Effect of Prior Experimental Human Enteropathogenic Escherichia coli Infection on Illness following Homologous and Heterologous Rechallenge

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Enteropathogenic Escherichia coli (EPEC) strains are one of several categories of pathogenic E. coli strains that cause diarrhea. EPEC infections are prevalent on six continents (5, 22–24, 28, 43). In many parts of the world, EPEC strains are the most common bacterial cause of diarrhea in infants (7, 21, 43). Disease due to EPEC can be severe, refractory to oral rehydration, protracted, and lethal (3, 14, 21, 45, 48).The pathogenesis of EPEC infection involves three distinct stages, initial adherence, signal transduction, and intimate attachment (12). Initial adherence is associated with the production of a type IV fimbria, the bundle-forming pilus (BFP) (20), that is encoded on the large EPEC adherence factor (EAF) plasmid (50). EPEC uses a type III secretion apparatus to export several proteins, including EspA, EspB, and EspD, that are required for tyrosine kinase-mediated signal transduction within the host cell (17, 25, 30, 31). This signaling leads to phosphorylation and activation of a 90-kDa protein that is a putative receptor for the bacterial outer membrane protein intimin (44). Intimin, the product of the eae gene, is required for intimate attachment of bacteria to the host cell membrane and for full virulence in volunteers (13, 26, 27). The interaction between EPEC and host cells results in the loss of microvilli and the formation of adhesion pedestals containing numerous cytoskeletal proteins (16, 33, 34, 39, 46). This interaction between bacteria and host cells is known as the attaching and effacing effect (40).One of the most striking clinical features of EPEC infections is the remarkable propensity of these strains to cause disease in very young infants. Rare reports of disease in older children and adults usually reflect common-source outbreaks that probably involve large inocula (47, 53). In contrast, in nosocomial outbreaks among neonates, EPEC spreads rapidly by person-to-person contact, apparently involving low inocula (54). The incidence of community-acquired EPEC infection is highest in the first 6 months after birth (4, 7, 21). EPEC infection is also more severe in younger children (8). Infants are more likely to develop diarrhea during the first episode of colonization with EPEC than they are during subsequent encounters (8). Whether the low incidence of EPEC diarrhea in older children and adults is due to acquired immunity or decreased inherent susceptibility is not known.The immune response to EPEC infection remains poorly characterized. It has previously been demonstrated that volunteers convalescing from experimental EPEC infection develop antibodies to the O antigen component of lipopolysaccharide (LPS) of the infecting strain, to intimin, and to type I-like fimbriae (13, 15, 29, 38). Antibodies to common EPEC O antigens are found more often in children of greater than 1 year in age than they are in younger children (42). Breast-feeding is protective against EPEC infection (2, 19, 43, 52). Breast milk contains antibodies against EPEC O antigens and outer membrane proteins and inhibits EPEC adherence to tissue culture cells (6, 9, 49).In an earlier study, it was reported that volunteers infected with EPEC developed antibodies to a 94-kDa outer membrane protein (38). Subsequently, it was determined that this antigen was intimin (26). Interestingly, the lone volunteer in that earlier study who did not have diarrhea after challenge with a wild-type EPEC strain had prechallenge serum antibodies to intimin. This led to the hypothesis that antibodies to intimin are protective against EPEC infection. To test this hypothesis and to test the more general hypothesis that EPEC infection induces protective immunity, two volunteer studies were performed. The first was a heterologous-challenge study performed in 1986, in which volunteers were infected with an O55:H6 EPEC strain and challenged, along with a naive cohort, with an O127:H6 EPEC strain. The second was a homologous-challenge study performed in 1991, in which veterans of a study comparing the virulence of a wild-type EPEC O127:H6 strain with that of an isogenic eae mutant (13) were rechallenged, along with a naive cohort, with the homologous wild-type strain. The availability of new purified antigens allowed us to analyze data from these studies in the context of humoral immune responses.     

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.     

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.     

CD1 Presents Antigens from a Gram-Negative Bacterium,Haemophilus influenzae Type b

Human CD1 is a family of nonpolymorphic major histocompatibility complex class I-like molecules capable of presenting mycobacterial lipids, including lipoarabinomannan (LAM), to double-negative (DN; CD4⁻ CD8⁻) as well as CD8⁺ T cells. Structural similarities between LAM and the capsular polysaccharides of gram-negative bacteria led us to consider the latter as candidate CD1 ligands. We derived two CD1-restricted DN T-cell populations which proliferated to Haemophilus influenzae type b (Hib) antigen. One T-cell population also proliferated to proteinase K-treated Hib antigen, suggesting that it recognized a nonpeptide. Our work thus expands the universe of T cell antigens to include nonpeptides distinct from mycobacterial lipids and suggests a potential role for CD1-restricted T cells in immunity to Hib.Human CD1 is a family of nonpolymorphic major histocompatibility complex (MHC) class I-like molecules (CD1a to CD1d) (4, 7, 15, 18). Although CD1 is encoded outside the MHC, its association with β2-microglobulin relates it structurally to MHC class I. CD1 molecules are expressed on immature thymocytes (19) and antigen-presenting cells (APC) including cytokine-activated macrophages (13), B cells (22, 23), and dermal dendritic cells (9). Recent studies have revealed that CD1 possesses the unique function of presenting nonpeptide antigen (Ag) to T cells (3, 17, 21, 24). A prototypic Ag presented in the context of CD1 is lipoarabinomannan (LAM), a mannose polymer substituted at one end with arabinose and at the other with a phosphatidic acid containing tubulostearic and palmitic acids. De-O-acylation of LAM totally abrogated T-cell responsiveness, suggesting that the lipid moiety was required for Ag recognition (21). Since gram-negative bacteria contain lipoglycans structurally analogous to LAM (2, 11, 14, 20), we sought to isolate CD1-restricted T cells which recognize antigens from Haemophilus influenzae type b (Hib), a representative gram-negative bacterium.     

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.     

Genetic and Serological Evidence for Multiple Instances of Unrecognized Transmission of Hepatitis C Virus in Hemodialysis Units

We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis. Screening and confirmatory assays for circulating antibodies to HCV became available (10, 27) after the molecular cloning of the HCV genome in 1989 (3). The second-generation enzyme immunoassay for detection of anti-HCV antibody has revealed a high prevalence of antibodies to HCV in hemodialysis (HD) patients (5). Most cases of HCV infection in HD patients are thought to be related to blood transfusions, but several reports from different parts of the world have also shown the presence of HCV infection in nontransfused HD patients as well (8, 22, 30), and the anti-HCV antibody-positive rates have been found to increase with the duration of dialysis (8, 22, 30). This suggests that nosocomial transmission of HCV occurs in HD units. Recently, patient-to-patient transmission of HCV in HD units has been demonstrated by molecular biology techniques, but the frequency of transmission was low (2, 20, 25). In these studies the sequences of HCV dominantly propagating in patients were determined and compared. Allander et al. (2) were the first to detect nucleotide sequence similarity in variable parts of the HCV genome in several HD patients. Sampietro et al. (20) and Stuyver et al. (25) found a rare HCV variant in several patients treated in the same HD unit by sequencing the relatively conserved region of the HCV genome, the 5′ untranslated region (5′-UTR), and the core region, respectively. However, no evidence of transmission had ever been demonstrated by sequence analysis of a minor population of HCV isolates.The HCV genome exhibits different variability of nucleotide sequences in different regions. There is a hypervariable region, hypervariable region 1 (HVR1), in the putative second envelope glycoprotein (E2) of the HCV genome (7). HVR1 consists of 27 amino acid residues located in the N-terminal region of E2 and is the most variable region in the HCV genome. Accordingly, it appeared that HVR1 might be useful for discriminating HCVs the same as a polymorphic marker for genetic fingerprinting and for detecting nosocomial transmission of HCV. It was thought that not only rare but also common genotypes of HCV might be found to be transmitted. Furthermore, most patients with chronic hepatitis C possess antibody against the HVR1 of their own isolates (9, 29), and anti-HVR1 antibody was also thought to be useful for demonstrating nosocomial transmission. In this study, we examined HCV transmission in HD units by performing phylogenetic analysis of HCV HVR1 sequences and testing for antibodies to HVR1 peptides. We also analyzed multiple sequences of HVR1 from each individual and investigated nosocomial transmission of HCV, including a minor population of HCV isolates transmitted in HD patients.     

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.     

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.