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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Murine Macrophages Use Oxygen- and Nitric Oxide-Dependent Mechanisms To Synthesize S-Nitroso-Albumin and To Kill Extracellular Trypanosomes

Reactive nitrogen intermediates were synthesized spontaneously in cultures of macrophages from Trypanosoma brucei brucei-infected mice by an inducible nitric oxide (NO) synthase. This was inhibited by the addition of nitro-l-arginine. In this paper, we report the kinetics of the fixation of macrophage-derived NO on bovine serum albumin by using an enzyme-linked immunosorbent assay. S nitrosylation was confirmed by the Saville reaction, using mercuric chloride. It is known that reactive oxygen intermediates (ROI) are also synthesized by stimulated macrophages. The fact that NO is able to bind cysteine only under aerobic conditions led us to investigate the role of macrophage-derived ROI in the formation of S-nitrosylated proteins by activated macrophages. The immunoenzymatic signal decreased by 66 and 30% when superoxide dismutase and catalase, respectively, were added to the culture medium of macrophages from infected mice. In addition, the decrease in S-nitrosylated albumin formation correlated with the protection of extracellular trypanosomes from the cytostatic and cytotoxic activity of NO. Melatonin, a hydroxyl radical scavenger resulting from the decomposition of peroxynitrous acid, had no effect. All these data support the concept that an interaction between NO and ROI promoted the production of S-nitroso-albumin by activated macrophages from infected mice.

Nitric oxide (NO) is an important bioregulatory mediator and possesses many physiological functions. NO and reactive nitrogen intermediates (RNI) are implicated in macrophage-derived cytostasis/cytotoxicity against tumor cells (15) and various intracellular and extracellular pathogens (1, 21, 22, 54) but also in the mechanisms of immunosuppression (45). NO circulates in plasma as S-nitrosothiols (16), mainly S-nitroso-albumin (49), and previous studies have elucidated the role of these intermediates in the long-distance effects of NO. Endothelium-derived relaxing-factor activities (19, 25, 34), inhibition of platelet functions (51), apoptosis (33), and anti-parasite activities of NO (36) are mediated through nitrosylated albumin. However, the mechanism of the in vivo formation of S-nitrosothiols remains unclear. It has been established that, under anaerobic conditions, NO does not react with cysteine, glutathione (26, 58), or serum albumin (6, 26). Therefore, RNI species seem to be needed for the nitrosylation process.Macrophages from Trypanosoma brucei brucei-infected mice produce high levels of NO (30). In addition, reactive oxygen intermediates (ROI), such as superoxide anion (O₂⁻) and O₂⁻-derived hydrogen peroxide (H₂O₂), are synthesized as a result of the oxidative burst (47) by macrophages from T. b. brucei-infected mice (12) or macrophages exposed to opsonized T. b. brucei (55). Since NO reacts with ROI (50), we investigated the interaction of the l-arginine→NO metabolism with the NADPH oxidase pathway leading to S nitrosylation of bovine serum albumin (BSA), resulting in the death of extracellular parasites.     

Diversity of Hemagglutination Phenotypes among P-Fimbriated Wild-Type Strains of Escherichia coli in Relation to papG Allele Repertoire

Data regarding the hemagglutination (HA) patterns of the three variants (classes I, II, and III) of the Escherichia coli adhesin PapG are conflicting. These HA patterns usually have been assessed for each papG allele separately with recombinant strains in slide HA assays. We rigorously evaluated an alternative microtiter tray HA assay and then used it to assess the HA of four erythrocyte types (human A₁P₁ and OP₁, rabbit, and sheep erythrocytes) by multiple wild-type E. coli strains representing the four naturally occurring combinations of the papG alleles, i.e., class I plus III, class III only, class II plus III, and class II only. The microtiter tray HA assay displayed significantly better reproducibility of intraobserver (83%) and interobserver (86%) results than did slide HA assays (39 and 73%, respectively). Novel findings from the study of 32 wild-type P-fimbriated strains included reproducible determinations of phenotypic diversity among different papG categories, among strains within each papG category, and from day to day for individual strains. There was also substantial overlap of phenotypes between papG categories I plus III and III only and between II plus III and II only. A class III papG recombinant strain’s HA pattern differed significantly from that of the wild-type class III strains. These data demonstrate that HA phenotypes of wild-type P-fimbriated E. coli strains can be reproducibly assessed by a microtiter HA assay and that they correspond broadly to papG genotype but in a more complex and varied fashion than previously recognized.P fimbriae, the adhesins most clearly implicated in the pathogenesis of extraintestinal infection due to Escherichia coli (6, 15), mediate Gal(α1-4)Gal-specific binding to host epithelial surfaces (3, 26, 28, 31–33) via the tip adhesin molecule PapG (9, 39). A possible explanation for the subtle differences in binding preferences among P-fimbriated E. coli strains that were noted following the discovery of P fimbriae (5, 7) came with the later discovery that PapG occurs in three molecular variants (38, 40, 42). The PapG variants, sometimes categorized in classes I, II, and III, bind preferentially to different Gal(α1-4)Gal-containing compounds (25, 60, 61) and are encoded by distinct alleles of the adhesin gene papG (42) (Table ​(Table1).1).

TABLE 1

Characteristics of the three PapG variants, as reported in the literature^a

Phenotypic and Genotypic Characterization of Clinical Strains of CDC Group IVc-2

CDC group IVc-2 is a gram-negative, oxidase-positive, nonfermentative bacillus that has been implicated in human infections, including septicemia and peritonitis. Biochemically it most closely resembles Bordetella bronchiseptica and Alcaligenes sp. Results of cellular fatty acid (CFA) and 16S rRNA gene analysis were combined with biochemical data to assist in identification and classification. The predominant CFAs were hexadecanoic acid (16:0), cis-9-hexadecanoic acid (16:1ω7c), cis-11-octadecanoic acid (18:1ω7c), and Δ-cis-9,10-methylenehexadecanoic acid (17:0cyc). Small amounts (2 to 5%) of 3-hydroxytetradecanoic acid (3-OH-14:0), tetradecanoic acid (14:0), 2-hydroxyhexadecanoic acid (2-OH-16:0), and Δ-cis-11,12-methyleneoctadecanoic acid (19:0cyc) were also consistently present. The highest 16S rRNA gene similarity was with Ralstonia eutropha and Ralstonia solanacearum. The CFA and 16S rRNA gene sequence data support the inclusion of CDC group IVc-2 in the recently created genus Ralstonia, which includes R. eutropha, R. pickettii, and R. solanacearum.CDC group IVc-2 is the vernacular name for a group of asaccharolytic, gram-negative, nonfermentative bacteria. The organism is found environmentally and is frequently associated with contaminated water sources (2, 3, 11, 13). Clinical cases of infection although rare, include 10 cases of bacteremia (1, 4, 5, 12, 17) and 2 cases of peritonitis (8, 25) in debilitated patients usually possessing indwelling catheters. Only a single case of infection in a patient lacking any predisposing pathologic condition has been reported (13).Biochemically, CDC group IVc-2 shares many features with Bordetella avium, Bordetella bronchiseptica, Oligella ureolytica (18), and Ralstonia eutropha (formerly Alcaligenes eutrophus) (16). Cellular fatty acid (CFA) content, however, indicates a close association with the rRNA group II pseudomonads, which include Burkholderia cepacia, Ralstonia pickettii (formerly Burkholderia pickettii), and Ralstonia solanacearum (formerly Burkholderia solanacearum) (22). In order to facilitate identification and to further clarify the taxonomic status of this organism, we evaluated the results of biochemical, CFA, and 16S rRNA gene sequence analysis.     

Molecular Cloning and Sequencing of Three Granulocytic Ehrlichia Genes Encoding High-Molecular-Weight Immunoreactive Proteins

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.Members of the genus Ehrlichia include species which have a tropism for mononuclear phagocytes (E. canis, E. chaffeensis, E. muris, E. sennetsu, and E. risticii) (33) and those which infect granulocytes (E. ewingii [33], E. phagocytophila [10, 33], E. equi [14, 26], and the recently discovered agent of human granulocytic ehrlichiosis [HGE] [3, 7]). Disease caused by granulocytic Ehrlichia (GE) is manifested by fever, lethargy, thrombocytopenia, and death, and many species from diverse geographical locations have shown evidence of natural infection, including horses (25, 27, 29, 38), dogs (24, 34, 35), small mammals (39, 40), and humans (4, 15).The similar host range and near identity of the 16S rRNA genes of E. phagocytophila, E. equi, and the HGE agent (7) have raised the possibility that these organisms represent a single species (2, 28). In addition, Dumler et al. (9) have shown that they share significant antigenicity by immunofluorescence and immunoblot assays. Objective methods for species classification, e.g., molecular genetic analysis, have not been readily available, primarily because of an inability to culture these ehrlichiae in vitro. However, we (reference 43 and unpublished data) and others (13) have recently demonstrated successful cultivation of GE isolates from dogs and humans, respectively.In this paper, we describe the use of purified GE obtained from in vitro culture of infected HL60 cells, a promyelocytic human cell line, to generate a genomic DNA library for expression screening with sera obtained from dogs experimentally infected with GE. The screening resulted in the isolation of recombinant clones containing complete genes encoding three putative proteins of GE, GE 160, GE 130, and GE 100 (named for apparent molecular mass in kilodaltons). One of these proteins, the 100-kDa protein, is similar in both glutamic acid content and repeated amino acid structure to an immunodominant 120-kDa E. chaffeensis protein (45). Both the 100- and 130-kDa granulocytic Ehrlichia proteins share some amino acid sequence homology to the 120-kDa E. chaffeensis protein.