In vitro lymphocyte responses of coccidioidin skin test-positive and -negative persons to coccidioidin, spherulin, and a coccidioides cell wall antigen.

The biological activity of C-ASWS, an alkali-soluble, water-soluble cell wall antigen isolated from mycelial-phase cells of Coccidioides immitis, was compared with that of a commercial coccidioidin (CDN; Cutter Laboratories); CDN-TS, a toluene-induced lysate of mycelial-phase cells; and spherulin, a spherule-derived extract of C. immitis. Lymphocytes obtained from healthy CDN skin test-positive donors (group I), healthy skin test-negative donors (group II), and patients with active coccidioidomycosis (group III) were assayed for lymphocyte transformation and production of macrophage inhibitory factor in response to the Coccidioides antigens. C-ASWS, CDN CDN-TS, and spherulin were each effective in eliciting blastogenic responses in lymphocytes of group I subjects. However, only C-ASWS and CDN-TS were effective in eliciting macrophage inhibitory factor production. The responses of group III subjects (patients) were depressed and, in most instances, were indistinguishable from those obtained in lymphocytes of group II subjects.     

Coccidioidin and merthiolate in previously sensitized animals.

The effect of merthiolate, which is used as a preservative in skin test materials, on skin test reactions was determined in guinea pigs. In four groups of animals, merthiolate in basal medium produced skin tests at 24 and 48 h characterized by erythema and/or induration in an intermediate region, i.e., 5 plus or minus 2.2 mm. One of the four groups of animals was a nonsensitized control group. The other three groups were subcutaneously sensitized with (i) merthiolate and saline, (ii) killed Coccidioides immitis arthrospores, and (iii) merthiolate with killed C. immitis arthrospores. Coccidioidin only and merthiolate in coccidioidin produced positive delayed results in groups 3 and 4, which were sensitized with arthrospores. A synergistic effect of merthiolate and coccidioidin was observed in animals of group 4 sensitized by merthiolate with killed C. immitis arthrospores. This effect was observed at 24 h when positive reactions of coccidioidin with merthiolate were significantly greater than skin tests with plain coccidioidin.     

Antigenic heterogeneity of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis.

The antigenic composition of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis, designated C-ASWS, was assessed by two-dimensional immunoelectrophoresis against goat antisera to C-ASWS and coccidioidin. The results established that C-ASWS from mycelia or spherule cell walls is heterogeneous in composition, containing two distinct antigenic components. One is present as a polymer that is antigenically identical to a polymeric antigen in coccidioidin, designated antigen 2. The other component detected in C-ASWS presented an unusual precipitin pattern in that a cathodal leg was demonstrable in the absence of an anodal leg. This incomplete precipitinogen was also detected in coccidioidin. In addition to the finding that C-ASWS is antigenically heterogeneous, the results provide evidence that the conformational and/or configurational structure of the C-ASWS antigen 2 (or antigen 2-like polymer) is altered during physicochemical extraction. This conclusion is based upon the finding that the immunoelectrophoretic profile of the C-ASWS polymer differs from that of coccidioidin antigen 2. The C-ASWS polymer is characterized by having a small cathodal precipitin peak connected to a large anodal peak, whereas coccidioidin antigen 2 is characterized by a predominant cathodal peak.     

Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody

The alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis (C-ASWS) mycelia and spherules was shown to react with anti-Coccidioides immunoglobulin M (IgM) precipitin antibody, both in the classical tube precipitin test and in the immunodiffusion assay for tube precipitin antibody (IDTP). The reactions obtained between C-ASWS and reference IgM precipitin antibody were identical to the reaction obtained when reference coccidioidin (CDN) was used. Definitive proof that C-ASWS extracts contain antigenic determinants that are reactive with IgM tube precipitin antibody was obtained by solid-phase immunoadsorption. Elution of reference IDTP antiserum over a column containing mycelium C-ASWS coupled to Sepharose 4B completely adsorbed precipitin antibody; i.e., reactivity in the IDTP was demonstrable in the column eluate but not in the column effluent fraction. The antigenic composition of C-ASWS extracts was evaluated and compared with that of CDN by two-dimensional immunoelectrophoresis against burro anti-CDN. The results established that both mycelium and spherule C-ASWS contain antigenic determinants in common with only one antigen present in CDN. The latter, designated antigen 2, is a large polymer which is predominant among the antigenic components in CDN. On a dry weight comparison, antigen 2 determinants were most concentrated in spherule C-ASWS, followed by mycelium C-ASWS and reference IDTP antigen. The finding that C-ASWS extracts are reactive with IgM tube precipitin antibody and are antigenically identical to antigen 2 in CDN suggests that antigen 2 is the biologically active component of CDN in tube precipitin assays.     

Experimental Induction of Anergy to Coccidioidin by Antigens of Coccidioides immitis

Failure to react to coccidioidin (anergy) often occurs in patients with disseminated coccidioidomycosis. One possible reason may be desensitization by excessive amounts of antigen. This was studied experimentally by injection of soluble and hyphal antigens of Coccidioides immitis into coccidioidin- and tuberculin-sensitive guinea pigs. Guinea pigs sensitized by injection of killed hyphal cells of C. immitis in complete Freund adjuvant were subsequently injected daily either with soluble coccidioidal antigen administered intraperitoneally or with hyphal antigen administered either subcutaneously or intraperitoneally. Gradual loss of cutaneous reactivity to coccidioidin occurred, but the reactivity to tuberculin remained unimpaired. The rapidity of desensitization was roughly proportional to the dose of antigen with desensitization occurring as early as 6 days after beginning injections. This anergic state was temporary, and reactivity returned several days after discontinuing injection of antigen. Injection of coccidioidal antigen led to production of coccidioidal complement-fixing antibody, but there was no consistent relationship between the antibody titer and state of cutaneous reactivity to coccidioidin. Peritoneal exudate or pulmonary alveolar cells from desensitized animals migrated freely in the presence of coccidioidin but were inhibited in the presence of tuberculin. Heat treatment did not impair the capacity of the soluble or hyphal antigen to induce anergy, thus suggesting that the antigen active in complement fixation was perhaps not involved in desensitization. Polysaccharide obtained by ethanol precipitation of dialyzed coccidioidin failed to induce anergy. Dialysis of the soluble coccidioidal antigen caused the loss of the desensitizing activity. Thus, specific desensitization could be induced by administration of large doses of coccidioidal antigen but dialyzable components appear important in this desensitization.     

Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum.

Detection of antibody to Coccidioides immitis F antigen is of proved value in the diagnosis of coccidioidomycosis. This antibody is demonstrable by use of an immunodiffusion assay with reference coccidioidin antigen and antiserum to C. immitis. Using a combination of lectin affinity and immunoaffinity chromatography, we isolated the F antigen from coccidioidin and prepared monospecific antibody to the purified antigen. The availability of these reagents will enable the development of a sensitive and specific assay for detecting serologic reactivity to this antigen.     

Course of coccidioidomycosis in intratracheally infected guinea pigs.

Two hundred Hartley-inbred guinea pigs were infected intratracheally with 50 viable arthrospores of Coccidioides immitis. At weeks 1 through 10 postinfection, groups of 20 guinea pigs were assayed for skin test, macrophage migration inhibitory factor (MIF), and lymphocyte transformation (LT) responses to coccidioidin. Forty-eight hours after skin testing and just before MIF and LT assays, blood was obtained for complement-fixing (CF) antibody titers and the animals were autopsied to assess the extent of fungal dissemination. Immunological assays established that skin tests and MIF responses converted within 3 weeks of infection. LT responses were not demonstrable until week 5. Dissemination of C. immitis to the liver or spleen was an early event, with 21% of guinea pigs positive by week 2 and 70% positive by week 5. CF antibody titers were demonstrable at week 5, increased logarithmically through week 7, then increased at a slower rate thereafter. Concomitant with the decreased rate of antibody production, guinea pigs began to clear C. immitis from their extrapulmonary tissues. Skin test responses peaked at 6 weeks postinfection when CF antibody titers were less than or equal to 1:16 and then plateaued with increased CF titers. Although this overall immunological profile is consistent with the disease in humans, there was not a direct correlation between CF antibody titer and dissemination to the liver or spleen, nor was there an inverse correlation between CF antibody titers and skin test or MIF responses. Rather, CF antibody titers and cell-mediated immune responses were equally demonstrable in guinea pigs with disseminated or nondisseminated disease.     

Reactivity to spherule-derived coccidioidin in the southeastern United States.

Delayed hypersensitivity skin tests with mycelium-derived (coccidioidin) or spherule-derived (spherulin) antigens (or both) can be used to identify patients who have been sensitized to the dimorphic fungus Coccidioides immitis. Prior studies suggest that coccidioidin and spherulin skin test antigens detect comparable numbers of reactors among exposed subjects. Studies in subjects residing in areas outside the United States where C. immitis is not endemic suggest that both antigens are specific for the fungus. The specificity and reactivity of coccidioidin and spherulin have not been compared in nonendemic regions of the United States in which the skin test antigens and an appropriate travel or exposure history are used to identify patients with possible C. immitis infection. A review of delayed cutaneous reactions to coccidioidin in 6,375 patients tested between 1970 and 1979 in the southeastern United States revealed 958 (15.0%) and 234 (5.7%) positive reactions (greater than or equal to 5 mm), respectively, at 24 and 48 h. Subsequent tests with spherulin in 2,775 patients tested in 1980 and 1981 revealed 866 (31.2%) and 288 (10.3%) positive reactions, respectively, at 24 and 48 h. False-positive immediate hypersensitivity reactions contributed to the large number of spherulin reactors at 24 h. Differences among the patients sampled, work exposure, and travel history were excluded as causes of this surprising and highly significant (P less than or equal to 0.0001) difference in the 48-h delayed cutaneous reaction. These observations suggest two possibilities: (i) spherulin is less specific than coccidioidin, or (ii) a surprising prevalence of C. immitis sensitization exists among patients in nonendemic regions of the United States.     

Antigenic identity of biologically active antigens in coccidioidin and spherulin.

We recently reported the isolation of three clinically relevant antigens from coccidioidin; viz., the antigen that is reactive in the immunodiffusion (ID) assay for detecting tube precipitin (TP) antibody (designated IDTP); the antigen that is reactive in the ID assay for detecting complement-fixing antibody (designated IDCF); and the heat-stable (HS) antigen which, when demonstrated in soluble extracts of fungal cultures by using the IDHS assay, establishes the mycologic identification of Coccidioides immitis. This investigation was undertaken to determine whether the IDTP, IDCF, and IDHS antigens isolated from coccidioidin are of antigenic identity with components in spherulin. By employing the purified coccidioidin antigens in line-immunoelectrophoresis with spherulin and by assaying coccidioidin and spherulin by tandem two-dimensional immunoelectrophoresis against antisera produced to the purified antigens, we report that these three coccidioidin-derived antigens are antigenically identical to precipitinogens in spherulin.     

Variation in the In Vitro Migration of Sensitized Guinea Pig Peritoneal Exudate Cells in the Presence of Coccidioidin

Guinea pigs were immunized by intramuscular injection of arthrospores from the M11 strain of Coccidioides immitis, and the peritoneal exudate cells were harvested 4 to 6 weeks later. After incubation with various concentrations of coccidioidin in tissue culture, the area of all migration was measured. Results of this study indicate that at a critical level, a variance of 0.1 mug of antigen per ml, determined the difference between approximately 88% migration and 5% migration as compared with control cells incubated in the absence of antigen. The concentration of antigen (ASU-9 stock coccidioidin concentrate) required to produce essentially complete inhibition of migration was determined to be 12.5 mug/ml.     

Suppression of T-lymphocyte response by Coccidioides immitis antigen.

Intravenous injection of BALB/c mice with coccidioidin or an alkali-soluble cell wall extract of Coccidioides immitis mycelia resulted in the induction of a splenic cell population(s) that suppressed delayed-type hypersensitivity response to coccidioidal antigen. To determine whether the levels of C. immitis antigen produced during the course of active coccidioidal disease might also cause suppression of T-lymphocyte response, BALB/c mice were infected by intranasal instillation of arthroconidia, and 2 weeks later, their sera were evaluated for suppression of T-lymphocyte response in syngeneic recipients. Intravenous transfer of sera, which were shown to contain high levels of coccidioidal antigen by an enzyme-linked immunoadsorbent assay, suppressed the delayed-type hypersensitivity response of recipients to immunization with coccidioidin. Solid-phase immunoadsorption of the sera with goat antibodies to C. immitis antigens removed the suppressive component(s). To determine whether the suppressive effect of circulating coccidioidal antigen(s) was associated with the activation of a splenic suppressor cell(s), as was observed in mice injected intravenously with coccidioidal antigen, spleen cell lysates were prepared from infected donors, and after filtration to remove viable fungi, the lysates were transferred to syngeneic mice. Recipients of lysates from infected but not noninfected donors were suppressed in their response to immunization with coccidioidin. Collectively, these results provide evidence that depressed T-cell responses observed in coccidioidomycosis are associated with, and may be attributable to, the activation of a suppressor cell or factor by circulating C. immitis antigens.     

Cryptococcal culture filtrate antigen for detection of delayed-type hypersensitivity in cryptococcosis.

Previous studies on a cryptococcal culture filtrate (CneF) antigen have shown that the antigen is useful in detecting delayed-type hypersensitivity and that it is specific for Cryptococcus. This study further defined one more parameter of specificity, showing that the CneF antigen does not elicit delayed-type hypersensitivity responses in Cryptococcus albidus-sensitized guinea pigs. When the crude CneF antigen was subjected to ultrafiltration fractionation, the skin test active components were found to be in the 50,000 or greater molecular weight range fraction. The concentrated retentates of the XM50 ultrafiltration membrane were more sensitive antigens than the crude CneF antigens. Further fractionation of the XM50 retentate using 3% acrylamide gel electrophoresis separated the antigen into two bands. One band, the P fraction, migrated only a short distance into the gel; the fraction was carbohydrate-like and did not elicit significant skin test responses in sensitized guinea pigs. The other band, G fraction, appeared with the tracking dye, was glycoprotein-like, and elicited significantly positive skin tests in sensitized guinea pigs. G fractions prepared using three different serotypes of Cryptococcus neoformans elicited similar size indurations when used in skin testing guinea pigs sensitized with either the homologous serotype isolated of C. neoformans or the heterologous serotype isolate.     

Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays.

The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli. The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies. The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody. The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID. The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin. The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera. Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.     

Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody.

Detection of immunoglobulin M (IgM) precipitin antibody to coccidioidin, the autolysate of mycelial-phase cells of Coccidioides immitis, is an important serologic aid in establishing a diagnosis of primary coccidioidomycosis. In the present study, the component of coccidioidin that reacts with IgM precipitin antibody was isolated by a combination of immunoaffinity and anion-exchange chromatography. Antigenic analysis of the purified antigen in two-dimensional immunoelectrophoresis against goat anti-coccidioidin revealed a precipitinogen characterized by a complete cathodal leg and a partial anodal leg. The reactivity of this incomplete precipitating antigen with anti-C. immitis IgM was established by serologic assays and by the adsorption of reference IgM precipitin antibody on solid-phase immunosorbents containing the purified precipitinogen. The isolation of the coccidioidin component that reacts with IgM precipitin antibody and the production of monospecific antibody will provide the necessary reagents for the development of a sensitive immunoassay for detecting this serodiagnostic response.     

Induction and expression of cell-mediated immune responses in inbred mice infected with Coccidioides immitis.

Comparisons of the course of coccidioidomycosis in two strains of inbred mice established that BALB/c mice are significantly more susceptible to pulmonary infection with Coccidioides immitis than are DBA/2 mice. The susceptibility of BALB/c mice does not reside in their inability to mount a delayed-type hypersensitivity response to C. immitis antigen. That is, BALB/c mice manifested footpad hypersensitivity to coccidioidin early during the course of disease, to a level comparable to that of DBA/2 mice. In contrast to the more resistant DBA/2 mouse strain, however, BALB/c mice developed anergy by day 15 postinfection. Suppression of the delayed-type hypersensitivity response was not specific for C. immitis antigen, as evidenced by the finding that BALB/c mice immunized with mycobacterial purified protein derivative prior to infection with C. immitis were suppressed in their footpad response to mycobacterial antigen at day 15 postinfection. Taken together, these results establish that genetically determined susceptibility to this fungus is associated with an acquired suppression of cell-mediated immune reactivity.     

Extraction of skin test activity from Coccidioides immitis mycelia by water, perchloric acid, and aqueous phenol extraction.

Water, perchloric acid extracts, and fractions of partially defatted whole mycelia of Coccidioides immitis contained delayed skin test activity when tested in C. immitis-infected guinea pits. Aqueous phenol extraction of these fractions resulted in partitioning of activity between aqueous-soluble and phenol-soluble fractions; activity was found to be water soluble after removal of phenol by extensive dialysis. Highest specific activity skin test antigen was invariably found in the phenol-soluble phase, water-soluble fraction. Material of equivalent activity could also be extracted directly from the defatted mycelia. Skin test active fractions contained glucose, mannose, 3-O-methylmannose, glucosamine, and amino acids.     

Production and characterization of a monoclonal antibody to the complement fixation antigen of Coccidioides immitis.

Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgG1) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Ag14 myeloma cells, and the fusion products were screened for IgG antibody to coccidiodin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgG1 Mab was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60 degrees C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, alpha-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.     

Isolation and characterization of an extracellular proteinase of Coccidioides immitis.

A proteinase isolated from the respiratory pathogen, Coccidioides immitis, was shown to have collagenolytic and elastinolytic activity, as well as the ability to cleave human serum immunoglobulin G and secretory immunoglobulin A. Proteolytic activity was demonstrated with a bovine casein digestion assay in conidial culture exudates, mycelial and spherule culture filtrates, conidial and spherule wall material, and Sephacryl S-300 fractions of the isolated soluble conidial wall material described previously. One of the latter fractions (fraction 2) demonstrated high proteolytic activity. The proteinase was purified from this chromatographic fraction by cold acetone extraction followed by Sephadex G-50 gel filtration and was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By means of tandem two-dimensional immunoelectrophoresis, the proteinase was identified as antigen 11 on the basis of its reaction in the coccidioidin/anticoccidioidin reference system. The proteinase is characterized by a broad substrate specificity, optimal activity at 35 to 40 degrees C (pH 8.0) in the presence of human collagen, elastin, or hemoglobin, an isoelectric point of pH 4.5, and inhibition by organofluorides, N-tosyl-L-phenylalanine chloromethyl ketone, chymostatin, and alpha-1-antitrypsin. These features of the enzyme are comparable to those of chymotrypsinlike serine proteinases. Demonstration that the proteinase can cleave human immunoglobulins and digest ubiquitous tissue structural proteins (e.g., collagen and elastin) suggests that it may play a role in the virulence of the fungal pathogen.     

Genetic Vaccination against Coccidioides immitis: Comparison of Vaccine Efficacy of Recombinant Antigen 2 and Antigen 2 cDNA

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.     

Isolation of antigens with proteolytic activity from Coccidioides immitis.

Three antigens with proteolytic activity have been isolated from crude, water-soluble fractions of the saprobic phase of the fungal pathogen Coccidioides immitis. Two proteinases, identified in our immunoelectrophoresis reference system as Ag11 and AgCS, were isolated from the soluble conidial wall fraction (SCWF). Ag11 was previously shown to be a serine proteinase and was characterized in this study as a 60-kilodalton (kDa) fraction by gel filtration (GF). The purified proteinase demonstrated little or no reactivity with 21 serum samples from coccidioidomycosis patients in the enzyme-linked immunosorbent assay; this may be due to limited presentation of this antigen to the host during the course of coccidioidomycosis. AgCS was separated by GF chromatography into two fractions identified by molecular masses of 39 and 19 kDa. Most proteolytic activity was shown by substrate gel electrophoresis to be associated with the lower-molecular-mass fraction. AgCS was reactive with 18 of the 21 serum samples and shown to be the major component of a heat-stable antigen previously reported to be immunospecific for C. immitis. The third antigen with proteolytic activity was isolated from the 5-day mycelial culture filtrate and identified by GF as a 56-kDa fraction. Uniformly high levels of immunoreactivity between 18 of the 21 patient sera and the 56-kDa antigen were demonstrated. Antigens with proteolytic activity may play important roles in fungus-host interactions as well as morphogenesis of the pathogen.