Host-Pathogen Interactions : XXXIII. A Plant Protein Converts a Fungal Pathogenesis Factor into an Elicitor of Plant Defense Responses

This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms.     

Host-Pathogen Interactions: I. A Correlation Between alpha-Galactosidase Production and Virulence

Resistance or susceptibility of Red Kidney, Pinto and Small White beans (Phaseolus vulgaris) to the alpha, beta, and gamma strains of Colletotrichum lindemuthianum was either confirmed or established. These fungal strains secrete α-galactosidase, β-galactosidase and β-xylosidase when grown on cell walls isolated from the hypocotyls of any of the above bean varieties. These enzymes effectively degrade cell walls isolated from susceptible 5-day old hypocotyls but degrade only slightly the walls isolated from resistant 18-day old hypocotyls. The amounts of the β-galactosidase and β-xylosidase secreted by the 3 fungal strains are relatively low and are approximately equivalent. The secretion of these 2 enzymes is not dependent upon the bean variety from which the hypocotyl cell walls used as a carbon source were isolated. However, the fungal strains secrete greater amounts of α-galactosidase when grown on hypocotyl cell walls isolated from susceptible plants than when grown on walls from resistant plants. Virulent isolates of the fungus, when grown on hypocotyl cell walls isolated from a susceptible plant, secrete more α-galactosidase than do attenuated (avirulent) isolates of the same fungal strain grown under the same conditions. The α-galactosidase secreted by each of the fungal strains is capable of removing galactose from the hypocotyl cell walls of each bean variety tested. Galactose is removed from the cell walls of each variety at the same rate regardless of whether the cell walls were isolated from a susceptible or resistant plant.     

Host-Pathogen Interactions: VIII. Isolation of a Pathogen-synthesized Fraction Rich in Glucan That Elicits a Defense Response in the Pathogen's Host

A polysaccharide from the fungal pathogen Colletotrichum lindemuthianum causes browning and phytoalexin production when applied to the cut surfaces of bean (Phaseolus vulgaris) cotyledons and hypocotyls. The application of an amount of polysaccharide equivalent to less than 100 ng of glucose will elicit this response in the bean tissues. The polysaccharide has been isolated both from culture filtrates and from the mycelial walls of the fungus. Purification of the polysaccharide involved anion and cation exchange chromatography and gel filtration. The polysaccharide has an apparent molecular weight between 1,000,000 and 5,000,000 daltons, and consists predominantly of 3- and 4-linked glucosyl residues.     

Attenuated Bordetella pertussis Protects against Highly Pathogenic Influenza A Viruses by Dampening the Cytokine Storm

The threat of a pandemic spread of highly virulent influenza A viruses currently represents a top global public health problem. Mass vaccination remains the most effective way to combat influenza virus. However, current vaccination strategies face the challenge to meet the demands in a pandemic situation. In a mouse model of severe influenza virus-induced pneumonitis, we observed that prior nasal administration of an attenuated strain of Bordetella pertussis (BPZE1) provided effective and sustained protection against lethal challenge with two different influenza A virus subtypes. In contrast to most cross-protective effects reported so far, the protective window offered upon nasal treatment with BPZE1 lasted up to at least 12 weeks, suggesting a unique mechanism(s) involved in the protection. No significant differences in viral loads were observed between BPZE1-treated and control mice, indicating that the cross-protective mechanism(s) does not directly target the viral particles and/or infected cells. This was further confirmed by the absence of cross-reactive antibodies and T cells in serum transfer and in vitro restimulation experiments, respectively. Instead, compared to infected control mice, BPZE1-treated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration, and strong suppression of the production of major proinflammatory mediators in their bronchoalveolar fluids (BALFs). Our findings thus indicate that protection against influenza virus-induced severe pneumonitis can be achieved through attenuation of exaggerated cytokine-mediated inflammation. Furthermore, nasal treatment with live attenuated B. pertussis offers a potential alternative to conventional approaches in the fight against one of the most frightening current global public health threats.Influenza virus pandemics are unpredictable but recurring events that can have severe consequences on societies worldwide. In the 20th century, three novel influenza virus strains emerged, causing the 1918, 1957, and 1968 pandemics, the most devastating being the 1918 Spanish flu that led to an estimated 50 million deaths (47). The recent spread of highly pathogenic avian influenza (HPAI) H5N1 virus across parts of Asia, Europe, and the Middle East, with an overall fatality rate of over 60% for humans, as well as the rapid pandemic spread of a novel influenza A virus of the H1N1 subtype, has caused worldwide concern about a potential remake of the 1918 disaster (8).Severe complications arising from pandemic influenza or HPAI H5N1 viruses are associated with rapid, massive inflammatory cell infiltration, resulting in acute respiratory distress, and reactive hemophagocytosis with multiple organ involvement. Both the 1918 Spanish influenza virus and HPAI H5N1 induce a cytokine storm characterized by an exaggerated production of inflammatory cytokines and chemokines in the serum and lungs caused by uncontrolled activation of the host''s innate immune system. This triggers massive pulmonary edema, primary and/or secondary pneumonia, and alveolar hemorrhage with acute bronchopneumonia (4, 12, 24, 27, 37, 40, 43, 44).The relationship between mortality, viral load, and immunopathology during influenza virus infection remains elusive and somewhat controversial. Some studies suggest that severe lung immunopathology is a direct consequence of a high viral load that the host is unable to resolve (12, 13), whereas others have reported that influenza virus-induced mortality is not a direct function of viral burden but a consequence of immune-mediated pathology (9, 11). Moreover, the picture is further complicated by the fact that different highly virulent influenza A viruses may induce distinct pathological signatures and lead to different courses of acute respiratory distress syndrome, refuting the hypothesis of a single, universal cytokine storm underlying all fatal influenza virus diseases (16).Currently, vaccination remains the cornerstone of influenza virus prevention. However, due to constant antigenic drift and shift of the two major viral surface proteins hemagglutinin (HA) and neuraminidase (NA) (7), influenza virus vaccines must be reformulated each year in order to match the circulating subtypes (41). The potential emergence of an influenza virus pandemic at any time, combined with limited vaccine supplies, has rendered global vaccination strategies difficult. Therefore, a universal influenza virus vaccine that can provide protection against different variants or strains and thus not require frequent updates is highly desirable.Here, we report that nasal administration of a recently developed live attenuated Bordetella pertussis vaccine strain, named BPZE1 (35), provides effective and sustained protection against lethal challenge with mouse-adapted H3N2 or H1N1 (A/PR/8/34) influenza A viruses. We demonstrate that the protective mechanism(s) does not target the viral particles or the infected host cells but controls the influenza virus-mediated inflammation by dampening the cytokine storm. As BPZE1 has recently entered phase I safety trials with humans (http://www.child-innovac.org), our observations support the potential application of this vaccine strain as a universal prophylactic treatment against highly pathogenic influenza A viruses.     

Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions

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CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses

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Host-Pathogen Interactions: XV. Fungal Glucans Which Elicit Phytoalexin Accumulation in Soybean Also Elicit the Accumulation of Phytoalexins in Other Plants

A β-glucan isolated from the mycelial walls of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgaris, and stimulate the accumulation of the phytoalexin, rishitin, in potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation in soybean, Glycine max.

Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accumulation of at least five fungistatic compounds. These compounds migrate during thin layer chromatography identically to the fungistatic compounds which accumulate in kidney beans which have been inoculated with Colletotrichum lindemuthianum, a fungal pathogen of kidney beans.

Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophthora megasperma var. sojae.

     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Host-Pathogen Interactions: VI. A Single Plant Protein Efficiently Inhibits Endopolygalacturonases Secreted by Colletotrichum Lindemuthianum and Aspergillus Niger

Endopolygalacturonases have been purified from the extracellular enzymes of Colletotrichum lindemuthianum and Aspergillus niger. A protein, purified from Red Kidney (Phaseolus vulgaris) beans for its ability to inhibit the endopolygalacturonase secreted by C. lindemuthianum, inhibits the A. niger endopolygalacturonase almost as efficiently as it inhibits the C. lindemuthianum enzyme.     

Host-Pathogen Interactions : XXII. A Galacturonic Acid Oligosaccharide from Plant Cell Walls Elicits Phytoalexins

Elicitors of phytoalexin accumulation in soybean (Glycine max L. Merr., cv Wayne) cotyledons were released from soybean cell walls and from citrus pectin by partial acid hydrolysis. These two hydrolysates yielded nearly identical distributions of elicitor activity when fractionated on anion-exchange columns. Chromatography of the pectin elicitor on gel filtration and high-pressure anion-exchange columns did not further purify the elicitor. Elicitor activity of the preparation was lost by treatment with either endo-α-1,4-polygalacturonase or pectate lyase. Glycosyl residue compositions of the purified elicitors from cell walls and pectin were both found to be approximately 98% galacturonosyl residues. Linkage analysis of the pectin elicitor showed that most, if not all, of the galacturonosyl residues were α-1,4-linked. The high-mass molecular ions detected by fast atom bombardment-mass spectrometry of the most active elicitor fractions from cell walls and pectin both corresponded precisely to a molecule composed of 12 galacturonosyl residues. These results suggest that dodeca-α-1,4-d-galacturonide is the active elicitor, but the possibility remains that the active component could be a slightly modified oligogalacturonide present, but not detected, in the purified fractions.     

Host-Pathogen Interactions: XI. Composition and Structure of Wall-released Elicitor Fractions

The structures of the four wall-released elicitor fractions isolated from the Phytophthora megasperma var. sojae mycelial walls have been examined. The results demonstrate that fraction I is primarily composed of a branched β-1,3-glucan, similar in structure to the extracellular elicitors described previously (Ayers, A., J. Ebel, F. Finelli, N. Burger, and P. Albersheim. 1976. Plant Physiol. 57: 751-759). Fractions II and IV are primarily composed of a highly branched mannan-containing glycoprotein, with fraction IV richer in protein than fraction II. Fraction III contains, attached to protein, a mixture of the two polysaccharide types found in fraction I and in fractions II and IV. The structural data presented here, in concert with the biological data presented in the previous two papers (Ayers et al. 1976. Plant Physiol. 57: 751-759; 760-765), demonstrate that the only compound produced by P. megasperma var. sojae which contains elicitor activity is the glucan. Evidence is presented that the terminal glycosyl residues of the glucan are required for elicitor activity. In addition, it is demonstrated that 90% of the glucan can be removed enzymically without any loss of biological activity. The active residue of the enzymic digestion is a highly branched 3- and 3,6-linked glucan containing about 4% mannosyl residues. The results presented suggest that the mannosyl residues of the glucan, which represent only about 1% of the undegraded glucan, are likely to participate in the active site of this molecule. The role of elicitors and phytoalexins in host-pathogen interactions is discussed. Evidence for the existence of and possible identity of another factor, which determines race specificity of host-pathogen interactions, is summarized.     

Bordetella pertussis Infection or Vaccination Substantially Protects Mice against B. bronchiseptica Infection

Although B. bronchiseptica efficiently infects a wide range of mammalian hosts and efficiently spreads among them, it is rarely observed in humans. In contrast to the many other hosts of B. bronchiseptica, humans are host to the apparently specialized pathogen B. pertussis, the great majority having immunity due to vaccination, infection or both. Here we explore whether immunity to B. pertussis protects against B. bronchiseptica infection. In a murine model, either infection or vaccination with B. pertussis induced antibodies that recognized antigens of B. bronchiseptica and protected the lower respiratory tract of mice against three phylogenetically disparate strains of B. bronchiseptica that efficiently infect naïve animals. Furthermore, vaccination with purified B. pertussis-derived pertactin, filamentous hemagglutinin or the human acellular vaccine, Adacel, conferred similar protection against B. bronchiseptica challenge. These data indicate that individual immunity to B. pertussis affects B. bronchiseptica infection, and suggest that the high levels of herd immunity against B. pertussis in humans could explain the lack of observed B. bronchiseptica transmission. This could also explain the apparent association of B. bronchiseptica infections with an immunocompromised state.     

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment

An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73 ^-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73 ^-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.     

Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans

The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants.     

A Mucosal Subunit Vaccine Protects against Lethal Respiratory Infection with Francisella tularensis LVS

Francisella tularensis (FT) is a highly virulent pathogen for humans and other mammals. Severe morbidity and mortality is associated with respiratory FT infection and there are concerns about intentional dissemination of this organism. Therefore, FT has been designated a category A biothreat agent and there is a growing interest in the development of a protective vaccine. In the present study, we determine the protective potential of a subunit vaccine comprised of the FT heat shock protein DnaK and surface lipoprotein Tul4 against respiratory infection with the live vaccine strain (LVS) of FT in mice. First, we establish an optimal intranasal immunization regimen in C57BL/6 mice using recombinant DnaK or Tul4 together with the adjuvant GPI-0100. The individual immunization regimens induced robust salivary IgA, and vaginal and bronchoalveolar IgA and IgG antigen-specific antibodies. Serum IgG1 and IgG2c antibody responses were also induced, indicative of a mixed type 2 and type 1 response, respectively. Next, we show that immunization with DnaK and Tul4 induces mucosal and systemic antibody responses that are comparable to that seen following immunization with each antigen alone. This immunization regimen also induced IFN-γ, IL-10 and IL-17A production by splenic CD4⁺ T cells in an antigen-specific manner. Importantly, over 80% of the mice immunized with DnaK and Tul4, but not with each antigen alone, were protected against a lethal respiratory challenge with FT LVS. Protection correlated with reduced bacterial burden in the lung, liver and spleen of mice. This study demonstrates the potential of DnaK and Tul4 as protective antigens and lends support to the notion of combining distinct, immunodominant antigens into an effective multivalent tularemia vaccine.