Francisella tularensis Schu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.Much of the recent interest in Francisella tularensis, the etiological agent of tularemia, is due to concern about its potential use as an agent of bioterrorism coupled with an incomplete understanding of the molecular basis of its pathogenicity. F. tularensis is highly pathogenic by the pneumonic route, causing disease in humans with an inoculum as small as 10 organisms, and infection by this route carries a mortality rate of 30 to 60% if untreated (43, 67). Due to its extreme virulence and ease of aerosol dissemination, several nations have weaponized F. tularensis and the U.S. Centers for Disease Control and Prevention have classified this organism as a category A select agent (20). F. tularensis has a remarkably broad host range: it is capable of infecting over 250 known species from across the entire phylogenetic tree, including amoebae, insects, small mammals (such as rodents and lagomorphs), and primates (51). Tularemia is primarily a zoonosis, and humans are thought to be accidental hosts (23). The majority of human infections, the pneumonic infections reported on Martha''s Vineyard in 2000 (21) being an notable exception, are cutaneous, lead to ulceroglandular disease, and ensue following exposure to infected animals or animal products (47). The ability of F. tularensis to infect such a wide range of eukaryotes suggests that this organism either co-opts cellular mechanisms common to all hosts, has the requisite virulence genes to adapt to many different intraorganismal environments, or both.Despite the infectivity of Francisella for disparate hosts, relatively little is known about its virulence genetics. F. tularensis invades and replicates within many cell types: phagocytes, such as primary macrophages (human monocyte-derived macrophages [MDMs] and mouse bone marrow-derived macrophages) and macrophage-like cell lines (J774A.1 and THP-1), as well as in nonphagocytic cells such as bronchial airway epithelial cells, hepatocytes, human umbilical vein endothelial cells, and epithelium-derived tissue culture cell lines (i.e., HEp-2, A549, HBE, and HepG2) (17, 24, 31, 40, 61). Macrophages are known to bind and phagocytose F. tularensis using at least three receptors: complement receptor 3 (CR3), which binds to complement 3b (C3b) protein deposited upon the bacterium when exposed to fresh serum (14) (60); mannose receptor (MR) (60); and scavenger receptor A (53). Once internalized, F. tularensis organisms are able to alter intracellular trafficking of their phagosomes and prevent fusion with the lysosome, acquiring late endosomal markers such as lamp-1 transiently and altogether avoiding endosomes containing cathepsin D and S (15). Shortly thereafter, F. tularensis escapes from the phagosome and grows in the macrophage cytosol. The majority of genes known to be important for phagosomal escape and intracellular growth lie within the duplicated Francisella pathogenicity island (FPI), an ∼30-kb region of the chromosome carrying the igl and pdp operons (46). Mutants with mutations in many of the genes in the FPI, such as iglABCD, vgrG, and iglI, as well as pdpA and pdpD, are defective for intracellular growth in both primary and tissue culture cells in vitro, and these genes may encode a novel type VI secretion system (4). Mutational analysis implicates genes such as mglA, sspA, fevR, fslA, pmrA, and migR in regulation of FPI genes (5-8, 10, 38). However, knowledge of other virulence genes residing outside the FPI that play important roles in intracellular growth is limited.We undertook a high-throughput approach to identify genes important for the growth of virulent F. tularensis Schu S4 in primary human macrophages. F. tularensis has two major biovars: type B (F. tularensis subsp. holarctica), which is found throughout the Northern hemisphere, and type A (F. tularensis subsp. tularensis), which is found exclusively within North America and typically causes a more severe disease in humans than type B isolates. These biovars are further subdivided into clades that exhibit differences in virulence phenotypes (45). A significant research effort to date has focused on the virulence properties of the non-human pathogens Francisella novicida and the F. tularensis live vaccine strain (LVS), an attenuated F. tularensis subsp. holarctica variant generated in the Soviet Union in the 1950s. Although both of these strains are avirulent in healthy humans, they can grow in some human cells and cell lines in vitro and are virulent in the mouse model of infection, properties that make them attractive models for studying the pathogenesis of tularemia. Several random transposon mutant libraries generated in F. novicida and F. tularensis LVS (41, 50, 55, 66, 68, 70) have been screened using both in vitro tissue culture models and mice. In addition, Qin and Mann generated an ∼700-member Tn5 (EZ-TN) mutant library in the type A strain F. tularensis Schu S4 and screened each clone through the hepatocyte-like cell line HepG2 to detect mutants defective in intracellular growth. Among the genes identified in this screen is FTT1236 (55).Lipopolysaccharide (LPS) is a major component in the outer membranes of Gram-negative organisms, and O antigens (O Ags) are known virulence factors of many pathogenic bacteria that function in part to protect against damage by serum complement and antimicrobial peptides as well as mask bacterial surface antigens (16). LPS is composed of a lipid A moiety that secures it to the Gram-negative outer membrane on which a core polysaccharide and O Ag are assembled. Whereas the LPS of most bacterial species is recognized as a pathogen-associated molecular pattern (PAMP) through MD-2/TLR4 and is a potent activating signal for the innate immune system, the LPS of F. tularensis is nearly inert (27). Of note, the lipid A of F. tularensis has an atypical structure, does not bind to LPS-binding protein (LBP), and is therefore not recognized by TLR2 or TLR4 (3, 28, 52). In addition, the F. tularensis O Ag is structurally distinct from that of F. novicida (43). Although our understanding of the genetics of O-Ag biosynthesis in F. tularensis is incomplete, it is known that wbt operon mutants of F. tularensis LVS do not express an O Ag. These strains bind C3b more avidly and are more sensitive to bile salts and to complement-mediated lysis than wild-type strains (13, 39). These mutants are also defective for intracellular growth in J774A.1 cells and exhibit reduced virulence and dissemination in mice (41, 56, 62, 69). Of note, an LVS FTL0706 (designated FTT1238 in F. tularensis Schu S4) mutant lacks an O Ag and exhibits decreased replication within, and is cytotoxic to, J774A.1 cells (41). A corresponding Schu S4 FTT1238 mutant also lacks an O Ag, but its virulence properties have not yet been described.In many pathogenic bacteria, capsular polysaccharides also contribute to serum resistance yet, unlike O Ags, diminish phagocytosis (16). Francisella has long been thought to have an extracellular structure that resembles that of a capsular polysaccharide. Observed by Hood in 1976 (30), the nature of this capsule has remained elusive. Acridine orange treatment was used to produce an undefined LVS mutant that appears to lack an extracellular polysaccharide structure (58). This strain was designated Cap⁻ (alternately termed “rough” in more recent work) and is serum sensitive (13, 62). Furthermore, expression of the capsular polysaccharide was observed to be increased by repeated passage of LVS on Chamberlain''s defined medium, which in turn increased virulence in mice (12). Recently, our group has identified a capsular polysaccharide using a new monoclonal antibody that recognizes this structure as distinct from LPS O Ag. This capsule has immunological properties distinct from those of purified LPS and is also a potential vaccine (2).Advances in transposon delivery systems in our lab and others allowed us to generate a near-saturating random transposon mutant library in strain Schu S4 that is capable of high-throughput screening using the transposon site hybridization (TraSH) system previously used by Weiss et al. for F. novicida (70). Here we describe the utilization of this genetic system to identify and characterize new virulence genes important for F. tularensis Schu S4 entry and growth in human MDMs. Among the genes we identified is a locus required for LPS O-Ag and capsular polysaccharide biogenesis and for serum resistance and intracellular growth within MDMs. We further show that defects in intracellular growth are due to their induction of premature macrophage death, which deprives mutant organisms of their replicative niche.     

Direct and indirect impairment of human dendritic cell function by virulent Francisella tularensis Schu S4

The gram-negative, facultative intracellular bacterium Francisella tularensis causes acute, lethal pneumonic disease following infection with only 10 CFU. The mechanisms used by the bacterium to accomplish this in humans are unknown. Here, we demonstrate that virulent, type A F. tularensis strain Schu S4 efficiently infects and replicates in human myeloid dendritic cells (DCs). Despite exponential replication over time, Schu S4 failed to stimulate transforming growth factor beta, interleukin-10 (IL-10), IL-6, IL-1beta, IL-12, tumor necrosis factor alpha, alpha interferon (IFN-alpha), and IFN-beta throughout the course of infection. Schu S4 also suppressed the ability of directly infected DCs to respond to different Toll-like receptor agonists. Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Lipopolysaccharide (LPS) from Schu S4 was present in Schu S4-conditioned medium. However, Schu S4 LPS was weakly inflammatory and failed to induce suppression of DCs at concentrations below 10 microg/ml, and depletion of Schu S4 LPS did not significantly alleviate the inhibitory effect of Schu S4-conditioned medium in uninfected human DCs. Together, these data show that type A F. tularensis interferes with the ability of a central cell type of the immune system, DCs, to alert the host of infection both intra- and extracellularly. This suggests that immune dysregulation by F. tularensis operates on a broader and more comprehensive scale than previously appreciated.     

Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpC and ΔpdpC ΔpdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.     

Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b^high macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.     

The Presence of CD14 Overcomes Evasion of Innate Immune Responses by Virulent Francisella tularensis in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.Daily insults of inhaled particulate and foreign antigens into the lungs could result in devastating inflammation. However, the lung counters these attacks by tightly regulating inflammatory responses. This regulation occurs both in the form of inhibitory molecules, such as surfactants that dampen macrophage and dendritic cell (DC) responsiveness, as well as production of immunosuppressive cytokines, such as transforming growth factor β (TGF-β) (1, 10, 15, 33, 34). Given the immunosuppressive nature of the lung environment, it is not surprising that pathogens capable of causing lethal disease following inhalation, such as Francisella tularensis, take advantage of this property for rapid replication while evading detection by the host immune response.Francisella tularensis is a Gram-negative, facultative intracellular bacterium and is the causative agent of tularemia. Pneumonic tularemia is an acute, lethal disease mediated by F. tularensis, following inhalation of as few as 10 to 15 bacteria in mice and humans (20, 49). Surprisingly, despite the rapidity by which pulmonary F. tularensis infections progress, there is little to no evidence of inflammation in the lung until the very end stage of infection (11, 20, 54). The mechanisms by which F. tularensis replicates within the lung while evading detection by the host are not well understood and represent an important hurdle for the development of novel therapeutics and vaccines directed against tularemia.Recently we have demonstrated that, similar to pulmonary cells, conventional human dendritic cells (hDC) derived from peripheral blood fail to secrete proinflammatory cytokines following infection with virulent F. tularensis strain SchuS4 (17). The absence of production of proinflammatory cytokines was not due to the inability of the cells to become infected or support replication of strain SchuS4, nor was it due to induction of apoptosis among infected hDC. There are multiple explanations for the lack of cytokine production in hDC and pulmonary cells following SchuS4 infection. One possibility is that these cells fail to detect F. tularensis during the initial phases of infection.CD14 is a glycosylphosphatidylinositol receptor that exists in both a membrane-bound form and a soluble form in vivo. CD14 is present on monocytes, most macrophages, fibroblasts, and neutrophils (39). Conventional human DC lose surface expression of CD14 following their differentiation from blood monocytes, primarily due to exposure to interleukin-4 (IL-4) (40). Similar to hDC, alveolar macrophages have been reported to have little to no CD14 present on their surface (9, 45). Soluble CD14 (sCD14) is abundant in serum. However, it is present at very low levels to nearly undetectable concentrations in the airways of mammals (47). CD14 (both in its soluble and membrane-bound forms) is best known as a coreceptor for lipopolysaccharide (LPS), facilitating optimal delivery of LPS to the Toll-like receptor 4 (TLR4)/MD-2 complex on the cell surface (35, 64). In addition to delivery of LPS to TLR4, CD14 has been described as an important coreceptor for delivery of other microbial antigens, including polyuronic acids from Pseudomonas, lipoteichoic acid (LTA) from Staphylococcus aureus, outer surface protein of Borrelia burgdorferi and WI-1 antigen of Blastomyces dermatitidis to TLR2 (reviewed in reference 58). Thus, CD14 is an important coreceptor for initiating inflammatory responses via TLR2 and TLR4 against a wide variety of bacterial and fungal diseases.In this report, we demonstrate that CD14 serves as a critical coreceptor for detection of F. tularensis SchuS4 during the initial stage of in vitro infection in hDC and primary human monocytes. Further, CD14 was also found to have an important role in the early in vivo detection and control of pulmonary infections with strain SchuS4. However, development of inflammatory responses associated with bacterial sepsis observed at late stages of SchuS4 infection were not dependent on the presence of CD14. Thus, in contrast to infections with other microbial pathogens where the presence of CD14 in the lungs is detrimental to control of infection, CD14 represents an important sensor to initiate protective host immune responses during pulmonary infections with strain SchuS4, but it is not required to elicit responses at the end of the disease process.     

Virulence of Streptococcus mutans: In Vivo Reversion of a Low-Virulence Mutant Results in Partial Displacement and Pathogenesis

A mutant of Streptococcus mutans 6715 wild type (WT), designated C4, has been shown previously to be defective in glucosyltransferase synthesis of insoluble glucan and to have low virulence in monoassociated gnotobiotic rats. The present investigation was concerned with the detection of WT-like variants of C4 in monoassociated rats, the supplantation of C4 by these WT-like organisms, and finally, the pathogenic potential of these WT-like organisms in gnotobiotic rats. In the first series of longitudinal studies with C4-monoassociated rats, WT-like organisms were detected at a low frequency (0.001%) in oral swab samples from only one of four cages of animals analyzed on day 7 after infection (age 27 days). The frequency of variants isolated from animals in the one cage increased, and by age 45 days these organisms represented approximately 1% of the mandibular plaque flora. After random redistribution of rats in the four cages (age 45 days), microbial analysis of oral swab samples (age 60 days) demonstrated the presence of variants in samples taken from rats in all four cages. The frequency of recoverable variants increased in older animals (age 90 days) and correlated with high caries activity. WT-like organisms were transmissible, since offspring (age 45 days) from these animals had high levels of variants as well as high caries activity. Similar results were obtained in a second longitudinal study; however, variants, although present in all four cages, were not detected until rats were 45 days old. All variant isolates exhibited morphological, biochemical, and in vivo virulence characteristics more similar to S. mutans 6715 WT than to C4. In vitro mixing experiments with C4 and either WT or a selected variant suggested that C4 was rapidly displaced by WT organisms. The results of this investigation demonstrate that the glucosyltransferase-defective, low-virulence C4 reverts to virulent WT-like organisms in vivo which compete more favorably for smooth surfaces than C4. Subsequently, these variants reached significant numbers in plaque which correlated with increased dental caries.     

Effects of CD14, TLR2, TLR4, LPB, and IL-6 Gene Polymorphisms on Chlamydia pneumoniae Growth in Human Macrophages In Vitro

Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro . The susceptibility of macrophages from healthy individuals to C. pneumoniae infection is highly variable. In this study, we evaluated the effects of innate immunity genes CD14 −260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL6 −174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 −260 C>T TT genotype cells and the difference to CC and CT genotypes was statistically significant ( P  = 0.032 and 0.022 respectively). The G-allele of the IL6 −174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes ( P  = 0.018). TLR2 Arg 753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.     

A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine

Francisella tularensis subsp. tularensis (type A) strain SCHU S4 is a prototypic strain of the pathogen that is highly virulent for humans and other mammals. Its intradermal (i.d.) 50% lethal dose (LD50) for mice is <10 CFU. We discovered a spontaneous mutant, designated FSC043, of SCHU S4 with an i.d. LD50 of >10(8) CFU. FSC043 effectively vaccinated mice against challenge with a highly virulent type A strain, and the protective efficacy was at least as good as that of F. tularensis LVS, an empirically attenuated strain which has been used as an efficacious human vaccine. Comparative proteomics was used to identify two proteins of unknown function that were identified as defective in LVS and FSC043, and deletion mutants of SCHU S4 were created for each of the two encoding genes. One mutant, the DeltaFTT0918 strain, failed to express a 58-kDa protein, had an i.d. LD50 of approximately 10(5) CFU, and was found to be less capable than SCHU S4 of growing in peritoneal mouse macrophages. Mice that recovered from sublethal infection with the DeltaFTT0918 mutant survived when challenged 2 months later with >100 LD50s of the highly virulent type A strain FSC033. This is the first report of the generation of defined mutants of F. tularensis subsp. tularensis and their use as live vaccines.     

Importance of PdpC,IglC, IglI,and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglI or ΔiglC mutants, although the former strain replicated. The ΔiglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.     

两株Wa株人源轮状病毒NSP4蛋白的表达及其致ICR小鼠腹泻的毒力比较

目的研究两株Wa株人源轮状病毒NSP4蛋白139位氨基酸位点的变异对毒力的影响。方法从2002～2004年昆明地区婴幼儿轮状病毒感染者大便样品中,通过RT-PCR,PCR扩增了22株轮状病毒株的NSP4全长基因、cDNA序列,并对cDNA序列测序,发现这些病毒株的NSP4氨基酸序列高度保守,仅在79位、139位氨基酸位点存在差异。选取139位氨基酸不同(V/I)的两株病毒株,在E.coli BL21中,用pGEX5X-1载体融合表达了两株病毒的86～175位氨基酸(GST-NSP4_(86-175);并酶切融合头得到两种NSP4_(86-175)蛋白,在ICR乳鼠中比较这4种不同蛋白致小鼠腹泻的差异。结果发现GST-NSP4_(86-175)蛋白毒性比NSP4_(86-175)蛋白强,而139位氨基酸不同(V/I)的两株病毒株的NSP4_(86-175)蛋白毒性没有明显的差异。结论139位氨基酸位点的变异并不影响NSP4蛋白的毒力改变。     

Interaction of a Lymphokine with Normal Human Macrophages Results in Release of a Suppressor Factor for Mitogen-Induced Immunoglobulin Synthesis

Normal human macrophage/monocyte cultures exposed to a suppressor factor produced by concanavalin A-activated T cells (T-SF), respond by releasing after 72 h a macrophage-derived suppressor factor (M phi-SF). The M phi-SF inhibits pokeweed mitogen-induced Ig synthesis but not T- or B-cell proliferation. Cycloheximide treatment of the macrophages does not interfere with generation of the M phi-SF, suggesting that de novo synthesis is not required. The factor is not preformed, for virgin macrophages do not contain M phi-SF, but it appears in macrophage cell lysates after exposure to T-SF. The production of the M phi-SF is inhibited by the presence of 2-mercaptoethanol. Both T-SF and M phi-SF are L-rhamnose inhibitable, and the M phi-SF appears to be released as a high molecular weight complex which is dissociable into a low molecular weight form of a size similar to the T-SF, i.e. approximately 20,000. The T-SF induced M phi-SF has some similarities with soluble immune response suppressor (SIRS) but differs from this factor in its lack of effect upon lymphocyte proliferation failure to induce conversion of T-SF to M phi-SF by treatment with H2O2.     

Impaired Antigen-Specific B-Cell Response and Altered Splenic Microstructure in Mice Following Continuous Administration of IL-4 In Vivo

The effect of long term in vivo administration of IL-4 on the induction of antigen-specific B cells, the splenic microenvironment and the yield of antigen-specific antibody producing hybridomas was studied. Immunization with DNP-KLH, followed by 12 weeks continuous IL-4 treatment resulted in increased numbers of total splenic (non-DNP) IgM and IgG AFC (antibody forming cells) on day 5 after booster, whereas the DNP-specific IgG and IgG1 AFC were reduced compared to age-matched control animals not treated with IL-4. In addition, an almost 300-fold increase in non-DNP IgE was found while the IgE anti-DNP response was minimal.
When the splenic cells were used in a fusion protocol, a relative decrease in yield of antigen-specific hybridomas was found in the long term IL-4 treated mice. Immunohistological staining of spleen sections from mice treated with IL-4 up until the time of booster revealed reduced B-cell follicle area and germinal centre numbers. These results show that extensive IL-4 treatment reduced antigen-specific B-cell formation and suggests a reduction in the number of B cells entering the memory B-cell pathway in the spleen.     

A Spontaneous Deletion within the Desmoglein 3 Extracellular Domain of Mice Results in Hypomorphic Protein Expression,Immunodeficiency, and a Wasting Disease Phenotype

Desmoglein 3 is a transmembrane component of desmosome complexes that mediate epidermal cell-to-cell adhesion and tissue integrity. Antibody blockade of desmoglein 3 function in pemphigus vulgaris patients leads to skin blistering (acantholysis) and oral mucosa lesions. Desmoglein 3 deficiency in mice leads to a phenotype characterized by cyclic alopecia in addition to the dramatic skin and mucocutaneous acantholysis observed in pemphigus patients. In this study, mice that developed an overt squeaky (sqk) phenotype were identified with obstructed airways, cyclic hair loss, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. Single-nucleotide polymorphism–based quantitative trait loci mapping revealed a genetic deletion that resulted in expression of a hypomorphic desmoglein 3 protein with a truncation of an extracellular cadherin domain. Because hypomorphic expression of a truncated desmoglein 3 protein led to a spectrum of severe pathology not observed in mice deficient in desmoglein 3, similar human genetic alterations may also disrupt desmosome function and induce a disease course distinct from pathogenesis of pemphigus vulgaris.Tissues experiencing mechanical stress are held together by supramolecular desmosome complexes composed of type I transmembrane glycoproteins from the desmoglein (Dsg) and desmocollin (Dsc) families of epithelial cadherins.¹ The extracellular protein domains of the desmogleins and desmocollins consist of four approximately 110–amino acid homologous cadherin domains (EC1 to EC4) and a proximal extracellular anchor domain. The desmosomal cadherins are differentially expressed in different cellular layers of select tissues. For example, desmoglein 1 (Dsg1) is expressed at high levels in the suprabasal outer layer of skin epidermis (stratified squamous epithelia) and thymus.^2,3 Desmoglein 2 (Dsg2) is expressed ubiquitously in the basal layers of the epidermis and desmosome-enriched cardiac tissues.^2,3 Desmoglein 3 (Dsg3) is primarily expressed by epidermal keratinocytes in the basal and immediate suprabasal layers of skin and in the basal layer of the mucosal epithelium of the mouth, eyes, and trachea.^2,3 Six Dsg and three Dsc genes are found in mice, with four Dsg and three Dsc genes in humans.⁴ Homophilic and heterophilic interactions between the Dsg and Dsc proteins lead to the formation of tightly packed desmosomal complexes.⁵The desmogleins are involved in human disease pathogenesis. Cleavage of the extracellular domain of Dsg1 by Staphylococcus aureus exfoliating toxin results in bullous impetigo in children, manifesting as skin blisters due to detachment (acantholysis) of the outer layer of epidermis.⁶ Inactivation of either the Dsg2 or Dsc3 gene is embryonic lethal.^7,8 The development of circulating IgG autoantibodies against Dsg1 or Dsg3 can result in the human autoimmune blistering disorders pemphigus foliaceus and pemphigus vulgaris due to reduced desmoglein expression on the cell surface.⁹ In patients with pemphigus foliaceus, acantholysis within the superficial layers of the epidermis results in clinical lesions that resemble those observed in lupus erythematosus and seborrheic dermatitis patients. Pemphigus foliaceus patients experience no oral involvement and have no associated mortality. By contrast, patients with pemphigus vulgaris experience acantholysis within the deep basilar and parabasilar portions of the epidermis, which results in lesions that may resemble toxic epidermal necrolysis. With pemphigus vulgaris, there is significant oral and skin involvement and untreated patients experience considerable mortality.Although mutations in the human Dsg3 gene have not been described, gene inactivation in Dsg3^−/− mice leads to fragility of the skin and oral mucous membranes, analogous to those found in pemphigus vulgaris patients,¹⁰ along with runting and progressive hair loss.¹¹ Two independent spontaneous mutations within mouse chromosome 18 affecting exons encoding the Dsg3 cytoplasmic domain also ablate protein expression and lead to a Dsg3^−/− phenotype.^10,12,13 Herein, a spontaneous gene mutation was identified in mice that develop an overt squeaky (sqk) phenotype with cyclic hair loss, obstructed airways, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. This phenotype was mapped to a partial exon deletion in the Dsg3 gene that results in hypomorphic expression of a truncated Dsg3 protein, which leads to a severe spectrum of pathology not observed in Dsg3^−/− mice.     

Effect of an Extract Based on the Medicinal Mushroom Agaricus blazei Murill on Release of Cytokines, Chemokines and Leukocyte Growth Factors in Human Blood Ex Vivo and In Vivo

An immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM) has been shown to stimulate mononuclear phagocytes in vitro to produce pro-inflammatory cytokines, and to protect against lethal peritonitis in mice. The present aim was to study the effect of AbM on release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days in healthy volunteers. The 17 signal substances examined were; T helper 1 (Th1) cytokines [interleukin (IL)-2, interferon (IFN)-γ and IL-12], T helper 2 cytokines (IL-4, IL-5 and IL-13), pleiotropic (IL-7, IL-17), pro-inflammatory [IL-1β, IL-6, tumour necrosis factor (TNF)-α (mainly produced by Th1 cells)] – and anti-inflammatory (IL-10) cytokines, chemokines [IL-8, macrophage inhibitory protein (MIP)-1β and monocyte chemoattractant protein (MCP)-1] and leukocyte growth factors [granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor]. After stimulation of whole blood ex vivo with 0.5–5.0% of a mushroom extract, AndoSan™ mainly containing AbM , there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-α). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1β (97%), TNF-α (84%), IL-17 (50%) and IL-2 (46%). Although not significant, there was a trend towards reduced levels for IL-8, IFN-γ and G-CSF, whilst those of the remaining nine cytokines tested, were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large, complex and bioactive β-glucans across the intestinal mucosa to the reticuloendothelial system and blood.     

Frameshift Mutations in a Single Novel Virulence Factor Alter the In Vivo Pathogenicity of Chlamydia trachomatis for the Female Murine Genital Tract

Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans. Infection of ocular and genital mucosal columnar epithelial cells causes blinding trachoma and sexually transmitted infection (STI), respectively. Trachoma is the leading cause of preventable blindness, afflicting an estimated 84 million individuals worldwide (29), and is recognized as one of the world''s most neglected infectious diseases (15). Chlamydial STIs are epidemic globally, with an estimated 92 million new cases occurring each year (30). Chlamydial STI can result in pelvic inflammatory disease and tubal factor infertility and is an important risk factor in the transmission of HIV (11). Persistent infection and reinfection of human chlamydial strains drive irreversible inflammatory responses, fibrosis, and damaging scarring. Control of these diseases will require the development of a vaccine, for which there has been nominal progress. Regrettably, a vaccine will not be forthcoming until the pathophysiology of these globally important diseases is better understood.Understanding the pathophysiology and immunology of human chlamydial infection and disease has been hampered by the lack of a suitable small-animal model for human C. trachomatis strains. There are excellent guinea pig and murine models that utilize naturally occurring chlamydial challenge strains. However, rodents are exceptionally resistant to genitourinary tract infection by human C. trachomatis isolates, where infections are characteristically acute and cleared rapidly by murine-specific innate immune mechanisms (17). Previous comparative pathogenomic studies highlighted the major differences between human and mouse gamma interferon (IFN-γ)-mediated antimicrobial effectors and portrayed how human and mouse chlamydial strains evade these host-specific defense mechanisms by utilizing a unique set of pathogen-specific genes that reside in the chromosome''s plasticity zone (1, 4, 17). Little, however, is known about other C. trachomatis genes that influence virulence for the mouse. Human isolates are commonly cultivated in vitro prior to infection of animals; therefore, virulence genes might be lost as a consequence of negative selective pressure. Identifying such genes could lead to the development of a more practical murine model that utilizes human challenge isolates. We tested this hypothesis and identified human strains following in vivo infection of C3H/HeJ female mice that exhibited marked differences in virulence for the female mouse genital tract. We show that the in vivo-selected virulent strain exhibits superior infection and disease-causing characteristics that mimic those in human infection. A comprehensive genomic analysis of attenuated and virulent isolates shows that mutations in a single gene are responsible for altering the pathogenesis of C. trachomatis infection.