Passive intranasal monoclonal antibody prophylaxis against murine Pneumocystis carinii pneumonia

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.     

Identification and characterization of a Candida albicans-binding proteoglycan secreted from rat submandibular salivary glands.

A previously identified Candida albicans-binding glycoprotein secreted from rat submandibular glands (RSMG) has been further purified from an aqueous RSMG extract by ion-exchange chromatography and gel filtration. Biochemical analysis of the glycoprotein revealed high levels of uronic acid and sulfate, suggesting that it was a proteoglycan. Its amino acid and carbohydrate compositions were similar to those observed for other proteoglycans and differed significantly from those of RSMG mucin, the major secretory glycoprotein of RSMG. In addition, the apparent molecular weight of the glycoprotein was reduced following treatment with either chondroitinase ABC or heparitinase, demonstrating the presence of chondroitin sulfate and heparan sulfate. On the basis of its structure and anatomical source, the glycoprotein is referred to as submandibular gland secreted proteoglycan 1 (SGSP1). SGSP1 also binds monoclonal antibody 1F9, which recognizes the human blood group A carbohydrate epitope found on RSMG mucin. Hence, SGSP1 appears to be a hybrid molecule with carbohydrate structures found in both proteoglycans and RSMG mucin. Enzymatic digestion of SGSP1, followed by its interaction with a radiolabelled C. albicans strain in a filter-binding assay, demonstrated that binding to this strain appears to be mediated primarily via the heparan sulfate side chains of SGSP1 and not via the blood group A oligosaccharide.     

Epitope mapping of a protective monoclonal antibody against Pneumocystis carinii with shared reactivity to Streptococcus pneumoniae surface antigen PspA

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the M(r) of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.     

Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa

Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis.     

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ΔnirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.The ubiquitous gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for both acute and chronic infections. P. aeruginosa is commonly an etiologic agent in ear infections (8), infections of burn wounds (27), corneal keratitis (21, 30), and pulmonary infections in patients with cystic fibrosis (16) and ventilator-associated pneumonia (5). P. aeruginosa is frequently resistant to conventional antibiotic therapy and the antimicrobial effector mechanisms of phagocytes (9), particularly in the biofilm mode of growth (15).The establishment of P. aeruginosa infection is accompanied by the synthesis of a diverse array of virulence factors composed of various exoproteins, such as elastase and exotoxin A, as well as the mucoid exopolysaccharide alginate (20). The type III secretion system (T3SS), a mechanism whereby cytotoxic effector proteins are directly secreted into the host cell cytoplasm following contact of the bacterium with a target cell (11), has also been identified as a virulence determinant of P. aeruginosa that contributes significantly to the pathogenesis of acute infection (45, 46). The P. aeruginosa T3SS plays a major role in triggering cell death in phagocytes and epithelial cells (17) and is composed of at least 20 proteins, including (i) a secretory apparatus, (ii) machinery devoted to the direct translocation of effectors into the host cell cytoplasm, and (iii) four effector proteins, ExoS, ExoT, ExoU, and ExoY, that contribute to host cell toxicity (19, 46). Regulation of virulence factor expression in P. aeruginosa is hierarchical and highly complex, involving quorum sensing (37, 44) and responses to environmental signals, including stresses applied via host defense mechanisms (46).P. aeruginosa is capable of growing anaerobically by using nitrate as a terminal electron acceptor through the denitrification pathway. The pathway and the operons (47) encoding the enzymatic steps are shown in Fig. ​Fig.1.1. The pathway intermediate nitric oxide (NO) is highly reactive with iron-containing prosthetic groups of proteins such as heme and Fe-S clusters (18). NO detoxification by NO reductase during anaerobic growth is therefore important for maintenance of cell function. In addition, NO is a potent antimicrobial effector produced by phagocytes (4), and P. aeruginosa flavohemoglobin has an important role in NO detoxification under aerobic conditions (1). Hence, P. aeruginosa must control NO toxicity produced both internally and by the host in order to survive under different environmental conditions.Open in a separate windowFIG. 1.Schematic of the denitrification pathway in P. aeruginosa. The representative operons are in italics.NO is also widely appreciated as an important signaling molecule in many biological systems (7). We recently demonstrated roles for membrane nitrate reductase and nitrite reductase, the first two enzymes in the denitrification pathway, in swarming motility, biofilm formation, and virulence in the surrogate nematode host Caenorhabditis elegans (43).These processes were assayed under aerobic conditions, indicating that the roles played by nitrate reductase and nitrite reductase were distinct from their contribution to the bioenergetics of P. aeruginosa anaerobic growth (43). The finding that similar phenotypes were displayed by a nitrate reductase (ΔnarGH) mutant and a nitrite reductase (ΔnirS) mutant suggested that nitrite or NO served as a signaling molecule in these processes, since downstream NO reductase and nitrous oxide reductase mutants behaved comparably to wild-type P. aeruginosa.In this study, we used a set of denitrification pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of phagocyte interaction by using the human monocytic cell line THP-1 (41). We show that both membrane nitrate reductase and nitrite reductase enzyme complexes are important for virulence during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, the ΔnarGH and ΔnirS deletion mutants were defective in the expression of T3SS components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous NO-generating compounds to ΔnirS mutant cultures restored production of T3SS phospholipase ExoU production, whereas nitrite addition had no effect. These data provide genetic and biochemical evidence indicating that NO generated via nitrite reductase contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.     

Oral and esophageal Candida albicans infection in hyposalivatory rats.

The opportunistic fungus Candida albicans is a major cause of oral and esophageal infections in immuno-compromised patients, individuals on drug therapy, and the chronically ill. Because it has been observed that persons suffering from hyposalivation have an increased prevalence of oral candididiasis, we developed an animal model of infection based on hyposalivation. The objectives of our studies were to understand the mechanisms by which C. albicans causes oral disease and to begin to elucidate the role played by saliva in controlling C. albicans in the oral cavity. Our results showed that (i) oral Candida infection was established by a small challenge inoculum, (ii) mucosal lesions developed in the oral cavities and esophagi of infected rats, and (iii) transmission of oral Candida infection from an inoculated rat to uninoculated cagemates occurred rapidly. In addition, we compared the abilities of a clinical isolate and a spontaneously derived morphological mutant from that isolate to infect hyposalivatory rats and to induce disease. Infection was induced by the morphological mutant in hyposalivatory rats; however, the morphological mutant took significantly longer to transmit oral infection to uninoculated cagemates than did the parental strain.     

Purification and characterization of stage-specific glycoproteins from Trypanosoma cruzi

Four developmentally regulated glycoproteins were purified from detergent solubilized cell membranes of Trypanosoma cruzi. Three trypomastigote specific glycoproteins each migrated as single bands under denaturing conditions with approximate Mr of 90,000, 85,000, and 55,000 and pI values of 4.3-5.0, 8.5-9.1, and 8.2, respectively. The fourth, epimastigote specific, protein had an approximate Mr of 72,000 and a pI of 4.8-5.1. The Mr of all four glycoproteins changed by 5-50% upon endoglycosidase F treatment. The Mr 72,000 antigen was the only one that reacted strongly with anti-epimastigote sera raised in rabbits. Sera from a Chagasic patient reacted strongly with the three trypomastigote specific glycoproteins and very weakly with the Mr 72,000 glycoprotein.