, New Zealand, the Republic of Georgia, South Africa, Switzerland, and the United States of America, a significant proportion of Y. enterocolitica isolates obtained from patients with diarrhea belong to biotype 1A (3, 6, 17, 32, 34, 39, 46, 47). In addition, a prospective case control study with Chilean children showed that biotype-1A strains were significantly associated with diarrhea (30), and a clinical study in Switzerland demonstrated that the illness associated with biotype-1A strains of Y. enterocolitica was indistinguishable from that due to classical virulent biotypes (6).If biotype-1A strains of Y. enterocolitica are able to cause disease, their pathogenic mechanisms are not clear because they lack the well-established virulence markers of primary pathogenic strains of Y. enterocolitica. Some Y. enterocolitica strains produce variants of YST-a, known as YST-b and YST-c (20, 52, 53), but their prevalence and contribution to disease are not known. In addition, a biotype-1A strain of serogroup O:6 was reported to produce a novel heat-stable enterotoxin, termed YST-II. This toxin differs from YST-a in a number of respects, including its mechanism of action, which does not appear to involve activation of guanylate cyclase (41). Other putative virulence determinants in this or other biotype-1A strains have not been investigated or reported.As biotype-1A strains of Y. enterocolitica are so heterogenous and occupy such a diverse range of environmental niches, we hypothesized that there may be a subset of these bacteria which are capable of causing disease but which lack the classical virulence markers of Yersinia species and therefore cannot be identified by assays for these markers. The aim of this study was to test this hypothesis by examining a collection of biotype-1A strains of clinical and nonclinical origins for virulence-associated determinants.     

Increased Levels of Intracellular Calcium Are Not Required for the Formation of Attaching and Effacing Lesions by Enteropathogenic and Enterohemorrhagic Escherichia coli

Elevated concentrations of intracellular calcium ([Ca]_i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]_i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]_i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]_i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]_i are not required for A/E lesion formation by EPEC and EHEC.Enteropathogenic Escherichia coli (EPEC) strains remain an important cause of severe infantile diarrheal disease in many parts of the developing world (8). EPEC colonizes the intestinal mucosa and produces attaching and effacing (A/E) lesions, which are characterized by localized destruction of brush border microvilli, intimate bacterial adhesion, and formation of a complex cytoskeletal structure beneath intimately attached bacteria (23). Accumulation of polymerized actin beneath bacteria results in the formation of cup-like pedestal structures in the apical enterocyte membrane (22). EPEC produces morphologically similar A/E lesions in a variety of cultured cell lines (21). The determinants of A/E lesion formation have been localized to a large pathogenicity island on the EPEC chromosome termed the locus of enterocyte effacement or (LEE) (27). Encoded within the LEE are a type III secretion system (16); an outer membrane protein adhesin, intimin (17); a translocated intimin receptor, Tir (18); and three EPEC secreted proteins (Esps), EspA, EspB, and EspD, each of which has been shown to be required for signal transduction to host cells and A/E lesion formation (12, 20, 25). Enterohemorrhagic E. coli (EHEC) strains, which cause a hemorrhagic colitis, possess a homologous LEE and also produce A/E lesions (27).In vitro studies employing defined mutants support a three-stage model of A/E lesion formation (10). Stage 1 involves an initial nonintimate bacterial adhesion to epithelial cells, which is thought to be promoted by a plasmid-encoded bundle-forming pilus (BFP). In stage 2 signal transduction in host cells is triggered by Esps, resulting in cytoskeletal rearrangements, microvillous effacement, and tyrosine phosphorylation of Tir following translocation to the host cell membrane (18). Stage 3 involves intimin binding to tyrosine-phosphorylated Tir to produce the characteristic intimate attachment; pedestal formation results from further cytoskeletal reorganization and polymerization beneath intimately attached bacteria.Early studies of EPEC signal transduction examined second messengers and demonstrated increased concentrations of intracellular calcium ([Ca]_i) in EPEC-infected epithelial cells (2, 11). The subsequent demonstration of inositol phosphate fluxes, including increased levels of inositol triphosphate (11, 13), suggested possible stimulation of the classical phospholipase C pathway and release of calcium from inositol triphosphate-sensitive intracellular stores. Activation of brush border villin by calcium, disruption of the microvillous actin cytoskeleton, and vesiculation of the microvillous membrane provided a plausible model for EPEC-induced brush border effacement (4). One difficulty with such a model, however, was how such a calcium signal could give rise to the highly localized cytoskeletal changes seen during EPEC infection: microvillous effacement occurs only at sites of intimate bacterial adhesion. We hypothesized that if calcium signalling is important, calcium signals should be equally localized within the cell to sites of A/E lesion formation. To test this hypothesis and analyze further the calcium responses to infection of HEp-2 cells by EPEC (and EHEC), we employed calcium-imaging fluorescence microscopy, which not only allows real-time measurements of [Ca]_i in infected cells but also allows the spatial distribution of [Ca]_i to be determined and thus correlated with A/E lesion formation. Here we report that not only were we unable to detect any significant elevation in [Ca]_i at sites of A/E EPEC adhesion, but we were unable to detect any significant alterations in [Ca]_i in EPEC- and EHEC-infected HEp-2 cells compared to uninfected cells.     

Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic Escherichia coli Linked to Human Diarrhea

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.Escherichia coli is one of the major causes of human infectious diseases, partly because of the wide variety of virulence mechanisms and pathotypes (15), and new pathotypes continue to be described. A new pathotype was proposed by Gunzburg et al. after examining diarrheal pathogens in a prospective community-based study among Australian Aboriginal children (22). One group of isolates was significantly (P < 0.05) associated with diarrhea, and these isolates were particularly common among children younger than 18 months. The isolates did not produce any recognized enterotoxin or classic enteric virulence factor, although they exhibited diffuse or aggregative adhesion in a modified adhesion assay (15). All isolates were able to detach HEp-2 cell monolayers and were termed “cell-detaching E. coli.” This property was shown to be mediated by alpha-hemolysin, and we demonstrate below that all cell-detaching E. coli strains produce alpha-hemolysin and that some may also produce cytotoxic necrotizing factor 1 (CNF1) and other toxins. However, neither alpha-hemolysin nor CNF1 has been clearly demonstrated to be an enteric virulence factor, and the role of hemolysin in particular is controversial. We will refer to these isolates as diarrhea-associated hemolytic E. coli (DHEC) isolates.Alpha-hemolytic E. coli strains have been associated with human enteric disease, especially among young children (8, 10–12, 20–22), and the related enterohemolysin of E. coli O157 (35) appears to be involved in enteric disease. There has, however, been no large prospective case-controlled epidemiologic study of the association of alpha-hemolysin with human diarrhea. Alpha-hemolytic bacteria are also associated with enteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33, 36, 44, 45). Porcine diarrheal strains are almost universally hemolytic (23a), and alpha-hemolysin in these isolates enhanced virulence and colonization (37) but was not itself diarrheagenic. More recent studies have found that Hly⁺ CNF1⁺ strains caused fluid accumulation in piglets (33) and that neonatal pigs were susceptible to challenge with Hly⁺ CNF⁺ strains, which caused bloody diarrhea, enterocolitis, and systemic disease (45).In contrast, some earlier studies were unable to demonstrate a role for hemolysin in enteric disease, since neither hemolytic bacteria nor their supernatants caused fluid accumulation in ileal loops (10, 14, 37). Hemolytic strains may be isolated from the feces of asymptomatic people (26), and, among humans, hemolysin is more commonly associated with strains causing extraintestinal infections (5, 26).The genetics and in vitro mechanisms of alpha-hemolysin are well known. The hlyCABD operon encodes the structural 110-kDa hemolysin protein (HlyA) and proteins involved in processing and export (42). Once secreted, hemolytic activity is short-lived, and this has complicated studies of hemolysin toxigenicity (42). Hemolysin does not require a receptor to bind to target cells, inserting instead into the target cell membrane to form a pore that allows the free flow of cations, sugars, and water. This leads to leakage of intracellular contents and affects the cytoskeleton and metabolism (4, 9, 42, 43). In extraintestinal infections, hemolysin has multiple effects and roles, including resistance to host defense, tissue damage, and lethality, either by direct action or by stimulation of inflammatory mediators and signal transduction pathways (7, 9, 16, 42).CNF is a 114-kDa protein with homology to a family of dermonecrotic toxins (18) and is encoded by the monocistronic cnf gene, which lies just downstream of hly. The CNF1 toxin causes HeLa cells to become large and multinucleated as a result of actin disassembly, which results from activation of Rho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1 in diarrhea remains unclear. CNF1-producing strains have been isolated from diarrheal stools and have been associated with several outbreaks in humans (8, 10) and animals (13, 33, 44). Unfortunately, no large, prospective, case-controlled studies have been performed, and the best evidence for the pathogenicity of CNF1-toxigenic isolates is the marked virulence in piglet challenge experiments (45), outlined above. Purified CNF1 did not show enterotoxic potential in the suckling mouse or induce fluid accumulation in the rabbit ileal loop (10, 14), in contrast to the related CNF2, which is linked to enteric disease in animals (13, 14, 30). Both CNF toxins are extremely lethal, and have a variety of in vivo effects including tissue necrosis and edema (12–14).In this paper, we characterize DHEC isolates that were obtained from a study where alpha-hemolysin was significantly associated with disease (22) and show that they are able to cause disease in rabbits. Using molecular genetics, we attempt to analyze the role of each gene in pathogenesis.     

Listeria monocytogenes Virulence Factors That Stimulate Endothelial Cells

Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L. monocytogenes or L. monocytogenes mutants; then surface expression of E-selectin and neutrophil adhesion were measured. The results showed that Δhly and prfA mutants were the most crippled, requiring 100-fold more mutant bacteria than wild-type bacteria for analogous stimulation. By comparison, L. monocytogenes mutants with deletions of actA, inlA, inlB, inlAB, plcA, and plcB resembled their parent strains, and a ΔplcA ΔplcB mutant displayed decreased intracellular growth rate but only a minor decrease in stimulation of E-selectin or neutrophil adhesion. Other experiments showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but internalization and increased surface E-selectin and intercellular adhesion molecule-1 expression were profoundly inhibited. However, cytochalasin D had no effect on the HUVEC response to stimulation with lipopolysaccharide or tumor necrosis factor alpha. These data suggest that listeriolysin O production by infecting L. monocytogenes contributes to increased expression of surface E-selectin and intercellular adhesion molecule-1, but neither it nor intracellular replication are directly responsible for this event. Nonetheless it is possible that listeriolysin O potentiates the effect(s) of an other molecule(s) that directly triggers this response. Additionally, cellular invasion by L. monocytogenes appears to be critical for initiating the HUVEC response, potentially by providing a signal which results in upregulation of the necessary bacterial genes.Interactions between vascular endothelial cells and pathogenic bacteria are common events in many infectious diseases and often result in endothelial cell stimulation and enhance leukocyte adhesion to infected cells (1). Such interactions are comprised of two components: endothelial cell stimulation by bacterial products and direct microbial infection of the endothelial cell. Bacterial products can stimulate endothelial cells in the absence of cellular infection, or the two processes can act in concert when bacteria invade endothelial cells. Bacterial products that stimulate cells without infection include the gram-negative cell wall component, lipopolysaccharide (LPS), the phospholipase C and perfringolysin O of Clostridium perfringens, and listeriolysin O (LLO) and the phosphoinositol-specific phospholipase C of Listeria monocytogenes (4, 16, 27, 33, 40, 41). As mentioned above, several different pathogenic bacteria have been shown to bind or invade endothelial cells and to stimulate them in the process (9, 14, 15, 38, 39, 44, 50). Products that could stimulate cells during binding and invasion include the outer membrane protein A of Borrelia burgdorferi, peptidoglycan from Leptospira icterohemorhagiae, and certain bacterial heat shock proteins (9, 21, 48, 49). Endothelial cell stimulation by either of these processes has profound effects on expression of endothelial cell adhesion molecules as well as cytokine and chemokine production and ultimately plays a critical role in the inflammatory process and host defenses.L. monocytogenes is a pathogenic facultative intracellular bacterium able to invade and replicate within mammalian cells (14, 18, 35). Several L. monocytogenes genes involved in cellular invasion and intracellular parasitism have been identified and their function and products studied in detail (reviewed in reference 36). These include the pleiotropic regulator of the virulence gene cluster prfA, members of the gene cluster (plcA, hly, mpl, actA, and plcB), and the inl family of invasion genes (5, 19). Products with roles in phagosomal lysis and escape into the cytoplasm include LLO, a pore-forming toxin encoded by hly, and two C-type phospholipases, a phosphoinositol-specific phospholipase C encoded by plcA and a broad-spectrum phospholipase C encoded by plcB that cleaves phosphatidylcholine (PC-PLC) (18, 30, 35, 42). These enzymes act with LLO to facilitate phagosomal escape and cell-to-cell spread and also may be involved in stimulating intracellular signaling in the eukaryotic target. The mpl gene encodes an enzyme that processes the immature form of PC-PLC into a mature form (10, 30, 32). Intracellular motility and subsequent cell-to-cell spread is dependent upon the ActA protein, which is essential for polymerization of host F-actin (11, 26). The recently described inl family of genes encode internalin A and internalin B proteins that are involved in binding and invasion of eukaryotic cells (13, 14, 20, 29).As a pathogenic microbe, L. monocytogenes is a well-known cause of bacteremia and central nervous system infections of immunocompromised humans and of domesticated animals (22, 31). The predilection of L. monocytogenes to invade the central nervous system from the bloodstream led to the hypothesis that infection of vascular endothelial cells was an important event in the pathophysiology of listeriosis (2, 14, 37). Previous work from this laboratory showed that L. monocytogenes can infect and replicate within human umbilical vein endothelial cells (HUVEC) (14). In response to infection, there was upregulated surface expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) and stimulation of neutrophil (polymorphonuclear leukocyte [PMN]) adhesion to infected monolayers (15). Induction of this inflammatory phenotype and function did not occur following infection with the nonpathogenic Listeria innocua and Listeria welshimeri or following incubation of infected HUVEC with uninfected cells separated by a permeable membrane or with sterile-filtered supernatants from infected cells. These results suggested that specific bacterial virulence factors and direct contact of L. monocytogenes with HUVEC were required to trigger the HUVEC response. The experiments presented here studied the roles of specific virulence factors as stimuli for endothelial cell adhesion molecule expression and PMN adhesion.     

Two new insulator proteins,Pita and ZIPIC,target CP190 to chromatin

Insulators are multiprotein–DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.Insulators in the Drosophila and vertebrate genomes have been identified based on their ability to disrupt the communication between an enhancer and a promoter when inserted between them (Raab and Kamakaka 2010; Ghirlando et al. 2012; Herold et al. 2012; Matzat and Lei 2013; Chetverina et al. 2014; Kyrchanova and Georgiev 2014). The growing amount of data show that insulator proteins fulfil an architectural function in mediating inter- and intrachromosomal interactions and in contacting regulatory elements such as promoters or enhancers (Maksimenko and Georgiev 2014).The best studied Drosophila insulator proteins, dCTCF (homolog of vertebrate insulator protein CTCF) and Su(Hw) are DNA-binding zinc-finger proteins (Herold et al. 2012; Matzat and Lei 2013; Kyrchanova and Georgiev 2014). Binding sites for dCTCF have been identified in the insulators that separate functional regulatory domains of the bithorax complex and in many promoter regions (Moon et al. 2005; Holohan et al. 2007; Mohan et al. 2007; Nègre et al. 2010, 2011; Ni et al. 2012). The Su(Hw) protein more frequently associates with intergenic sites (Adryan et al. 2007; Bushey et al. 2009; Nègre et al. 2010, 2011; Soshnev et al. 2012, 2013). As shown in a transgenic assay, dCTCF and Su(Hw) binding sites can support specific distant interactions (Kyrchanova et al. 2008a,b), which suggests a key role for these proteins in organizing chromatin architecture.The Su(Hw), dCTCF, and BEAF-32 proteins interact with Centrosomal Protein 190 kD, named CP190 (Pai et al. 2004; Gerasimova et al. 2007; Mohan et al. 2007; Bartkuhn et al. 2009; Oliver et al. 2010; Liang et al. 2014). CP190 (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc-finger motifs, and a C-terminal E-rich domain (Oliver et al. 2010; Ahanger et al. 2013). The BTB domain of CP190 forms stable homodimers that may be involved in protein–protein interactions (Oliver et al. 2010; Bonchuk et al. 2011). In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis (Butcher et al. 2004). It has been shown that CP190 is recruited to chromatin via its interaction with the Su(Hw) and dCTCF proteins (Pai et al. 2004; Mohan et al. 2007). Inactivation of CP190 affects the activity of the dCTCF-dependent insulator Fab-8 from the bithorax complex (Gerasimova et al. 2007; Mohan et al. 2007; Moshkovich et al. 2011) and the gypsy insulator, which contains 12 binding sites for the Su(Hw) protein (Pai et al. 2004). Binding of Su(Hw) and CP190 at gypsy-like sites is mutually dependent, indicating a stabilizing role of CP190 in these cases (Schwartz et al. 2012).Recent genome-wide ChIP-chip studies provide evidence for an extensive overlap of the CP190 distribution pattern with dCTCF, BEAF-32, and Su(Hw) insulator proteins and the promoters of active genes (Bartkuhn et al. 2009; Bushey et al. 2009; Nègre et al. 2010, 2011; Schwartz et al. 2012; Soshnev et al. 2012). Very recently, it has been demonstrated that CP190 bridges DNA-bound insulator factors with promoters (Liang et al. 2014). These data support the model that CP190 has a global role in the function of insulator proteins. However, there are a number of sites in the Drosophila genome where CP190 does not colocalize with any known insulator DNA binding protein (IBP), suggesting that there may be some other proteins that recruit CP190 to chromatin (Schwartz et al. 2012).To identify new factors that associate with CP190, we purified the FLAG-tagged CP190 protein from S2 cells and identified two zinc-finger proteins, CG7928 and Pita, which were shown to interact with CP190 in vivo and in vitro. Genome-wide identification of binding sites for Pita and CG7928 in S2 cells revealed their extensive colocalization with CP190, providing evidence for direct interactions between these proteins, which was supported by binding and in vivo functional assays. Based on these results we termed CG7928 the “zinc-finger protein interacting with CP190” (ZIPIC).