Antiphagocytic Activity Research Articles

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea. At 72 h postadministration and after, some animals showed mild to moderate dyspnea and tachypnea. Autopsies of all animals were performed, including a histopathological examination of the lungs. At 72 h after administration, two distinct morphological groups were identified histopathologically. Severe necrotizing pneumonia characterized by the presence of abundant fungal hyphae was seen in animals that died spontaneously within 48 h postadministration and rats with bronchopneumonia and was characterized by the presence of numerous fungal spores. There was an obvious difference in pathogenicity among the strains of A. fumigatus. Strains D and K were more pathogenic, and only the rats exposed to these strains showed the presence of fungal hyphae in the lungs. The mycotoxin gliotoxin that is produced by A. fumigatus and has antiphagocytic activity was not detected in the spores from any of the A. fumigatus strains.

Replication of donor lymphocytes in recipients is not essential for the passive transfer of allergic encephalomyelitis

Gliotoxin is a fungal metabolite belonging to the class of epipolythiodioxopiperazines which possesses both immunomodulating and anti-phagocytic activities. We have examined the effect of gliotoxin on passively induced allergic encephalomyelitis and report here that pulse treating activated experimental allergic encephalomyelitis (EAE) effector lymphocytes with gliotoxin inhibits, in a dose-dependent manner, their ability to transfer disease. Cells treated with 300 ng/ml are unable to replicate in vitro in response to concanavalin A stimulation, nor did they produce interleukin-2 (IL-2) following stimulation. Furthermore this concentration of gliotoxin also causes complete fragmentation of genomic DNA in treated cells, yet these cells are still capable of transferring clinical EAE. These data suggest that replication of donor lymphocytes in the recipient is not essential for the development of EAE.

Demonstration of capsule in a strain of Staphylococcus hyicus by an electron microscope.

A compact type variant designated as strain ST67V was isolated by a high-temperature subculture method from strain ST67P of Staphylococcus hyicus, which was a diffuse type in serum-soft agar. The parent strain was relatively virulent in mice and resisted ingestion by mouse peritoneal cells. The variant strain, however, was avirulent in mice and no antiphagocytic activity was observed in the peritoneal cavity. In ultra-thin sections of the organisms treated with anti-ST67P rabbit anti-serum conjugated with ferritin, the outermost layer of the cell wall of the parent strain was covered with a well-defined capsule while no capsule was shown in the variant strain.

Demonstration of an outer membrane protein with antiphagocytic activity from Pasteurella multocida of avian origin.

A strain of Pasteurella multocida of avian origin was found to inhibit phagocytosis of Candida albicans by mononuclear phagocytes in vitro. Whole-cell lysates of P. multocida showed this effect, as did a 50-kilodalton (kDa) protein eluted from sodium dodecyl sulfate-polyacrylamide gels obtained by electrophoresis of whole-cell lysates. Heat, digestion with trypsin, and antibody specific for this 50-kDa protein neutralized the antiphagocytic effects of P. multocida, of the whole-cell lysates, and of the 50-kDa protein itself. Evidence that this protein was in the outer membrane of the bacterial cell included the findings that (i) treatment of encapsulated or unencapsulated P. multocida with trypsin reduced the antiphagocytic effect; (ii) whole-cell lysates prepared from trypsinized, unencapsulated P. multocida had reduced antiphagocytic activity; and (iii) antibody to outer membrane proteins neutralized the antiphagocytic effect. Turkeys given antibodies specific for the 50-kDa outer membrane protein were protected against lethal challenge with P. multocida.

Interaction of Escherichia coli with polymorphonuclear leukocytes in pathogenesis of urinary tract infection in mice.

Two type 1 fimbria-producing strains of Escherichia coli, 31-B and K12W1-3, and two type 1 fimbriae-defective mutants derived from 31-B, BH5 and BH9, were compared for their capacity to induce vesical infection in mice undergoing water diuresis and to interact in vitro with murine peritoneal exudate polymorphonuclear leukocytes (PMN). Strains 31-B and BH5 caused rapid bacterial multiplication in the bladder wall after being inoculated intrabladderly; their log-phase cells grown at 37 degrees C, in striking contrast to their stationary-phase or 17 degrees C-grown cells, resisted phagocytic killing by PMN in the presence of normal murine serum. Strains K12W1-3 and BH9 failed to cause vesical infection, and their cells were always susceptible to the opsonophagocytic killing by PMN irrespective of the growth conditions. Nevertheless, the log-phase cells of the three isogenic strains, 31-B, BH5, and BH9, grown at 37 degrees C gave almost the same chemiluminescent response patterns during incubation with PMN in normal serum. The phagocytic resistance in strains 31-B and BH5 was eliminated by briefly treating bacterial cells with EDTA. These results suggest that the two virulent strains may express an antiphagocytic activity during their growth in the bladder and continue to stimulate the oxidative metabolic burst of PMN without being ingested and killed, and that the antiphagocytic activity may be related to a bacterial surface component(s) that is removed by EDTA.

Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H.

Isolated complement components were used to study the regulation of the alternative complement pathway C3 convertase (EC 3.4.21.47), also called C3b,Bb, on M protein-carrying (M+) and M protein-lacking (M-) streptococci. Neither M- nor M+ streptococci directly affected the formation or dissociation of the surface-bound C3b,Bb or the inactivation of surface-bound C3b by factor I. However, the activity of the serum control protein of the alternative complement pathway, factor H, in controlling streptococcus-bound C3b and C3b,Bb was 6-8 times stronger on M+ organisms than on M- organisms. Furthermore, M+ streptococci of different serotypes and purified streptococcal M6 protein were shown to selectively bind factor H, the dissociation constants ranging from 4.5 X 10(-6) M to 6 X 10(-7) M. We conclude that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H. Binding of a regulatory protein appears to be a previously unrecognized route by which a pathogen is able to evade alternative pathway activation.

The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.

Suppression of yeast ingestion by dexamethasone in macrophage cultures: Evidence for a steroid-induced phagocytosis inhibitory protein

The mechanism by which glucocorticoid steroids suppress yeast phagocytosis in cultures of resident and thioglycollate-elicited murine peritoneal macrophages was examined. Time course and dose-response studies demonstrated that the phagocytic capacity of resident macrophages was suppressed by dexamethasone to the same extent in both newly established cultures and cultures that were incubated for several days. In contrast, relative to newly established cultures of elicited cells that were treated with the drug, elicited macrophages that were incubated for at least 1 day prior to exposure to dexamethasone, exhibited enhanced sensitivity to the action of the steroid. Steroid-induced phagocytic inhibitory responses were blocked by the metabolic inhibitors cycloheximide and actinomycin D. The suppression of phagocytosis by dexamethasone was mediated by a factor, present in the cellular homogenates of steroid-treated macrophages, that was partially purified by Sephadex G-25 chromatography. Since the phagocytic inhibitory activity in these homogenates was destroyed following exposure to heat and trypsin, the factor has been named phagocytosis inhibitory protein (PIP). The antiphagocytic activity of PIP was neutralized by treatment with RM23, a monoclonal antibody directed against lipocortin. These results support the hypothesis that the suppression of yeast ingestion is mediated by the action of PIP, which is induced in dexamethasone-treated macrophage cultures. Moreover, PIP appears to belong to the lipocortin family of phospholipase inhibitory proteins.

Species specificity of Haemophilus pleuropneumoniae hemolysin demonstrated in the cytocidal and anti-phagocytic effects.

Hemolysin was prepared from the culture supernatant of Haemophilus pleuropneumoniae, and its cytocidal and anti-phagocytic effects were investigated with pulmonary macrophages (PMP) obtained from the pig, mouse, guinea pig, or cotton rat. The hemolysin demonstrated both cytocidal and anti-phagocytic activities with the cotton rat, guinea pig, and pig PMP cells, but not with the mouse PMP cells. The species specificity in the cytocidal and anti-phagocytic effects with the PMP cells may be important in analyzing for the pathogenesis of the organisms to various animal species.

Size variation of the M protein in group A streptococci.

In addition to the type-specific antigenic variation that is a well-known characteristic for the group A streptococcal M protein, we have now found that the M molecules vary with respect to their molecular size, both between M types and within an M type. By the use of an M6 monoclonal antibody, which crossreacts with 20 different M protein types, and antibodies to the N-acetyl glucosamine determinant of the cell wall, we have been able to identify the M protein molecules released from the streptococcal cell wall with muralytic enzymes, particularly group C phage-associated lysin. Immunoblot analysis of the cell extract identified M protein molecules bound to various cell wall fragments, suggesting a peptidoglycan linkage for the M molecule. M protein extracted from 20 different streptococcal serotypes revealed size variations from 41,000 to 80,000 in molecular weight. This extreme variation is unusual for related proteins. Similar size variations in the M molecule were also found in random clinical isolates of type 6 streptococci. No size change was seen in M6 protein isolated from: (a) strains within a limited epidemic, (b) a strain passaged in mice 192 times, and (c) a strain passaged in the laboratory for 156 generations, suggesting that the observed variation is not a rapid process. The results indicate that, within the broad limits observed in this study, the size of the M protein may not be critical to the antiphagocytic activity of the molecule.

Identification of an Agent in Cultures of Aspergillus fumigatus Displaying Anti-phagocytic and Immunomodulating Activity in vitro

When cultured in vitro, Aspergillus fumigatus generated a metabolite(s) with anti-phagocytic activity as tested by macrophage adherence to plastic and phagocytosis of particulate matter. The metabolite(s) appeared after 3 d culture and reached a peak concentration after 5-6 d. The action of the anti-phagocytic agent(s) was rapid (5-15 min) and appeared not to alter membrane permeability or cause rapid cell death. Treatment of stimulator spleen cells with the agent(s) inhibited their ability to induce alloreactive and major histocompatibility complex restricted cytotoxic T cells. The metabolite(s) was chloroform-soluble and separated into three biologically active compounds on thin-layer chromatography. These compounds were purified greater than 1000-fold and one of them was identified as gliotoxin, a known metabolite of A. fumigatus, based upon NMR and IR spectroscopy, mass spectrometry, biological properties and other data.

Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus.

Colonies which varied in opacity were isolated from the four strains of Vibrio vulnificus. Opaque and translucent colonial types of the strains were distinguished from the corresponding parent strains. Variation in the opacity of colonies formed by each strain was accompanied by variation of capsular material formation, which was clarified by electron microscopy of the organisms stained with ruthenium red. The opaque-type colonies of the strains had capsular materials. On the other hand, three translucent-type colonies had no observable capsular materials, and one had incomplete capsular materials, in contrast to the corresponding opaque type. The corresponding opaque and translucent types of the strains were compared for points of virulence in mice and guinea pigs. By having capsular materials, the bacterial strains acquired resistance to serum bactericidal action, antiphagocytic activity, high lethality for mice, and strong invasiveness in the subcutaneous tissue of guinea pigs. Capsular materials of V. vulnificus were considered to be important for the expression of virulence.

THE GENETIC CONTROL OF VIRULENCE IN GROUP A STREPTOCOCCI

Recently, we reported that conjugal transfer of plasmid pERL1, determining i.a. erythromycin resistance (Emr), into group A streptococci could trigger the expression of anti-phagocytic activity, adhesiveness, opacity factor and capacity to bind immunoglobulin Fc-parts and beta 2-microglobulin. In the present study, ethidium bromide treatment of Emr transconjugants allowed the selection of "cured", erythromycin sensitive (Ems) mutants. This procedure did not affect the expression of the abovementioned characteristics. However, when plasmid pERL1 was again transferred to two such mutants, the "secondary", Emr transconjugants obtained showed lack of each of these properties. Our experiments thus demonstrated a "switch-on" as well as a "switch-off" effect, exerted by the same plasmid, pERL1, on some major pathogenic properties of group A streptococci.

THE GENETIC CONTROL OF VIRULENCE IN GROUP A STREPTOCOCCI

The erythromycin-resistance marker of two plasmids, pSM19035 and pERL1, could be transferred by conjugation in matings within group A streptococci and between group H and group A streptococci, with a frequency of 10(-5) to 8 X 10(-7). Two of the recipient strains used, 22v-1 and 22v-2, showed traces of opacity factor (OF) activity. This activity increased markedly upon transfer of both plasmids to the strains, whereas a number of other recipients, viz. 22h-21, 12Teiko-1 and Challis 6-1, remained OF-negative after conjugation. Furthermore, the transconjugants resulting from matings using strains 22v-1 and 22v-2 as recipients expressed anti-phagocytic activity. Attempts were made to type the transconjugants but they did not belong to type M12, M22 (the types from which the parents were derived) M3 or M1. Concomitant with the expression of anti-phagocytic activity, IgG and IgA Fc-receptor activity occurred in the transconjugants of 22v-1 and 22v-2, whereas the donor and recipient cells were without receptors. It was not possible to demonstrate extrachromosomal DNA in transconjugants possessing anti-phagocytic, OF or Fc-receptor activity, although they retained their ability to serve as donors of the Emr marker. It is suggested that the triggering by plasmids of anti-phagocytic activity, OF and IgG and IgA Fc-receptors is due to insertion of plasmid DNA into the chromosome.

THE GENETIC CONTROL OF VIRULENCE IN GROUP A STREPTOCOCCI

Transfer of five plasmids was studied in 30 different mating systems, involving streptococci of groups A, D and H. The erythromycin resistance marker (Emr) of all five plasmids was transferable at frequencies ranging from 8 X 10(-7) to 2 X 10(-4). All original donor strains except SM60 (pERL1) contained phage activity, but evidence was obtained favouring conjugation over transduction as the mechanism of transfer. The process was rec-independent. The transconjugants were able to serve as donors of the Emr-marker in secondary matings. A shortened derivate (pSM10) of the plasmid pSM10419 could also be transferred. Finally, the presence of the plasmid pSM15346 influenced the expression of the capacity to adhere to human epithelial cells and the resistance to UV-light of the host, whereas anti-phagocytic activity and production NADse and of opacity factor was unaffected by the plasmid.

Spikes and fimbriae: alpha-helical proteins form surface projections on microorganisms.

Two basic alpha-helical plans appear to characterize portions of the proteins projecting from the surface of many microorganisms. Structure A is related to that of the antibody-binding protein A of Staphylococcus aureus, in which a single polypeptide chain doubles back to form an Anti-parallel domain; the other, structure M, is related to the M proteins of group A streptococci, in which Multiple parallel alpha-helical chains interlock in a coiled-coil conformation. A projection about 100 A long may be organized on either plan, but very long projections are likely to be of the M pattern. The alpha-helical motif may account for the length of these spikes, with other functions such as anchoring or antiphagocytic activity located in domains of different structure.

Streptococcal M protein extracted by nonionic detergent. III. Correlation between immunological cross-reactions and structural similarities with implications for antiphagocytosis.

Three immunologically cross-reactive and non-cross-reactive streptococcal M proteins were analyzed by a chromatographic tryptic peptide mapping system. The results indicate that cross-reactions correlate with the extent of structural similarity among the M protein molecules analyzed. The data also reveal that free lysine is released by the action of trypsin from these three M proteins, suggesting a common lys-lys or arg-lys sequence. In addition, only one peptide has been found to be common within all three M types. This limited structural relatedness among the three M proteins examined indicates that sequence variation plays a major role in the immunological specificity of the M antigens. However, despite sequence variation, all M protein molecules have a common antiphagocytic activity. The fact that no common opsonic antibody has yet been found, even against limited M types, argues against this biological activity being solely the result of a common sequence. Based on these data, it is suggested that the antiphagocytic effect of M protein may be due to a conformationally created environment on the surface of the molecule which is selected by both immunological and biological pressure.

Ethylenediaminetetraacetic acid-sensitive antiphagocytic activity of Neisseria gonorrhoeae

Colonial types of Neisseria gonorrhoeae were examined for the presence of pilus-independent antiphagocytic activity. Type 3 and depiliated type 1 gonococci had a shearing- and protease-resistant antiphagocytic activity that was eliminated by treatment with ethylenediaminetetraacetic acid (EDTA) and that was not present on type 4 bacteria. Incubation of EDTA-treated bacteria 37 degrees C for 90 min resulted in fas prevented by antibiotics that block the final assembly of cell wall macromolecules that depend on the C55-isoprenoid carrier for export. These include both lipopolysaccharide and peptidoglycan. Restoration was, however, unaffected by drugs that interfere with the synthesis of peptidoglycan, but not that of lipopolysaccharide, and by inhibitors of protein synthesis. These data suggested that gonococci have an antiphagocytic mechanism in addition to the previously described determinant (presumably pili) that was removed by blending or by treatment with proteases. Of the two antiphagocytic activities, type 1 had both, type 3 had only the EDTA-sensitive component, and type 4 had neither.

Antiphagocytic Effect of Slime from a Mucoid Strain of Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa produce a viscid slime when grown on the surface of agar media. These strains are known to colonize persistently the tracheobronchial tree of children with cystic fibrosis. Colonization may result from inhibition of phagocytosis due to slime produced by the organism. Slime separated from one mucoid strain was examined to determine whether it possessed antiphagocytic activity in vitro. Cells of P. aeruginosa, Escherichia coli, and Staphylococcus aureus were rapidly phagocytized by rabbit polymorphonuclear leukocytes when mixtures were rotated for 2 hr at 37 C in the absence of slime. The addition of relatively small amounts of slime to bacteria and leukocytes inhibited phagocytosis as measured by phagocytic killing of the organisms. Inhibition was found to be most complete with P. aeruginosa.

Mechanisms of bacterial virulence and infection

Abstract Extract The control of many infectious diseases of veterinary importance has contributed to the virtual elimination in advanced countries of the catastrophic effects which these diseases once produced on human as well as animal populations; and, although there is still much to be done in the underdeveloped areas of the world, the measures for control of infectious disease are known and have only to be applied effectively in these areas. The success in controlling infectious diseases has been parallelled by the equally successful recognition and description of their causative micro-organisms. However, the mechanisms whereby these bacteria, viruses, fungi and protozoa produce disease are still not clear. A good deal is known about the classical bacterial toxaemias—e.g., those produced by the Clostridia spp.—and some information has accumulated about the antiphagocytic activities of certain bacterial capsular and cell-wall substances; but apart from these areas, little is known about the mechanisms of microbial pathogenicity. A few examples of this lack of knowledge are as follows.