Isolation and preliminary characterization of the 14- to 18-kilodalton Candida albicans antigen as a phospholipomannan containing beta-1,2-linked oligomannosides.

Western blot (immunoblot) analysis of Candida albicans germ tube extracts has demonstrated the probable presence of beta-1,2-linked oligomannosides acting as epitopes distributed over a 14- to 18-kDa antigen unreactive to concanavalin A. These conclusions about the existence of these non-mannan-associated oligomannoside species were reinforced in the present study by the demonstration of reactivity of factor serum 5 (Iatron Laboratories) with the same antigen. A monoclonal antibody which reacted in an enzyme immunoassay with beta-1,2-linked oligomannosides converted into neoglycolipids and in Western blotting with the 14- to 18-kDa antigen from yeast and germ tubes, through metaperiodate-sensitive epitopes, was used for further characterization of the molecule. Reducing agents and strong protease digestion, which have deleterious effects on C. albicans proteins and mannoproteins, affected neither the antigenicity nor the relative molecular weight of the molecule. Western blots performed after migration of protease-treated extracts in polyacrylamide gels without sodium dodecyl sulfate (SDS) showed that the 14- to 18-kDa antigen could be negatively charged, whereas metabolic radiolabeling demonstrated that these charges could originate, at least in part, from the presence of phosphorus within the molecule. Chloroform-methanol-water extraction of protease-resistant material led to purification of the 14- to 18-kDa antigen, as determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Metabolic radiolabeling with mannose confirmed the presence of these sugar residues within the purified 14- to 18-kDa antigen (despite its nonreactivity to concanavalin A), whereas radiolabeling with palmitic acid demonstrated its lipopolysaccharidic nature. Together, these results led to the conclusion that the 14- to 18-kDa antigen is a phospholipomannan.     

Beta-1,2-linked oligomannosides from Candida albicans act as signals for tumor necrosis factor alpha production.

Different cell wall components from Candida albicans have been shown to stimulate murine macrophages for tumor necrosis factor alpha (TNF-alpha) secretion. All of these molecules contain beta-1,2-oligomannosides. In order to examine their role in TNF-alpha production, acid-labile oligosaccharides, released from C. albicans VW32 cell wall phosphopeptidomannan by mild acid hydrolysis, and previously shown to correspond to homopolymers of beta-1,2-linked mannopyranosyl units, were separated by gel filtration chromatography according to their degree of polymerization. Murine macrophages incubated with purified oligomannosides (M2 to M8) released TNF-alpha to an extent which was dependent on, although not directly correlated with, the length of the mannosyl chain. Slight activity was observed with M4 and M5; M6 and M7 had virtually no effect, whereas M8 was associated with strong TNF-alpha release. This effect of M8 was dose dependent and was not altered by polymyxin B, known to interfere with lipopolysaccharide-induced TNF-alpha production. These results suggest that stimulation of TNF-alpha release by C. albicans glycoconjugates containing beta-1,2-linked oligomannosides may be due, at least in part, to the presence of these components.     

Beta-1,2- and alpha-1,2-linked oligomannosides mediate adherence of Candida albicans blastospores to human enterocytes in vitro

Candida albicans is a commensal dimorphic yeast of the digestive tract that causes hematogenously disseminated infections in immunocompromised individuals. Endogenous invasive candidiasis develops from C. albicans adhering to the intestinal epithelium. Adherence is mediated by the cell wall surface, a domain composed essentially of mannopyranosyl residues bound to proteins, the N-linked moiety of which comprises sequences of alpha-1,2- and beta-1,2-linked mannose residues. Beta-1,2-linked mannosides are also associated with a glycolipid, phospholipomannan, at the C. albicans surface. In order to determine the roles of beta-1,2 and alpha-1,2 oligomannosides in the C. albicans-enterocyte interaction, we developed a model of adhesion of C. albicans VW32 blastospores to the apical regions of differentiated Caco-2 cells. Preincubation of yeasts with monoclonal antibodies (MAbs) specific for alpha-1,2 and beta-1,2 mannan epitopes resulted in a dose-dependent decrease in adhesion (50% of the control with a 60- micro g/ml MAb concentration). In competitive assays beta-1,2 and alpha-1,2 tetramannosides were the most potent carbohydrate inhibitors, with 50% inhibitory concentrations of 2.58 and 6.99 mM, respectively. Immunolocalization on infected monolayers with MAbs specific for alpha-1,2 and beta-1,2 oligomannosides showed that these epitopes were shed from the yeast to the enterocyte surface. Taken together, our data indicate that alpha-1,2 and beta-1,2 oligomannosides are involved in the C. albicans-enterocyte interaction and participate in the adhesion of the yeasts to the mucosal surface.     

Probable presence of beta(1-2)-linked oligomannosides that act as human immunoglobulin G3 epitopes and are distributed over a Candida albicans 14- to 18-kilodalton antigen.

Kinetic analysis of candidosis patients' immunoglobulin G3 response has shown that reactivity towards beta(1-2)-linked mannan-derived oligomannosides was associated with the recognition through metaperiodate-sensitive epitopes of a 14- to 18-kDa Candida albicans antigen unreactive with concanavalin A.     

Mouse monoclonal IgA binds to the galectin-3/Mac-2 lectin from mouse macrophage cell lines

The beta-galactoside-binding S-type lectin galectin-3/Mac-2, expressed among several other cell types on activated macrophages, is known to bind IgE, but not other Ig classes. We report in this paper that the single major constituent in a detergent lysate from the J774 mouse macrophage cell line bound to a mouse monoclonal IgA-affinity column. This fraction has been identified by mass spectrometry analysis as galectin-3. Binding of both mouse IgA and IgE to galectin-3 coated plates was inhibited by lactose and asialofetuin. Furthermore, three different monoclonal IgAs bound also to purified recombinant hamster galectin-3 coated plates in a concentration dependent manner. The potential functional significance of IgA binding to galectin-3 within macrophages and possibly other cell types is discussed.     

Biomphalaria glabrata embryonic cells express a protein with a domain homologous to the lectin domain of mammalian selectins

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined. Received: 26 August 1998 / Accepted: 20 November 1998     

Contribution of phospholipomannan to the surface expression of beta-1,2-oligomannosides in Candida albicans and its presence in cell wall extracts

beta-1,2-Oligomannosides (beta-1,2-Man) derived from Candida albicans mannan have been shown to act as adhesins and to induce protective antibodies. We used monoclonal antibodies specific for beta-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of beta-1,2-Man epitopes. These monoclonal antibodies were also used for Western blotting of cell surface extracts to study the nature of the molecules expressing the beta-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of beta-1,2-Man epitopes at the cell wall surfaces of yeasts grown on solid media. PLM was present in intercellular matrixes of colonies grown on agar and was detected as a contaminant in mannan batches prepared by conventional methods.     

Amplification of the hyphal wall protein 1 gene to distinguish Candida albicans from Candida dubliniensis

The authors developed a new, simple, and reliable PCR/restriction fragment length polymorphism technique, using amplification of the hyphal wall protein 1 gene of Candida albicans and its gene homologue in Candida dubliniensis, to differentiate the two species of Candida. Performed with a new primer set, CRR-f/CRR-r, PCR produced two different fragments: one of 1,180 bp for C. albicans, and one of 930 bp for C. dubliniensis.     

The 86-kilodalton antigen from Schistosoma mansoni is a heat-shock protein homologous to yeast HSP-90

We report the sequence of a cDNA clone encoding an 86-kDa polypeptide antigen (p86) from Schistosoma mansoni. Fusion proteins made in Escherichia coli are recognized by human infection sera. The reading frame of this antigen is highly homologous to those of the large heat-shock proteins of Saccharomyces cerevisiae (HSP90) and Drosophila melanogaster (HSP83). mRNA encoding p86 increases in response to heat shock of adult worms, as does HSP70. Comparisons of the sequences of HSP70 and HSP83 homologues show that these two families of heat-shock proteins are not significantly related except for the last four amino acid residues, which are Glu-Glu-Val-Asp in every case. This sequence is not found at the carboxy terminus of any other protein in the current databases.     

Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae.

A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.     

Pseudomonas aeruginosa and Candida albicans interaction in the respiratory tract: from pathophysiology to a therapeutic perspective

Several studies evidenced a pathogenic interaction between P. aeruginosa and C. albicans. P. aeruginosa is one of the most frequent microorganisms responsible for ventilator-associated pneumonia (VAP) and C. albicans usually colonize tracheo-bronchial tract of patients undergoing mechanical ventilation in the intensive care unit. In vitro, P. aeruginosa exploits filamentous fungi resulting in fungal killing and limitation of C. albicans growth in the host. Biofilm also play an important role as a persistent source of infectious material for recurrent episodes of VAP. Indeed, Candida spp and P. aeruginosa are the most common pathogens retrieved from endotracheal tube biofilm and tracheal secretions in patients with VAP. Finally, it has been demonstrated that previous tracheo-bronchial C. albicans colonization enhanced the incidence of P. aeruginosa pneumonia in a murine model. A recent study performed in patients requiring intubation and mechanical ventilation identified Candida spp tracheo-bronchial colonization as a risk factor for P. aeruginosa VAP. In addition, a retrospective study suggested that antifungal treatment might reduce P. aeruginosa VAP or tracheo-bronchial colonization in intubated patients with Candida spp tracheo-bronchial colonization. These interactions have major environmental and medical consequences. Experimental studies providing a better understanding of the mechanisms of interaction and clinical studies evaluating the necessity of a antifungal treatment might improve the management of these opportunistic infections.     

Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family.

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the beta-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.     

Expression of the C3d-binding protein (CR2) from Candida albicans during experimental candidiasis as measured by lymphoblastogenesis.

The complement C3d-binding protein (CR2) of Candida albicans has been purified by immunoaffinity chromatography, and its specificity has been characterized by immunoblotting with monoclonal antibodies to the C. albicans CR2 and the mammalian CR2. Recent studies with immunoelectron microscopy indicated that the CR2 was expressed during a systemic infection in a murine model of candidiasis. As a continuation of these observations, the immunogenicity of the C. albicans CR2 was investigated in a lymphoblastogenesis assay. Lymph node cells as well as splenic lymphocytes from mice infected subcutaneously with viable blastoconidia of C. albicans reacted to the C. albicans CR2 to a significantly greater extent than did lymphocytes from uninfected mice (P less than 0.01). The maximum stimulation of splenic lymphocytes by the purified receptor occurred at a concentration of 0.54 micrograms of protein per ml after 72 h of incubation of lymphocytes and receptor. Also, splenocytes from infected or CR2-immunized mice exhibited significantly reduced responses to the T-cell-dependent mitogen phytohemagglutinin (P less than 0.01). These data indicate that lymphocytes from infected mice respond to the C. albicans CR2 in a lymphoproliferation assay to a greater extent than do lymphocytes from uninfected mice, indicating that the CR2 is expressed in vivo.     

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56.

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.     

Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.

Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.     

The lppC gene of Streptococcus equisimilis encodes a lipoprotein that is homologous to the e (P4) outer membrane protein from Haemophilus influenzae

We report the cloning, sequencing, and analysis of a novel chromosomal gene of Streptococcus equisimilis strain H46A that codes for a membrane lipoprotein, designated LppC. The lppC gene is located 3′ adjacent to, and co-oriented with, the unrelated gapC gene that encodes the previously characterized glyceraldehyde-3-phosphate dehydrogenase. Sequencing of lppC revealed an 855-bp open reading frame that predicted a 32.4-kDa polypeptide possessing a potential lipoprotein signal sequence and modification site (VTGC). Signal sequence processing of LppC synthesized in the homologous host or expressed from plasmid pLPP2 in Escherichia coli was sensitive to globomycin, a selective inhibitor of lipoprotein-specific signal peptidase II. Subcellular localization of LppC using polyclonal antibodies raised to the hexahistidyl-tagged protein proved LppC to be tightly associated with the cytoplasmic membrane of S. equisimilis and with the outer membrane of E. coli JM109 (pLPP2). Southern, Northern and Western analyses indicated that lpp was conserved in S. pyogenes, and transcribed independently of gap as monocistronic 0.9-kb mRNA from a σ ⁷⁰-like consensus promoter. Database searches found homology of LppC to the hel gene-encoded outer membrane protein e (P4) from Haemophilus influenzae to which it exhibits 58% sequence similarity. However, unlike the hel gene, lppC was unable to complement hemA mutants of E. coli for growth on hemin as sole porphyrin source in aerobic conditions. Furthermore, neither the wild type nor an lppC insertion mutant of S. equisimilis could grow on hemin in iron-limited medium. These results, together with findings indicating that S. equisimilis H46A had no absolute requirement for iron, led us to conclude that lppC, in contrast to hel, is not involved in hemin utilization and has yet to be assigned a function. Received: 7 March 1997     

Characterization of the gene encoding a 26-kilodalton protein (OMP26) from nontypeable Haemophilus influenzae and immune responses to the recombinant protein

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.     

Ca3 fingerprinting of Candida albicans bloodstream isolates from the United States,Canada, South America,and Europe reveals a European clade

It was previously demonstrated by a cluster analysis that 26 unrelated U.S. isolates of Candida albicans separated into three distinct groups (groups I, II, and III) while South African isolates separated into four distinct groups (groups I, II, III, and SA). To verify the absence or underrepresentation of SA isolates in North America, and to identify which groups are represented in Europe and South America, collections of bloodstream isolates from each geographical locale were analyzed by cluster analyses based on genetic fingerprinting with the Ca3 probe. The results verify that North America is almost devoid of SA isolates (2%). However, the results reveal a new clade, designated group E, relatively specific to Europe. While 26% of a European collection of 46 isolates was composed of group E isolates, only 2% of the 164 North American isolates, 5% of 22 South American isolates, and 1% of 361 South African isolates were composed of group E isolates. The North American collection proved to be the least-diverse collection in regard to group representation. In a comparison of collections from the Northeast, Midwest, and Southwest regions of the United States, Canada, and South America, it was demonstrated that both the U.S. Southwest and the South American collections were devoid of group II isolates. Together these results identify for the first time a European-specific clade and demonstrate clear distinctions in the representations of the five demonstrated clades (groups I, II, III, SA, and E) in different geographical locales.