Infection and Immunity

Published by American Society for Microbiology
Online ISSN: 1098-5522
Print ISSN: 0019-9567
Plasmids, phages, and strains used in this study 
In situ gel hybridization of DNA from clinical isolates. Total DNAs were digested with EcoRI and hybridized with the 841-bp SmaI-Pstl slt-IIA probe. The clinical isolates from which DNAs were obtained were J-2 (lane 1), H19 (lane 2), B2F1 (lane 3), B2387 (lane 4), B1409-C1 (127) (lane 5), B1375-GSC15 (107) (lane 6), E32511 (lane 7), CL8 (lane 8), A8959-C7 (lane 9), A9047-CS1 (lane 10), EDL933 (lane 11), and CL40 (lane 12). Numbers to the right are marker DNA fragments in kilobase pairs. 
Antibodies used in this study 
Clinical isolates of E. coli that contain DNA hybridizing with slt-I and slt-II probes or both No. of probe-positive 
Comparison of the nucleotide and predicted amino acid sequences of the processed A and B subunits of the slt-IIc genes with those of the SLT-II family % Homology oft: 
Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli O157:H- strain E32511. One copy of the operon was found to be essentially identical to slt-II; it differed from slt-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon slt-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-II showed that SLT-IIc was antigenically related to but distinct from SLT-II.
Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.
Focal necrosis (arrow) in cerebellar pec (see legend to Fig. 1). Necrotic focus is demarcat( hemorrhage and is associated with arteriolar necrosi Bar, 100 ,um. 
Arteriole in cerebellar peduncle of pigle Fig. 1). Endothelial cell appears regenerative with nucleus and basophilic cytoplasm (arrowhead). Myc media are necrotic (arrow). Bar, 25 p.m. 
Gnotobiotic piglets inoculated with Escherichia coli serotype O157:H7 strains that produced Shiga-like toxin II developed brain lesions similar to those observed in edema disease of swine, including arteriolar necrosis and malacia. Loss of ability to produce Shiga-like toxin II resulted in loss of ability to cause brain lesions.
Slide agglutination assay for the whole cells of four C. tropicalis strains with anti-Candida PFAbs 
Chemical compositions and specific rotations of mannans Fr 0199 and Fr 1647 obtained from C. tropicalis strains 
'H chemical shifts (anomeric region) of oligosaccharides (ct anomer) obtained from C. tropicalis mannans Fr 0199 and Fr 1647 
We conducted a structural analysis of the cell wall mannans isolated from two Candida tropicalis strains, IFO 0199 and IFO 1647, exhibiting strong agglutinabilities against anti-Candida factor sera 5 and 6. The products released from these mannans by acid treatment were identified as the oligosaccharides, from biose to pentaose, consisting solely of beta-1,2-linked mannopyranose units corresponding to common epitopes of Candida albicans serotypes A and B (factor 5). Mild acetolysis of acid- and alkali-treated mannans produced large amounts of hexaose and heptaose, Man rho beta 1-2Man rho beta 1-2Man rho alpha 1-2Man rho alpha 1-2Man rho alpha 1-2Man and Man rho beta 1-2Man rho beta 1-2Man rho beta 1-2Man rho alpha 1-2 Man rho alpha 1-2Man, corresponding to the C. albicans serotype A-specific epitopes (factor 6). However, the homologous pentaose, Man rho beta 1-2Man rho alpha 1-2 Man, was not generated by this procedure. The oligosaccharides (biose to hexaose) obtained from the mannans by conventional acetolysis were composed exclusively of alpha-1,2-linked mannopyranose units. Therefore, the mannans of C. tropicalis IFO 0199 and IFO 1647 do not have the alpha-1,3-linked mannopyranose units previously observed in the mannans of C. albicans and Candida stellatoidea. The results of this study and previous findings indicate that the similarity of the antigenicities of three Candida species, C. albicans serotype A, C. stellatoidea type II, and C. tropicalis, reside in the beta-1,2 and alpha-1,2 linkages containing oligomannosyl side chain (factor 6) in the cell wall mannan.
Photograph of a 1.5% agarose gel showing ␤ -actin, IFN- ␥ , IL-4, and IL-10 bands identified by RT-PCR. Fifty microliters of whole blood from each patient was diluted to 200 ␮ l using RPMI 1640 and cultured in duplicate in the absence (lanes 1 to 4) or in the presence (lanes 5 to 8) of PPD-RT23 (100 U/ml) at 37°C in a CO 2 incubator. After 48 h, cultures were harvested, duplicates were pooled, total RNA was extracted, and cDNA was synthesized and diluted to 100 ␮ l. Five microliters each of this cDNA was used as a template to amplify the ␤ -actin, IFN- ␥ , IL-4, or IL-10 gene using sequence-specific primers. The results were documented in a Kodak EDAS 120 system, and band intensity was measured as pixels. The cytokine band intensity was normalized to the ␤ -actin band intensity using the formula net intensity of cytokine/net intensity of ␤ -actin ϫ 100. Lane M, standard marker ( ␾ 174 digested with Hae III). 
Comparison of basal (no-antigen control) and PPD-RT23-induced IFN-, IL-4, and IL-10 expression in pulmonary tuberculosis patients (n 43) and controls from areas of endemicity (n 44) from south India, studied by the whole-blood culture method. Fifty microliters of whole blood diluted to 200 l with RPMI medium was incubated for 48 h in the presence or absence of antigen in a CO 2 incubator. Ninety-one percent of the controls and 71% of the patients expressed IFN-in the no-antigen control mixture, while all of the patients and controls expressed IFN-in the presence of PPD. In contrast, only certain individuals (responders, positives), produced IL-4 and/or IL-10 in either the presence or absence of PPD. The values of cytokine expression (percentage of-actin expression) of a sample in the absence and presence of PPD are connected by lines, and the paired t test (PT) or WRT was applied to determine the significance.
Correlation between BCG scar status and IL-10 and/or IL-4 expression status in pulmonary tuberculosis patients and controls
Nested classification analysis of three factors (DRB102, BCG, and IL-10 status) and their interactions in pulmonary tuberculosis patients (n 71)
HLA DRB1*02 and its subtypes predispose individuals for a far-advanced sputum-positive pulmonary tuberculosis transcending ethnic boundaries. Mycobacterium bovis BCG does not afford the desired protection against adult pulmonary tuberculosis, and a spectrum of immune reactivity exists in controls and hospital contacts. All of these findings have been identified and demonstrated in areas of endemicity. Skewing of immunity from protective to pathogenic may involve a shift in the Th1-Th2 paradigm. To elaborate these ideas, we studied gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 cytokine expression in 71 adult pulmonary tuberculosis patients and 74 controls from areas of endemicity in south India by 48-h microculture and reverse transcription-PCR. Most of the patients and controls expressed IFN-gamma de novo, and in the presence of purified protein derivative (PPD), all of them expressed significantly higher levels of IFN-gamma, suggesting a PPD-specific recall memory. HLA DRB1* allele-dependent IFN-gamma expression was identified only in controls, suggesting a skewing of the immune response in patients. In contrast to the case for IFN-gamma, only some patients and controls expressed IL-4 or IL-10 (Th2 profile); thus, the Th1 profile was identifiable only by a nonexpression of IL-4 or IL-10 in this area of endemicity. The Th2 profile was associated with HLA non-DRB1*02 and BCG scar-negative status in patients, attributing a significant risk (odds ratio = 2.074; 95% confidence interval = 0.612 to 7.07). It is possible that Mycobacterium tuberculosis (PPD)-specific IL-10 is expressed preemptively in unvaccinated (BCG scar-negative) individuals with a non-DR2 genetic background by chronic exposure in this area of endemicity and leads to pulmonary tuberculosis of adults.
IFN- ␥ production by spleen cells from HHD mice immunized with pCI-MPT51. The IFN- ␥ production by splenocytes from HHD mice immunized with the pCI-MPT51 plasmid in response to 1 of 26 overlapping peptides (5 ␮ M) covering the MPT51 molecule or medium alone ( Ϫ ) was evaluated. Splenocytes from naı  ̈ve HHD mice were also examined as a control. The data are representative of the results of three independent experiments. 
Identification of a T-cell epitope in the MPT51 p53-62 peptide and the T-cell subset recognizing the epitope in HHD mice. (A) Levels of IFN- ␥ -producing T-cell subsets in spleens of HHD mice immunized with the pCI-MPT51 plasmid. Three-color flow cytometric analysis was performed for detection of intracellular IFN- ␥ and cell surface CD4 and CD8 molecules after immune splenocytes were cultured in the presence of the MPT51-derived peptides p51-70 (20-mer peptide), p21-29 (FLAGGPHAV), p53-61 (TLAGKGISV), and p53-62 (TLAGKGISVV). The data are the percentages of IFN- ␥ -producing CD4 ϩ or CD8 ϩ cells in the total CD4 ϩ or CD8 ϩ cells after 4 h of stimulation with peptides. The 
MPT51 p53-62 is a dominant T-cell epitope in HHD mice. The IFN- ␥ production by splenocytes from HHD mice immunized with the pCI-MPT51 plasmid in response to twofold serially diluted doses of candidate peptides MPT51 p53-62 (TLAGKGISVV), p53-61 (TLAGKGISV), and p21-29 (FLAGGPHAV) was evaluated. The data are representative of the results of three independent experiments. 
MPT51 p53-62 peptide binds to cell surface HLA-A * 0201 molecules and can be recognized by immune T cells in the context of 
Detection of MPT51 p53-62-specific memory T cells in PBMCs of HLA-A * 0201 ϩ PPD-reactive healthy subjects. (A) Flow cytometric analyses to detect MPT51 p53-62-specific memory T cells in PBMCs of HLA-A * 0201 ϩ PPD-reactive healthy subjects using MPT51 p53-62/HLA- A * 0201 tetramer. PBMCs of the healthy subjects were prepared and cultured for 10 days together with mitomycin C-treated, MPT51 p53-62-pulsed autologous PBMCs and then subjected to flow cytometric analysis after treatment with PE-conjugated MPT51 p53-62/HLA-A * 0201 tetramer and FITC-conjugated anti-human CD8 MAb staining (graphs A and B). HLA-unmatched PBMCs were used as a negative control (graph C). Representative flow cytometry patterns are shown. The percentages of tetramer-positive cells in the total CD8 ϩ cells are indicated. (B) IFN- ␥ production by PBMCs of HLA-A * 0201 ϩ PPD-reactive healthy subjects stimulated with MPT51 p53-62 (TLAGKGISVV)- or p53-61 (TLAGKG ISV)-pulsed autologous PBMCs for 10 days as evaluated by an IFN- ␥ ELISA. Samples A and C correspond to graphs A and C in panel A. 
CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-γ) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-γ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-γ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.
Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.
Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) in resistance of NTHI to oxidants and persistence in vivo. Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.
Genetic organization of an A. baumannii capsule cluster containing polymerization and transport genes. Eps I polysaccharide export outer membrane protein (epsA; locus tag ABBFA003459; E. coli ortholog, wza). Low-molecular-weight protein tyrosine phosphatase (ptp; locus tag ABBFA003460; E. coli ortholog, wzb). Protein tyrosine kinase (ptk; locus tag ABBFA003461; E. coli ortholog wzc). An FK506-binding protein-type peptidyl-prolyl cis-trans isomerase (fkpA) is outside the capsule gene cluster. Ruler numbering indicates the genomic coordinates (NCBI accession number CP001172) (1). 
Percentages of bacterial cells bound by MAb 13C11 (anti-LPS core) and 13D6 (anti-K1 capsule) as assessed by flow cytometry 
Confirmation that AB307.30 and AB307.45 are capsule deficient by Western analysis. Composite Western blotting was performed on proteinase K-treated whole-cell lysates; the lysates were then probed with MAb 13D6, which recognizes the capsular polysaccharide epitope, and MAb 13C11, which recognizes a deep LPS core epitope. Binding of MAb 13D6 (A) and of MAb 13C11 (B) to AB307-0294 (lane 1), AB307.30 (lane 2), AB307.30/pNLAC1::ptk (lane 3), AB307.30/pNLAC1 (ptk mutant with the pNLAC1 plasmid without the insert, capsule minus) (lane 4), AB307.45 (lane 5), AB307.45/pNLAC1::epsA (lane 6), AB307.45/pNLAC1 (epsA mutant with the pNLAC1 plasmid without the insert, capsule minus) (lane 7), AB307::Tn17 (lane 8), AB307::Tn17/pSS11 (complemented lpsB mutant, wt LPS, capsule positive) (lane 9), and AB307::Tn17/pNLAC1 (lpsB mutant with the pNLAC1 plasmid without the insert, deep LPS core mutation, capsule positive) (lane 10). The positions of molecular mass standards are shown to the right. 
Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection.
Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.
Properties of adherence of S. greenside serotype 050 to trophozoites of E. histolyticaa 
Carbohydrate-binding activity present on the Entamoeba histolytica cell surfaces was found to mediate the adherence of two types of bacteria, Escherichia coli serotype 055 and Salmonella greenside 050. Adherence was inhibited by low-molecular-weight carbohydrates (10 mg/ml) such as galactose, lactose, and N-acetylgalactosamine, as well as by asialofetuin and the lipopolysaccharide extracted from E. coli 055. Mild periodate oxidation of the bacteria inhibited their adherence, whereas heat inactivation, glutaraldehyde fixation, or gamma-irradiation had no effect. On the other hand, pretreatment of trophozoites with glutaraldehyde, cytochalasin B, or cold (5 degrees C) abolished adherence. None of these treatments, however, affected the attachment of bacteria that contain on their cell surface type I pili with mannose-binding capacity. These findings lend further support to our earlier observations on how amoebae interact with bacteria.
Chemical compositions and specific rotations of Fr K, Fr K-b, and Fr K-e 
We conducted a structural analysis of the antigenic cell wall mannoprotein (mannan) isolated from Candida kefyr (formerly Candida pseudotropicalis) IFO 0586. The result of two-dimensional homonuclear Hartmann-Hahn analysis of this mannan indicates that the molecule is constructed from alpha-1,2- and alpha-1,6-linked mannopyranose residues. Upon alkali treatment (beta-elimination reaction), this mannan released two alpha-1,2-linked mannooligosaccharides, biose and triose. The structure of the alkali-stable mannan (outer chain) moiety was investigated by acetolysis. The structures of the resultant oligosaccharides, biose and triose, from the outer chain moiety were found to be the same as those of the alkali-released ones. Further, the treatment of the parent mannan with an Arthrobacter GJM-1 exo-alpha-mannosidase gave a linear mannan consisting solely of alpha-1,6-linked mannopyranose residues. These results indicate that the mannan forms the long backbone of the alpha-1,6 linkage, with a large number of short alpha-1,2-linked oligomannosyl side chains forming a comblike structure. Moreover, we investigated the serological properties of this mannan by performing an inhibition assay of a slide agglutination reaction with mannooligosaccharides and polyclonal factor sera (Candida Check; Iatron). The result indicates that the factor 1 serum preferentially recognizes the alpha-1,2-linked oligomannosyl side chains in this mannan. On the other hand, the fact that the mannan does not contain an antigenic determinant(s) corresponding to factor 8 suggests that the epitope(s) of this factor resides in other molecules on the cell surface of this strain.
Structural and antigenic heterogeneity has been noted among lipopolysaccharides (LPS) produced by Pseudomonas aeruginosa within serogroups previously considered to be serologically homogeneous. We characterized murine monoclonal antibodies (MAbs) and immunization-induced human polyclonal antibodies reactive with one or more of five structurally variant LPS subtypes belonging to serogroup 06 of the International Antigenic Typing System. Analyses of five different MAbs employing purified LPS or whole patterns of subtype specificity, ranging from recognition of a single subtype to reactivity with all five. MAb-mediated opsonophagocytic killing and in vivo protection against live challenge in mice correlated, in general, with differential binding to various LPS subtypes. In comparison, sera from human vaccinees immunized with LPS-derived high-molecular-weight polysaccharide from P. aeruginosa Fisher immunotype 1, one of five serogroup 06 subtypes, exhibited LPS binding and opsonic activity against all five subtypes. Antibodies in the human sera effectively inhibited binding to all five LPS subtype antigens of the cross-reactive MAb, LC3-2H2, suggesting the existence of a common serogroup-related epitope. These findings emphasize the importance of defining subtype-associated variations in LPS antigenicity and corresponding differences in antibody specificity and function as a basis for designing immunoprophylactic or therapeutic strategies which target P. aeruginosa LPS.
Anthrax lethal toxin (LT) activates the NLRP1b (NALP1b) inflammasome and caspase-1 in macrophages from certain inbred mouse strains, but the mechanism by which this occurs is poorly understood. We report here that similar to several NLRP3 (NALP3, cryopyrin)-activating stimuli, LT activation of the NLRP1b inflammasome involves lysosomal membrane permeabilization (LMP) and subsequent cytoplasmic cathepsin B activity. CA-074Me, a potent cathepsin B inhibitor, protects LT-sensitive macrophages from cell death and prevents the activation of caspase-1. RNA interference knockdown of cathepsin B expression, however, cannot prevent LT-mediated cell death, suggesting that CA-074Me may also act on other cellular proteases released during LMP. CA-074Me appears to function downstream of LT translocation to the cytosol (as assessed by mitogen-activated protein kinase kinase cleavage), K+ effluxes, and proteasome activity. The initial increase in cytoplasmic activity of cathepsin B occurs at the same time or shortly before caspase-1 activation but precedes a larger-scale lysosomal destabilization correlated closely with cytolysis. We present results suggesting that LMP may be involved in the activation of the NLRP1b inflammasome.
Chlorinated hydrocarbon 2,2-bis(parachlorophenyl)-1,1,1-trichloroethane (DDT) and its derivative 2,2-bis(parachlorophenyl)-1,1-dichloroethane (o,p'-DDD) protected the cells of human embryonic intestine, Henle strain, against the cytotoxic effects induced by staphylococcal enterotoxin B (SEB). The hydrocarbons were incorporated in the culture medium, and the cytotoxicity of SEB was measured by cell destruction and by inhibition of cell growth during incubation for 48 hr. When DDT at nontoxic levels (4 to 25 mug/ml) was added to cultures, 24 or 4 hr prior to or 7 hr after the administration of toxin (100 mug/ml), the cells did not show the characteristic cytotoxicity. A therapeutic effect was also observed with o,p'-DDD added to cultures 9 hr after SEB. Because the cells treated with DDT for a period of 24 hr remained resistant to the toxin when DDT was removed from the cultures, it appears that DDT acts upon the cells rather then upon enterotoxin B. The protective effect of DDT was also observed against the toxin-induced skin erythemas and necrosis in guinea pigs treated with DDT.
Age-related loss of the Lyt-1,2 subset 
C58 mice (aged greater than or equal to 5 months) are susceptible to age-dependent polioencephalomyelitis, a paralytic central nervous system disease induced by lactic dehydrogenase virus. Susceptibility results from the loss of protective T cells. Data are presented showing a positive correlation between the age-related loss of Lyt-1,2 cells and the development of susceptibility to neuroparalytic lactic dehydrogenase virus infection.
In order to identify the branches containing both beta-1,2 and alpha-1,2 linkages as the serotype A-specific epitope(s) in the mannans of Candida albicans, serotype A strains with oligosaccharides constituting the beta-1,2 linkage, the alpha-1,2 linkage, and both the beta-1,2 and the alpha-1,2 linkages were prepared from the mannans of C. albicans serotype A strains (NIH A-207 and J-1012) and tested for their inhibitory effects in the precipitin and slide agglutination assays. The results indicated that two oligosaccharides containing both beta-1,2 and alpha-1,2 linkages, Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Manp alpha 1-2Man and Manp beta 1-2Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Manp alpha 1-2Man, served as epitopes participating in the serotype A specificity of C. albicans strains.
Comparison of the B. abortus (Ba) Cgt cyclic ␤ -1,2-glucan transporter protein with the A. tumefaciens (At) ChvA and S. meliloti (Sm) NdvA proteins. Conserved amino acids are indicated by gray boxes. Three typical ABC transporter motifs, Walker A and B and the SGG(Q) ABC signature, are indicated by boxes. The percentages of identity with the amino acid sequence of the ABC transporter protein (Cgt) from B. abortus are indicated in parentheses. The alignment was performed with the MegAlign program. 
Virulence and nodulation complementation assays. (A) Al- falfa plants inoculated with different strains of S. meliloti . (1 and 2) F34; (3 and 4) LI1; (5 and 6) LI1 (pBB4 cgt ); (7 and 8) noninoculated control. (B) Kalanchoe leaf inoculated with different strains of A. tumefaciens. The virulence and nodulation assays were carried out as described in Materials and Methods. Photos were taken 4 weeks posti- noculation in both assays. 
Complementation of Sinorhizobium and Agrobacterium mutants Strain Motility a Virulence b Nitrogen fixation c Anionic glucan d
Intracellular replication of different strains of B. abortus . Monolayers of HeLa (A and B) and J774 (C) cells were inoculated with 10 7 and 5.10 6 CFU of bacteria, respectively. After 1 h of incuba- tion at 37 ° C, cells were washed as described in Materials and Methods and streptomycin and gentamicin were added. Numbers of CFU were determined at the indicated times. Values are the means Ϯ standard deviations of the results from one independent experiment out of two performed in duplicate; the P value is Ͻ 0.05). Statistical analysis was performed with a t test. 
Brucella persistence in spleens of mice inoculated with different strains of B. abortus 2308 and S19. Mice were inoculated intraperitoneally with 10 4 CFU of B. abortus . At the indicated times postinfection, 5 mice per group were killed and their spleens were removed. The numbers of CFU in spleen tissues were determined as indicated in Materials and Methods. (A) B. abortus 2308 strains: re- covery of viable bacteria from spleens at 4 or 8 weeks postinfection. (B) B. abortus S19 strains: recovery of viable bacteria from spleens at 2 or 4 weeks postinfection. (C) B. abortus S19 strains: weight of spleens of infected mice at 2 or 4 weeks postinfection. Values are expressed as means Ϯ standard errors of the means ( n ϭ 5); the P value is Ͻ 0.05. Statistical analysis was performed with a t test. ء , mice inoculated with the B. abortus cgt mutant complemented with plasmid pBB4522 showed values not statistically different from those observed with the same strain complemented with pBB4 cgt . 
The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic β-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic β-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic β-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic β-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.
TLC of cyclic ␤ -1,2-glucans accumulated by different B. abortus strains. Cyclic ␤ -1,2-glucans were extracted and submitted to TLC as described in Materials and Methods. Lanes: 1, B. abortus 2308; 2, B. abortus 2308 cgs mutant (BvI129 strain); 3, B. abortus strain BvI129 complemented with plasmid pBA19; 4, B. abortus S19; 5, B. abortus S19 cgs mutant (BAI129 strain); 6, B. abortus strain BAI129 complemented with plasmid pBA19. ء and ءء , migration of charged and neutral B. abortus cyclic ␤ -1,2-glucans, respectively. 
Western blot of different B. abortus strains. Whole-cell ly- sates of different B. abortus strains were electrophoresed and trans- ferred, and LPS O antigen was detected as described in Materials and Methods. Lanes: 1, B. abortus 2308 (wild-type strain); 2, B. abortus 2308 BvI129 ( cgs mutant); 3, B. abortus S19 (vaccinal strain); 4, B. abor- tus S19 BAI129 ( cgs mutant); 5, B. abortus RB51. 
Brucella persistence in spleens of mice inoculated with different strains of B. abortus . Mice were inoculated intraperitoneally as described in Materials and Methods with 10 4 CFU of B. abortus . (A) Shaded bars, B. abortus 2308 (wild-type strain); open bars, B. abortus 2308 BvI129 ( cgs mutant). (B) Shaded bars, B. abortus S19 (vaccinal strain); open bars, B. abortus S19 BAI129 cgs mutant. At different times postinfection (2, 4, or 12 weeks) five mice per group were killed and their spleens were removed. The numbers of CFU in spleen tissues were determined as indicated in Materials and Methods. 
Quantitation of IFN- ␥ in spleen of infected mice. BALB/c mice were infected with 10 4 CFU of B. abortus S19 (shaded bars) or B. abortus cgs mutant (open bars). At different times postinfection, spleens were removed and total RNA was extracted and analyzed separately. (A) Quantitation of IFN- ␥ mRNA was carried out by RT-PCR as described in Materials and Methods. Error bars indicate standard deviations. (B) Agarose gel of the amplicon products from three mice at each time point. After the gel dried, radioactivity was detected by autoradiograph. 
Pattern of immunoglobulins elicited by B. abortus S19 or the B. abortus BAI129 cgs mutant. KELA results for pooled sera from five mice inoculated with 7 ϫ 10 8 CFU of B. abortus S19 (A) or B. abortus BAI129 (B) obtained at 1, 2, or 4 weeks postinfection are shown. IgG1 plus IgG2a give total KELA units. 
Null cyclic β-1,2-glucan synthetase mutants (cgsmutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuatedB. abortus S19 strain. Although the cgsmutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected withB. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by thecgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.
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.
β-1,2-Oligomannosides (β-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 β-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of β-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 β-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of β-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.
In previous articles, we reported the presence of phosphate-bound beta-1,2-linked oligomannosyl residues in the mannans of strains of Candida albicans serotypes A and B and Candida stellatoidea. To identify the antigenic factor corresponding to this type of oligomannosyl residue, a relationship between chemical structure and antigenic specificity in the mannans of C. albicans NIH B-792 (serotype B, B-strain) and C. albicans J-1012 (serotype A, J-strain) was investigated by using a combination of two-dimensional 1H nuclear magnetic resonance spectroscopy of H-1, H-2, and H-5 regions in the mannans and an enzyme-linked immunosorbent assay that employed concanavalin A-coated microtiter plates. It was shown in the present 1H nuclear magnetic resonance study that an examination of chemical shifts not only in the H-1 region but also in the H-5 region was useful for the quantitative determination of the phosphate-bound beta-1,2-linked oligomannosyl residues. In the enzyme-linked immunosorbent assay using concanavalin A-coated plates, it was revealed that, of factor sera 1, 4, and 5, only factor serum 5 showed a reactivity proportional to the densities of the beta-1,2-linked oligomannosyl residues of the mannan subfractions of different phosphate contents that had been prepared from the bulk B-strain mannan by DEAE-Sephadex chromatography. The above results indicate that the phosphate-bound beta-1,2-linked oligomannosyl residues, Manp beta 1----(2Manp beta 1----)n2Man (n = 0-5), correspond to antigenic factor 5.
Effect of growth conditions on the expression of ␤ -1,2- linked mannose epitopes on C. albicans glycoconjugates. C. albicans VW32 (serotype A) was preincubated and then incubated for 24 h at 28 ° C (lanes 1), 37 ° C (lanes 2), 39 ° C (lanes 3), or 41 ° C (lanes 4) on Sabouraud ’ s agar. The preparation in lanes 5 was preincubated at 41 ° C and then incubated at 28 ° C. Electrophoretic separation of the AERC extracts was performed on 5 to 15% acrylamide mini-gels. (A) Con- tents revealed with MAb DF9-3; (B) contents revealed with MAb 5B2. 
Western blot analysis of AERC extracts from C. albicans serotypes A and B grown at 28 ° C (lanes 1) or 37 ° C (lanes 2) for 24 h on Sabouraud ’ s agar. Reference strains and strains isolated from pos- itive blood cultures were used. The serotype A strains used were C. albicans NIH A 207 (A), C. albicans 4224 (B), C. albicans 517 (D), C. albicans ATCC 10261 (F), C. albicans ATCC 32354 (G), and C. albi- cans VW32 (I). The serotype B strains used were C. albicans 6780 (C), C. albicans 8252 (E), and C. albicans ATCC 90028 (H). AERC extracts were obtained by the standard procedure. Large 7 to 20% acrylamide gels were used to allow better resolution of the samples, and ␤ -1,2- mannosylation of glycoconjugates was revealed with MAb DF9-3. 
Comparison of cell wall extracts from C. albicans strains VW32 (serotype A) (A) and NIH B 792 (serotype B) (B) grown at 28 ° C (lanes 1) or 37 ° C (lanes 2) in Sabouraud ’ s broth. Cells were fi rst sensitized for 30 min with 0.3 M ␤ -mercaptoethanol and then treated with Zymolyase 20T (40 U/g [wet weight] of cells) for 45 min at 37 ° C. Acrylamide mini-gels (7 to 20% acrylamide) and MAb DF9-3 were used for separation and detection of glycoconjugates, respectively. 
Comparative analysis of the reactivities of MAbs 5B2 (A), DJ2-8 (B), DF9-3 (C), 10G (D), and AF1 (E) speci fi c for ␤ -1,2- oligomannosides in AERC extracts from C. albicans strain VW32 (serotype A) (lanes 1), strain NIH A 207 (serotype A) (lanes 2), and strain NIH B 792 (serotype B) (lanes 3). Cells were grown at 28 ° C on Sabouraud ’ s agar, and 7 to 20% acrylamide gels were used. 
Divergent reactivities of MAb B6.1 with PLM from sero- type A and B strains. (A) MAb DJ2-8, used as a control, reacted with PLM from both serotype A strain VW32 (lane 1) and serotype B strain NIH B 792 (lane 2). (B) MAb B6-1 reacted with PLM from the serotype B strain (lane 2) but not with PLM from the serotype A strain (lane 1). (C) Con fi rmation of the selective reactivity of MAb B6-1 with PLM from serotype B strains (lane 6, strain ATCC 90028; lane 7, strain 6780; lane 8, strain 8252). PLM from serotype A strains were unreac- tive (lane 1, strain VW32; lane 2, strain NIH A 207; lane 3, strain ATCC 32354; lane 4, strain ATCC 10261; lane 5, strain 8252). Cells were grown at 37 ° C on Sabouraud ’ s agar. The panel A and B blots were revealed by using the enhanced chemiluminescence technique (Pierce, Rockford, Ill.) and exposure on ECL hyper fi lm. The panel C blot was revealed by using standard conditions (8). 
Increasing the growth temperature from 28 to 37°C reduced the expression of β-1,2-oligomannoside epitopes on mannoproteins of Candida albicans serotypes A and B. In contrast, β-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (Mr) of this compound. At all growth temperatures investigated, serotype A PLM displayed an Mr and an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.
Blots from SDS-PAGE extracts of C. albicans germ tubes. Lanes 1 and 2, Western blot of an alkali extract of C. albicans germ tubes obtained under reducing conditions. Oxidation with periodate prior to immunodetection with MAb DF9-3 (lane 1) resulted in complete disappearance of reactivity compared with a control strip incubated in acetate buffer (lane 2). Lanes 3 and 4, Western blots of an Fp extract of C albicans germ tubes run in a standard SDS-PAGE and probed, respectively, with ConA-HRP and MAb DF9-3. Lane 5, Western blot of the same Fp extract, which was run in SDS-PAGE without mercaptoethanol and probed with MAb DF9-3. 
Purification of the 14-to 18-kDa antigen from an Fp extract. (A) Crude Fp extract; (B) CMW (10:10:3) extract from proteinase K-resistant material. Lanes 1, silver staining of polyacrylamide gel; lanes 2, Western blot stained with ConA-HRP; lanes 3, Western blot stained with MAb DF9-3. 
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 mannosides (beta-Mans) are believed to contribute to Candida albicans virulence. The presence of beta-Mans has been chemically established for two molecules (phosphopeptidomannan [PPM] and phospholipomannan) that are noncovalently linked to the cell wall, where they correspond to specific epitopes. However, a large number of cell wall mannoproteins (CWMPs) also express beta-Man epitopes, although their nature and mode of beta-mannosylation are unknown. We therefore used Western blotting to map beta-Man epitopes for the different families of mannoproteins gradually released from the cell wall according to their mode of anchorage (soluble, released by dithiothreitol, beta-1,3 glucan linked, and beta-1,6 glucan linked). Reduction of beta-Man epitope expression occurred after chemical and enzymatic deglycosylation of the different cell wall fractions, as well as in a secreted form of Hwp1, a representative of the CWMPs linked by glycosylphosphatidylinositol remnants. Enzyme-linked immunosorbent assay inhibition tests were performed to assess the presence of beta-Man epitopes in released oligomannosides. A comparison of the results obtained with CWMPs to the results obtained with PPM and the use of mutants with mutations affecting O and N glycosylation demonstrated that both O glycosylation and N glycosylation participate in the association of beta-Mans with the protein moieties of CWMPs. This process, which can alter the function of cell wall molecules and their recognition by the host, is therefore more important and more complex than originally thought, since it differs from the model established previously with PPM.
Experimental design used in the study.  
Effect of conditioned medium from-1,2-oligomannoside-stimulated J774 cells on LPS-induced TNF-production. Supernatants from J774 cells incubated for 18 h in the presence of different concentrations of-1,2-oligomannosides were transferred onto fresh J774 cells for 3 h (s), 1 h (OE), or 0 h (F) before addition of LPS. After 5 h of further incubation, TNF-production in cell-free supernatants was measured.
Candida albicans β-1,2-oligomannosides stimulate macrophage tumor necrosis factor alpha (TNF-α) but not NO release. This stimulation desensitized macrophages by altering β-1,2-oligomannoside-dependent TNF-α production and lipopolysaccharide-dependent TNF-α and NO secretion. Desensitization was not related to tyrosine phosphorylation signal transduction but was transferred by culture supernatants in which arachidonic acid derivatives were evidenced.
Confocal laser scanning microscopy of Caco-2 monolayers at 30 min postinfection with C. albicans blastospores. Monolayers sectioned in vertical planes ( x - z ) were processed for double labeling using Alexa fl uor 568-phalloidin (red) and MAb 5B2 (green), and a merged image is presented. The apical region of the cell is identi fi ed by strong reactivity of phalloidin with the actin network of the brush border. Yeast cells are present at the surfaces of the cells. Bar, 5 ␮ m. 
Effects of MAbs 5B2 (anti- ␤ -1,2 mannose), EBCA1 (anti- ␣ -1,2 mannose), and A255 (control antibody) on attachment of C. albicans blastospores to Caco-2 monolayers. Yeast cells were incu- bated with 3, 6, and 60 ␮ g of antibodies/ml or with PBS alone prior to and during adhesion. Attachment is expressed as a percentage of that of the controls to which no antibody was added. The values are the means plus standard deviations of three independent experiments, including testing in duplicate wells. 
Immuno fl uorescence confocal microscopy analysis of ␤ -1,2 oligomannosidic epitopes after a 30-min (A) or 12-h (B) culture of Caco-2 cells with C. albicans . The monolayers were processed for double labeling using Alexa Fluor 568-phalloidin (red) and MAb 5B2 (green), and merged images are presented. (A) Islands of material reactive with MAb 5B2 (arrowheads) in the vicinity of C. albicans (arrows). (B) Analysis in the vertical plane shows that ␤ -1,2 oligom- annosidic epitopes shed by C. albicans (long arrow) are present at the apical surfaces of the cells (short arrows). Bars, 5 ␮ m. 
Double-labeling experiment with MAbs 5B2 and EBCA1 on Caco-2 monolayers incubated for 12 h with C. albicans . (A to C) The same microscopic fi eld was examined under phase-contrast microscopy (A), immuno fl uorescence microscopy for reactivity of MAb 5B2 with ␤ -1,2 oligomannoside (green) (B), and immuno fl uorescence microscopy for reactivity of MAb EBCA1 with ␣ -1,2 oligomannoside (red) (C). Both MAbs react with C. albicans blastospore and germ tube and with clusters of antigenic material at the surface of the cells. (D) Merged image of panels B and C. The secondary antibodies were Alexa Fluor 488 goat anti-mouse IgM (B) and Alexa Fluor 594 goat anti-rat IgM (C). Bars, 5 ␮ m. 
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.
Historically, sunlight has seemed to fortify antituberculosis resistance. Evidence is presented here suggesting a role for vitamin D in this effect. The active metabolite of this photosynthesized vitamin, 1,25-dihydroxy-vitamin D3 (1,25D), promotes maturation and activation of human monocytes and macrophages (MPs). Therefore, it was tested for ability to protect MPs against virulent tubercle bacilli. MPs were derived by 7-day culture from blood monocytes, infected with the bacilli, and exposed to 1,25D in several regimens. Their inhibition of bacilli was measured by lysing samples of the cultures at 0, 4, and 7 days after infection and making bacillary CFU counts from serial dilutions of the lysates. 1,25D enabled MPs to slow or stop bacillary replication. Autologous serum supported the 1,25D-induced protection because the vitamin was not effective in medium supplemented with a serum substitute and was less effective in a heterologous AB serum than in autologous serum. The protection developed rapidly and could be induced even when 1,25D was added 3 days after infection. A concentration on the order of 4 micrograms/ml was needed for protection by the regimens used in these experiments. That is considerably higher than normal circulating concentrations of 1,25D but could be reached in infectious granulomas, because MPs can make 1,25D from precursor 25-hydroxyvitamin D3. The precursor circulates at levels 10(3) higher than those of 1,25D and is directly influenced by dietary intake or photosynthetic production of vitamin D. These results identify 1,25D as an immunomodulator which can reproducibly activate human MPs to express tuberculoimmunity. They connect vitamin D, sunlight, and tuberculoimmunity and suggest that vitamin D should be considered a vital factor in the practical control of tuberculosis.
RNI production by original HL-60 cells (a) and HL-60 hca cells (b). Cells (10 5 in 100 l of medium) were stimulated for 48 h with medium alone ( ), LPS (100 ng/ml) (), PMA (10 ng/ml) ( ), or LPS (100 ng/ml) plus PMA (10 ng/ml) ( ). (c) Inhibitory effect of L-NMMA on RNI production by HL-60 hca cells after 6 days of stimulation with LPS (100 ng/ml) (F). s, background level of RNI production by unstimulated cells. Data are means SEMs for three experiments in each case.
(a) Accumulation of RNI with time in culture wells containing 3 10 6 HL-60 hca cells in 2 ml of medium stimulated with LPS (100 ng/ml). Data represent means SEMs for nine experiments. (b) Daily rate of [ 14 C]citrulline formation from [ 14 C]arginine, using 1.5 10 5 cells per well containing 100 l of medium and stimulated with 100 ng of LPS per ml. Results are means SEMs for six experiments.
(a) RT-PCR for NOS1 (lanes 1), NOS2 (lanes 2), NOS3 (lanes 3), or a GAPDH control (lanes G) with RNA extracted from HL-60 hca cells stimulated with LPS (100 ng/ml) for 0, 24, or 96 h and from unstimulated HUVEC controls. (b) Northern analysis of NOS2 gene expression in HL-60 hca cells after various durations of stimulation with LPS (100 ng/ml). Kinetics over the first 24 h are shown by the graph, representing the ratio of NOS2 mRNA to ␤ -actin mRNA as quantitated on a digital image as described in text. Longer kinetics are depicted in the inset, which compares NOS2 mRNA (Northern blot) with total RNA stained with ethidium bromide (EtBr). The molecular size of the NOS2 band is approximately 4.5 kb. 
(a) Induction of RNI in HL-60 hca cells after 6 days of stimulation with 1,25-D 3 ( ᮀ ), ergocalciferol ( E ), or cholecalciferol ( ‚ ). Filled circles denote experimental controls with medium alone, LPS (100 ng/ml), or LPS (100 ng/ml) plus L -NMMA (1 mM). Data points are means Ϯ SEMs from four experiments. (b) RT-PCR for NOS1 (lanes 1), NOS2 (lanes 2), NOS3 (lanes 3), or a GAPDH control (lanes G) with RNA extracted from HL-60 hca cells stimulated with 10 Ϫ 8 M 1,25-D 3 LPS for 0, 24, or 96 h and from unstimulated HUVEC controls. 
Electron transmission micrograph of HL-60 hca cells, demonstrating infection with M. tuberculosis after 3 days of culture. Magnifications, ϫ 16,500 (left panel) and ϫ 32,500 (right panel). N, HL-60 hca cell nucleus. Arrows point to mycobacteria. 
Inducible synthesis of nitric oxide (NO) by macrophages is an important mechanism of the host defense against intracellular infection in mice, but the evidence for significant levels of inducible NO production by human macrophages is controversial. Here we report that the human promyelocytic cell line HL-60, when differentiated to a macrophage-like phenotype, acquires the ability to produce substantial amounts of NO on stimulation with LPS or 1, 25-dihydroxyvitamin D3 (1,25-D3) in the absence of activating factors such as gamma interferon. Expression of the inducible nitric oxide synthase (NOS2) was confirmed by sequencing of the reverse transcription-PCR product from stimulated HL-60 cells. Kinetic studies after lipopolysaccharide stimulation show that NOS2 mRNA levels rise within 3 to 6 h, that conversion of [14C]arginine to [14C]citrulline is maximal at 5 to 6 days, and that levels of reactive nitrogen intermediates stabilize at around 20 microM at 7 to 8 days. We find that 1,25-D3 acts to suppress the growth of Mycobacterium tuberculosis in these cells and that this effect is inhibited by NG-monomethyl-L-arginine, suggesting that vitamin D-induced NO production may play a role in the host defense against human tuberculosis.
A beta-glucosidase of Coccidioides immitis was identified in electrophoresis gel separations of the concanavalin A-bound mycelial culture-filtrate-plus-lysate preparation. p-Nitrophenol-beta-D-glucopyranoside was used as the substrate to visualize the enzymatically active fraction in nonreducing gels. The gel-isolated, chromatographically purified enzyme has an optimal pH of 8.0 and cleaves beta-1,3-glycosyl linkages. The alkaline beta-glucosidase was further characterized by a pI of 3.8 to 4.0, optimal activity at 37 to 40 degrees C, and molecular size of 120 kDa as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified beta-glucosidase is identical to a previously reported 120-kDa antigen (Ag) which reacts with immunoglobulin M (IgM) tube precipitin (TP) antibody in sera from patients with coccidioidomycosis. The TP-Ag was described as a valuable serodiagnostic reagent for detection of specific IgM in patients with early coccidioidal infections. The beta-glucosidase, like the TP-Ag, was localized in the cell wall and cytoplasmic vesicles of parasitic cells (spherules) by immunofluorescence and immunoelectron microscopy with specific antiserum raised against the purified enzyme. The boiled cell wall fraction isolated from these same young (presegmented) spherules was partially digested by the beta-glucosidase. Addition of a potent beta-glucosidase inhibitor, 1-deoxynojirimycin, to the parasitic-phase culture medium at a concentration of 200 microM blocked or retarded conversion of arthroconidia to spherules. Antibody was raised in guinea pigs against chromatographically purified 1-deoxynojirimycin which was conjugated with bovine serum albumin. The inhibitor was localized by immunofluorescence in the wall of the 1-deoxynojirimycin-treated cells. We suggest that the spherule wall-associated, alkaline hydrolase functions as a beta-1,3-glucanase to provide for wall plasticity as well as intussusception of newly synthesized wall polymers during the period of rapid diametric growth of parasitic cells of C. immitis.
Mast cells are sentinels for infection. Upon exposure to pathogens, they release their stores of proinflammatory cytokines, chemokines, and histamine. Mast cells are also important for the control of certain tick-borne infections. Anaplasma phagocytophilum is an obligate intracellular tick-transmitted bacterium that infects neutrophils to cause the emerging disease granulocytic anaplasmosis. A. phagocytophilum adhesion to and infection of neutrophils depend on sialylated and α1,3-fucosylated glycans. We investigated the hypotheses that A. phagocytophilum invades mast cells and inhibits mast cell activation. We demonstrate that A. phagocytophilum binds and/or infects murine bone marrow-derived mast cells (BMMCs), murine peritoneal mast cells, and human skin-derived mast cells. A. phagocytophilum infection of BMMCs depends on α1,3-fucosylated, but not sialylated, glycans. A. phagocytophilum binding to and invasion of BMMCs do not elicit proinflammatory cytokine secretion. Moreover, A. phagocytophilum-infected cells are inhibited in the release of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-13, and histamine following stimulation with IgE or antigen. Thus, A. phagocytophilum mitigates mast cell activation. These findings potentially represent a novel means by which A. phagocytophilum usurps host defense mechanisms and shed light on the interplay between mast cells and vector-borne bacterial pathogens.
Recent studies of the dimorphic fungal pathogens Histoplasma capsulatum and Paracoccidioides brasiliensis have suggested a role in virulence for the cell surface carbohydrate alpha-(1,3)-glucan. To investigate a possible basis for alpha-(1,3)-glucan in the pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis, we examined three genetically related strains of B. dermatitidis that differ in their virulence for mice: wild-type virulent strain ATCC 26199; mutant strain ATCC 60915, which is 10,000-fold reduced in virulence; and mutant strain ATCC 60916, which is avirulent. Immunologic quantitation of cell wall alpha-(1,3)-glucan revealed that the mutant yeasts were almost devoid of this sugar moiety, in contrast to the high concentration of alpha-(1,3)-glucan on the cell wall of the wild-type yeasts. These differences are discussed in relation to previous studies of yeast surface expression of the WI-1 antigen and recognition and binding of the related strains by human monocyte-derived macrophages.
Expression of surface [Gal-1,3-Gal] structures on cells from GalT wild-type mice but not GalT KO mice. Open histograms show data for unstained cells. Filled histograms show data for IB4FITC-stained cells. (A) Murine LLCa target cells. (B) GalT C57BL/6 splenocytes. (C) GalT KO splenocytes. Cells were stained with IB4-FITC to detect [Gal-1,3-Gal] structures and then analyzed by flow cytometry as described. A second experiment yielded identical results. 
Absorption with synthetic [Gal-1,3-Gal] inhibits IB4-FITC staining of E. coli O86:B7 and fecal bacterial isolates from orally inoculated GalT KO mice. Open histograms indicate results for unstained bacteria. Filled histograms indicate results for IB4-FITC-stained cells. (A) Fecal isolates from untreated GalT KO mice. (B) Fecal isolates from GalT KO mice treated only with streptomycin. (C) E. coli O86:B7 culture. (D) Fecal bacteria from inoculated GalT KO mice. (E and F) E. coli O86:B7 or fecal bacteria samples stained with IB4-FITC absorbed with synthetic [Gal-1,3-Gal] disaccharide (filled histogram) or lactose (open histogram). 
Distribution of anti-Gal IgG and IgM titers in orally inoculated GalT KO mice at day 14 postgavage. Anti-Gal antibody binding titer and isotype were determined by incubating GalT LLCa target cells with serially diluted sera from immunized GalT KO mice. Target cells were then incubated with goat anti-mouse IgM-PE and IgG-FITC antibodies and analyzed by using a FACSCAN flow cytometer as described earlier. Each symbol represents a serum sample from one individual mouse. No antibody binding was detected in test mice prior to oral inoculation or in control mice after streptomycin treatment. Symbols: , IgG titer; F, IgM titer. The bar indicates the median titer. 
Antibodies induced in GalT KO mice by oral inoculation are specific for [Gal-1,3-Gal]. Sera (1:5) were absorbed with 20 mM synthetic [Gal-1,3-Gal] disaccharide for 18 h at 4°C. Duplicate samples were incubated with 20 mM lactose [Gal-1,4-Glc] as a negative disaccharide control. Sera were then assayed for antibody binding to GalT target cells as described in the text. (A) Control mouse sera. The open histogram indicates results for the C57BL/6 serum. The filled histogram indicates results for the naive GalT KO serum. Target cells incubated only with secondary antibody yielded identical binding results. (B) Immunized GalT KO mice. The open histogram indicates results for the C57BL/6 serum control. The filled histogram indicates results for the serum from inoculated GalT KO mice. (C) Serum from inoculated GalT KO mice absorbed with 20 mM synthetic [Gal-1,3Gal] disaccharide (filled histogram) or 20 mM lactose (open histogram). Sera from nine representative immunized mice were assayed with similar results. 
Naturally occurring antibodies against [Gal α-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional α-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT+ enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT+ bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 1010 live GalT+ Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT+ target cells, with extensive cytolysis observed consistently at serum IgM titers of ≥1:320. Absorption with synthetic [Gal α-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT+ bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.
Coccidioides posadasii is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States. Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis. A 495-bp fragment of a C. posadasii gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen. The translated, full-length gene (GEL1) showed high sequence homology to a reported β-1,3-glucanosyltransferase of Aspergillus fumigatus (70% identity, 90% similarity) and was selected for further study. The GEL1 mRNA of C. posadasii was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores. BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of C. posadasii by either the intraperitoneal or intranasal route. In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice. The recombinant β-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis.
Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.
Quantitation of oa-(1,3)-glucan in H. capsulatum yeast cell walls: strain differences and culture conditions 
Yeast cells of H. capsulatum virulent strain G186AR and avirulent variant G186AS, reacted with MOPC 104E [anti-a-(1,3)-glucan] and stained with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin M. Top panels, Phase-contrast microscopy; bottom panels, fluorescence microscopy of the same field. Magnification, x425. 
Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to alpha-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less alpha-(1,3)-glucan than did their virulent parents. No alpha-(1,3)-glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and alpha-(1,3)-glucan has been described for Paracoccidioides brasiliensis. alpha-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.
Size-exclusion chromatography of anion-exchange peaks with high GT activity. (A through D) Size-exclusion chromatograms of anion-exchange peaks obtained at 0.10 (A), 0.125 (B), 0.175 (C), and 0.26 (D) M NaCl. The solid lines represent the protein concentration (A 280 ), and the dotted lines (-) represent the GT activity at various elution volumes.
Anti-Gal binding to GT-treated RBCs by FACS. RBCs were incubated with the peak of highest GT activity obtained post-size-exclusion chromatography of each anion-exchange peak (see Fig. 4) and then incubated first with anti-Gal and then with fluorescein isothiocyanate-linked goat anti-human Ig. (A through D) Data for RBCs incubated with active or heat-inactivated GT obtained post-size-exclusion chromatography of anion-exchange peaks obtained at 0.10 (A), 0.125 (B), 0.175 (C), and 0.26 (D) M NaCl. Affinity-purified anti-Gal had a low level of binding to untreated RBCs (3% of untreated RBCs bound anti-Gal [results not shown]).
SDS-PAGE analysis of GT fractions obtained post-size-exclusion chromatography of each anion-exchange peak (Fig. 4) and which were used in the FACS experiments shown in Fig. 5. Samples were subjected to SDS-PAGE under reducing conditions and then were stained with silver. (A through D) GTs that were obtained post-size-exclusion chromatography of anion-exchange peaks obtained at 0.10 (A), 0.125 (B), 0.175 (C), and 0.26 (D) M NaCl. Molecular mass markers are in the first lane on the left and represent molecular masses (from top to bottom) of 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. 
Humans have abundant circulating anti-alpha (1,3-di)-galactosyl (alpha Gal) antibodies (anti-Gal). Anti-Gal has been implicated in the clearance of senescent human erythrocytes (RBCs). The nature of the anti-Gal-binding RBC epitope has defied explanation, given that humans repress expression of the alpha 1,3 galactosyltransferase (alpha 1,3 GT) enzyme. This study explored whether alpha Gal epitopes on human RBCs might be synthesized by alpha 1,3 GTs of bacterial origin that are translocated into the circulation during commensal colonization of the gut by gram-negative bacteria. We found that an acellular Klebsiella pneumoniae sonicate could add 3H-UDP-Gal to human RBCs in the alpha configuration at 37 degrees C in the presence of 6 mM MnCl2 (pH 7.6). Gradient anion-exchange chromatography of the Klebsiella sonicate yielded four fractions that could catalyze the addition of 3H-Gal to human RBCs. Size-exclusion chromatography of these anion-exchange fractions yielded peaks of high GT activity for each, but only those derived from the first, third, and last anion-exchange fractions incorporated Gal such that the RBCs bound anti-Gal by fluorescence-activated cell sorter, suggesting that these three GTs are alpha 1,3 GTs. Thus, Klebsiella spp. make at least four GTs that can add an alpha Gal to human cell surface acceptor structures. Three of these GTs can form alpha 1,3 Gal structures on human RBCs that bind anti-Gal, thereby creating "autoimmune" senescence-associated RBC epitopes.
Variations in the distribution ofP. brasiliensis cell walls and their fractions with increasing amounts ofFCS in the medium 
In vitro subculturing of the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9 leads to the disappearance of alpha-1,3-glucan as a main component of its cell wall. However, the addition of fetal calf serum to the growth medium induces the synthesis of this polysaccharide. It is suggested that the synthesis of alpha-1,3-glucan in the cell wall of the yeastlike form of P. brasiliensis is induced by external factors.
The lipooligosaccharide (LOS) of Haemophilus ducreyicontains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.
E. coli strains used 
Molecular weights, ionic strength for elution in anion-exchange FPLC (EIS), and isoelectric points of the adhesin and the fimbrillins from cloned F antigens 
Hemagglutination and adhesive properties of FACs, fimbriae from which adhesins were removed, and purified adhesinsa 
Properties of monoclonal antibodies obtained by immunization with crude F71-specific adhesin Monoclonal Immunoglobulin Inhibition of ELISAI reactivity with: antibody subclass hemagglutination F71 adhesin F71 fimbrillin F8 adhesin F8 fimbrillin F13 adhesin F13 fimbrillin 
The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).
All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep3-phosphorylated 3-deoxy-d-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, d-glycero-d-manno-heptose (dd-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked β1→4, to the dd-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide dd-Hepα1→6Glcβ1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.
Adjuvant activity of synthetic Salmonella-type lipid A, its 4'-monophosphate analog, and reference natural products to increase number of anti-SRBC hemolytic PFC in the spleens of BALB/c mice by intraperitoneal injection with SRBCa
A synthetic lipid A (Salmonella type, compound 516), beta (1-6)-linked D-glucosamine disaccharide 1,4'-bisphosphate, with three acyloxyacyl groups and one hydroxyacyl group, i.e., (R)-3-hexadecanoyloxytetradecanoyl, (R)-3-hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3-tetradecanoyloxytetradecanoyl groups at the 2-amino, 3-hydroxyl, 2'-amino, and 3'-hydroxyl groups, respectively, was less biologically active than the synthetic Escherichia coli-type lipid A (compound 506), which has only two acyloxyacyl groups at the 2' and 3' positions and is substituted with a (R)-3-hydroxytetradecanoyl group at the 2-amino group. Compound 516 exhibited considerably weaker pyrogenic and leukopenic activity than compound 506, and it scarcely prepared rabbit skin for the Shwartzman reaction and lacked lethal toxicity on chicken embryos, although its lethal toxicity in galactosamine-loaded mice was as strong as that of compound 506. Compound 516 was also less active than compound 506 or natural E. coli lipid A (from Restrain F515) in other biological test systems, such as the Limulus test, stimulation of macrophages and lymphocytes, and interferon-inducing activity but not for interleukin-1 induction or complement activation. This observation suggests that there is an optimal number of acyloxyacyl groups on the glucosamine backbone for producing the biological activities of lipid A, especially the endotoxic activities. The 4'-monophosphate analog (compound 514) of compound 516 in general had significantly weaker activity than compound 516 in the above assays, most probably because of its greater hydrophobicity and consequently lower solubility in assay systems. Bacterial R595 lipid A derived from S. minnesota Re-mutant, which is a mixture of compounds 516 and 506, their 4'-monophosphate analogs and other compounds, exerted intermediate degrees of activity between compounds 506 and 516 in the various test systems employed.
We previously found that AC-1, an extracellular polysaccharide, produced by Acetobacter xylinum and composed of (1,4)-β-d-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha by macrophages in vitro via Toll-like receptor 4 (TLR-4) signaling. In the present study, we examined the effect of oral administration of AC-1 on protective immunity against Listeria monocytogenes. Mice were given AC-1 or phosphate-buffered saline (PBS) intragastrically 2 days before, on the day of, and 2 days after an intraperitoneal inoculation of L. monocytogenes. The survival rate of AC-1-treated mice was significantly improved and bacterial growth in AC-1-treated mice was severely retarded compared to those of PBS-treated mice after infection with L. monocytogenes. IL-12 p40 levels in serum and magnitudes of CD4+ Th1 and CD8+ Tc1 responses against Listeria antigen were significantly higher in AC-1-treated mice than in PBS-treated mice. The effect of AC-1 on antilisterial activity was diminished in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer through TLR-4, enhanced protective immunity against L. monocytogenes via augmentation of Th1 responses. These results suggest that infectious processes driven by intracellular microorganisms could be prevented to develop by the (1,4)-β-d-glucan.
N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.
The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries.
The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in β-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of β-1,6-glucan in the cell wall was decreased in the Cabig1Δ strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Δ mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Δ mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Δ mutant.
(A) Restriction enzyme sites within the 4.4-kb p10-S insert used to establish the copy number of the LDH gene. _, 0.5-kb HpaI fragment probe. (B) Southern blot of S. mutans JH1000 chromosomal DNA. JH1000 chromosomal DNA was digested with MluI (lane b), HindIlI (lane c), MluI plus HindIll (lane d), SpeI (lane e), EcoRV (lane f), and SpeI plus EcoRV (lane g). Lane a contains bacteriophage lambda DNA digested with HindlIl. The filter-bound DNA was probed with HindIll-digested lambda DNA and the 0.5-kb HpaI fragment (panel A) containing the active site of the S. mutans LDH gene. 
Previously, the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase gene of Streptococcus mutans JH1000 was cloned into Escherichia coli (J. D. Hillman, M. J. Duncan, and K. P. Stashenko, Infect. Immun. 58:1290-1295, 1990). In the present study, the nucleotide sequence of 1.29 kb of S. mutans DNA which contained the promoter and protein-coding region of the gene was determined. In vitro disruption of the gene was achieved by deletion of the promoter and a major portion of the protein-coding sequence. Subsequently, a tetracycline resistance gene from S. mutans was inserted at the deletion site as a marker for selection. In addition, evidence from Southern hybridization showed that S. mutans JH1000 contained a single copy of the lactate dehydrogenase gene.
To identify potential vaccine candidates for the prevention of infection with the filarial nematode Onchocerca volvulus, we screened an O. volvulus L3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed to O. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.
Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall β-glucans in stimulating inflammatory responses. Fungal β-glucans are comprised of D-glucose homo-polymers, containing β-1,3 linked glucose backbones with β-1,6 linked glucose side chains. Prior studies in Pneumocystis carinii (Pc)(‡‡) have characterized β-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for β-1,6 glucans in predominantly mediating host cellular activation. Accordingly, we sought to characterize β-1,6 glucans in the cell wall of Pneumocystis and establish their activity in lung cell inflammation. Immune staining revealed specific β-1,6 localization in Pc cysts walls. Homology-based cloning facilitated characterization of a functional Pckre6 β-1,6 glucan synthase in Pneumocystis, which when expressed in kre6 deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized β-1,6 glucan synthase inhibitors decreased the ability of isolated Pc preparations to generate β-1,6 carbohydrate. In addition, isolated β-1,6 glucan fractions from Pneumocystis elicited vigorous TNF-α responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane micro-domain function. Together, these studies indicate that β-1,6 glucans are present in the Pc cell wall and contribute to lung cell inflammatory activation during infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.
Top-cited authors
Joanne L Flynn
  • University of Pittsburgh
Arturo Casadevall
  • Johns Hopkins Bloomberg School of Public Health
Roy Curtiss
  • University of Florida
Vincent Bensan Young
  • University of Michigan
Renée M Tsolis
  • University of California, Davis