Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

Social amoebae establish a protective interface with their bacterial associates by lectin agglutination.

Both animals and amoebae use phagocytosis and DNA-based extracellular traps as anti-bacterial defense mechanisms. Whether, like animals, amoebae also use tissue-level barriers to reduce direct contact with bacteria has remained unclear. We have explored this question in the social amoeba Dictyostelium discoideum, which forms plaques on lawns of bacteria that expand as amoebae divide and bacteria are consumed. We show that CadA, a cell adhesion protein that functions in D. discoideum development, is also a bacterial agglutinin that forms a protective interface at the plaque edge that limits exposure of vegetative amoebae to bacteria. This interface is important for amoebal survival when bacteria-to-amoebae ratios are high, optimizing amoebal feeding behavior, and protecting amoebae from oxidative stress. Lectins also control bacterial access to the gut epithelium of mammals to limit inflammatory processes; thus, this strategy of antibacterial defense is shared across a broad spectrum of eukaryotic taxa.

Determination of degree of RBC agglutination for blood typing using a small quantity of blood sample in a microfluidic system.

Blood typing assay is a critical test to ensure the serological compatibility of a donor and an intended recipient prior to a blood transfusion. This paper presents a microfluidic blood typing system using a small quantity of blood sample to determine the degree of agglutination of red blood cell (RBC). Two measuring methods were proposed: impedimetric measurement and electroanalytical measurement. The charge transfer resistance in the impedimetric measurement and the power parameter in the electroanalytical measurement were used for the analysis of agglutination level. From the experimental results, both measuring methods provide quantitative results, and the parameters are linearly and monotonically related to the degree of RBC agglutination. However, the electroanalytical measurement is more reliable than the impedimetric technique because the impedimetric measurement may suffer from many influencing factors, such as chip conditions. Five levels from non-agglutination (level 0) to strong agglutination (level 4+) can be discriminated in this study, conforming to the clinical requirement to prevent any risks in transfusion.

High-throughput characterization of protein-protein interactions by reprogramming yeast mating.

High-throughput methods for screening protein-protein interactions enable the rapid characterization of engineered binding proteins and interaction networks. While existing approaches are powerful, none allow quantitative library-on-library characterization of protein interactions in a modifiable extracellular environment. Here, we show that sexual agglutination of Saccharomyces cerevisiae can be reprogrammed to link interaction strength with mating efficiency using synthetic agglutination (SynAg). Validation of SynAg with 89 previously characterized interactions shows a log-linear relationship between mating efficiency and protein binding strength for interactions with Kds ranging from below 500 pM to above 300 µM. Using induced chromosomal translocation to pair barcodes representing binding proteins, thousands of distinct interactions can be screened in a single pot. We demonstrate the ability to characterize protein interaction networks in a modifiable environment by introducing a soluble peptide that selectively disrupts a subset of interactions in a representative network by up to 800-fold. SynAg enables the high-throughput, quantitative characterization of protein-protein interaction networks in a fully defined extracellular environment at a library-on-library scale.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination.

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii.

Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P < 0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii.

Antibody blocks acquisition of bacterial colonization through agglutination.

Invasive infection often begins with asymptomatic colonization of mucosal surfaces. A murine model of bacterial colonization with Streptococcus pneumoniae was used to study the mechanism for mucosal protection by immunoglobulin. In previously colonized immune mice, bacteria were rapidly sequestered within large aggregates in the nasal lumen. To further examine the role of bacterial agglutination in protection by specific antibodies, mice were passively immunized with immunoglobulin G (IgG) purified from antipneumococcal sera or pneumococcal type-specific monoclonal human IgA (hIgA1 or hIgA2). Systemically delivered IgG accessed the mucosal surface and blocked acquisition of colonization and transmission between littermates. Optimal protection by IgG was independent of Fc fragment and complement and, therefore, did not involve an opsonophagocytic mechanism. Enzymatic digestion or reduction of IgG before administration showed that protection required divalent binding that maintained its agglutinating effect. Divalent hIgA1 is cleaved by the pneumococcal member of a family of bacterial proteases that generate monovalent Fabα fragments. Thus, passive immunization with hIgA1 blocked colonization by an IgA1-protease-deficient mutant (agglutinated) but not the protease-producing wild-type parent (not agglutinated), whereas protease-resistant hIgA2 agglutinated and blocked colonization by both. Our findings highlight the importance of agglutinating antibodies in mucosal defense and reveal how successful pathogens evade this effect.

HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates Gram-negative bacterial pathogens.

C-type lectins (CTLs) are Ca(2+)-dependent carbohydrate recognition proteins, which play important roles in the innate immunity of both vertebrates and invertebrates. In this study, we identified and characterized a C-type lectin (named HdhCTL1) from Pacific abalone, Haliotis discus hannai. HdhCTL1 is composed of 176 amino acid residues and shares low (23.9%) identity with the known CTL of abalone. HdhCTL1 possesses a putative signal peptide and a carbohydrate-recognition domain (CRD) typical of CTLs. The CRD of HdhCTL1 contains four disulfide bond-forming cysteine residues that are highly conserved in CTLs. HdhCTL1 mRNA was detected in a wide range of tissues and expressed abundantly in the digestive gland. Experimental infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhCTL1 expression in a time-dependent manner. Recombinant HdhCTL1 (rHdhCTL1) purified from Escherichia coli was able to agglutinate Gram-negative bacterial pathogens. The agglutinating ability of rHdhCTL1 was abolished in the presence of mannose. These results suggest that HdhCTL1 is a novel CTL which is likely to be involved in host defense against bacterial infection.

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling.

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in ß-sheets and forms quickly large aggregates (>µm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein-protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes.

The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis.

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Evidence for a novel biological role for the multifunctional ß-1,3-glucan binding protein in shrimp.

ß-1,3-Glucan binding proteins (ßGBPs) are soluble pattern recognition proteins/receptors that bind to ß-1,3-glucans from fungi cell walls. In crustaceans, ßGBPs are abundant plasmatic proteins produced by the hepatopancreas, and have been proved to play multiple biological functions. Here, we purified and characterized novel members of the ßGBP family from the hemolymph of two Brazilian shrimps, Farfantepenaeus paulensis (FpßGBP) and Litopenaeus schmitti (LsßGBP). As observed for other crustacean species, FpßGBP and LsßGBP are monomeric proteins (∼100kDa) able to enhance the activation of the prophenoloxidase system, a potent antimicrobial defense conserved in arthropods. More interestingly, we provided here evidence for a novel biological activity for shrimp ßGBPs: the agglutination of fungal cells. Finally, we investigated the modulation of the ßGBP gene in F. paulensis shrimps experimentally infected with a cognate fungal pathogen, Fusarium solani. From our expression data, ßGBP gene is constitutively expressed in hepatopancreas and not modulated upon a non-lethal fungal infection. Herein, we have improved our knowledge about the ßGBP family by the characterization of a novel biological role for this multifunctional protein in shrimp.

Sulphite : cytochrome c oxidoreductase deficiency in Campylobacter jejuni reduces motility, host cell adherence and invasion.

Campylobacter jejuni lacks the enzyme phosphofructokinase and, consequently, is incapable of metabolizing glucose. Instead, the pathogen uses a number of other chemicals to serve as electron donors. Like chemolithotrophic bacteria, C. jejuni is able to respire sulphite in the presence of a sulphite : cytochrome c oxidoreductase (SOR) that is encoded by the genes cj0004c and cj0005c; the former encodes a monohaem cytochrome c oxidoreductase and the latter a molybdopterin oxidoreductase. After screening of a transposon-based mutant library, we identified a mutant with an insertion in gene cj0005c that was strongly reduced in its capacity to infect Caco2 cells. Further characterization of a corresponding non-random knockout mutant together with a complemented mutant and the parental strain showed the cj0005c-deficient mutant to exhibit clearly reduced motility and diminished adherence to host cells. Furthermore, the transcription of genes responsible for the synthesis of, in particular, legionaminic acid was downregulated and the mutant had a reduced capacity to autoagglutinate. In contrast, neither the proliferation of the mutant, nor its intracellular ATP content, was altered compared to the parental strain.

More than one tandem repeat domain of the extracellular adherence protein of Staphylococcus aureus is required for aggregation, adherence, and host cell invasion but not for leukocyte activation.

The extracellular adherence protein (Eap) is a multifunctional Staphylococcus aureus protein and broad-spectrum adhesin for several host matrix and plasma proteins. We investigated the interactions of full-length Eap and five recombinant tandem repeat domains with host proteins by use of surface plasmon resonance (BIAcore) and ligand overlay assays. In addition, agglutination and host cell interaction, namely, adherence, invasion, and stimulation of proliferation, were determined. With plasmon resonance, the interaction of full-length Eap isoforms (from strains Newman and Wood 46) with fibrinogen, fibronectin, vitronectin, and thrombospondin-1 was found to be specific but with different affinities for the ligands tested. In the ligand overlay assay, the interactions of five single tandem repeat domains (D1 to D5) of Eap-7 (from strain CI-7) with fibronectin, fibrinogen, vitronectin, thrombospondin-1, and collagen I differed substantially. Most prominently, D3 bound most strongly to fibronectin and fibrinogen. Full-length Eap, but none of the single tandem repeat domains, agglutinated S. aureus and enhanced adherence to and invasion of host cells by S. aureus. Constructs D3-4 and D1-3 (in cis) increased adherence and invasiveness compared to what was seen for single Eap tandem repeat domains. By contrast, single Eap tandem repeat domains and full-length Eap similarly modulated the proliferation of peripheral blood mononuclear cells (PBMCs): low concentrations stimulated, whereas high concentrations inhibited, proliferation. Taken together, the data indicate that Eap tandem repeat domains appear to have distinct characteristics for the binding of soluble ligands, despite a high degree of sequence similarity. In addition, more than one Eap tandem repeat domain is required for S. aureus agglutination, adherence, and cellular invasion but not for the stimulation of PBMC proliferation.

Comparison of PCR for sputum samples obtained by induced cough and serological tests for diagnosis of Mycoplasma pneumoniae infection in children.

Passive agglutination (PA) and immunoglobulin M (IgM), IgA, and IgG enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Mycoplasma pneumoniae were compared with PCR testing of sputum samples obtained from children with lower respiratory tract infections. The sensitivity and specificity of PA were 80.3% and 92.3% at a titer of 1:80. ELISA was found to be less sensitive than PA.

[Identification of the autoagglutination factor of Hms- cells of the plague agent].

A search for cellular components responsible for autoagglutination (AA) in broth and salt solutions of Hms- cells of the plague agent Yersinia pestis was performed. The AA- mutants were obtained using vaccine strain Y. pestis EV76 derivative containing one species-specific plasmid pYP. The mutants were shown to differ from the parent strain by the decreased surface hydrophobicity, insensitivity to plague diagnostic L-413c bacteriophage and negative haemagglutination reaction with antibodies to F1 capsular substance of the plague agent. The mutants did not differ from the parent strain by electrophoretic mobility and immunochemical activity of LPS but were characterized by the absence of a 17 kDa protein on the cell surface. The AA+ cells that lost this protein after weak alkali extraction were less hydrophobic and failed to express AA in 0.5 M ammonium sulfate. After the extraction, the cells lost the ability to neutralize L-413c and to react with the anti-F1 antibodies, while both activities as well as 17 kDa protein were detected in the extracts. Thus, the 17 kDa protein is suggested to be a hydrophobic surface antigen which acts as a receptor of the L-413c bacteriophage and represents an AA factor of Hms- cells of Y. pestis.

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans.

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.

Cuantificación de la depresión alélica en el grup A1B / Quantification of the allelic depression in the Group A1B

Se midió la reactividad del antígeno A1 en dos grupos de hematies A1 y A1B mediante la técnica de aglutinación cuantitativa. Para ello se utilizó dos tipos de suero anti A con títulos de 1/2048 y 1/512. Se encontró una DH50 de 1/568 y 1/549, para los eritrocitos grupo A1 y una DH50 de 1/351 y 1/276 para los grupos A1B. Esto nos indica una depresión del antígeno A1, en el grupo A1B,1 de un 38 por ciento y un 50 por ciento, respectivamente.

Expression of the aggA locus of Pseudomonas putida in vitro and in planta as detected by the reporter gene, xylE.

In vitro agglutinability by Pseudomonas putida, isolate Corvallis, with a plant root surface agglutinin is correlated with rapid adhesion of cells of the fluorescent pseudomonad to bean (Phaseolus vulgaris) root surfaces. Agglutinability in P. putida cells is regulated by nutrient status as well as growth phase. Cells grown in three different nutrient complex media are agglutinable at early and mid-late logarithmic phase but become nonagglutinable at stationary phase. Cells grown in a minimal medium are weakly agglutinable, but the addition of lysine, aspartic acid, or histidine increases agglutinability. Cells in the same minimal medium supplemented with bean root surface components grow in a highly agglutinated state. Previous data indicate both agglutination and rapid adhesion to roots by P. putida Corvallis involves the aggA locus, which contains two putative open reading frames (ORF), ORF-AGG1 and ORFAGG2, on complementary strands. Sequence and deletion analyses suggest ORFAGG1 is the most probable ORF responsible for agglutination and adhesion. Chimeric fusion of an Escherichia coli lac promoter with ORFAGG1, but not with ORFAGG2, complemented agglutinability of an aggA::Tn5 P. putida Agg mutant, providing further evidence that ORFAGG1, not ORFAGG2, is responsible for agglutination. Heterologous expression of ORFAGG1 yields a 50-kDa precursor and a 48-kDa mature periplasmic protein. Fusions of ORFAGG1 and ORFAGG2 to the reporter gene, xylE, and detection of the reporter enzyme, catechol-2,3-oxygenase reveal an active promoter in the 5' noncoding region of ORFAGG1. The ORFAGG1 promoter is active during growth of the cells in liquid culture and is regulated by growth medium. Greatest activity of the catechol-2,3-oxygenase is observed in stationary phase when the cells are nonagglutinable. Expression of the ORFAGG1 promoter is detected in P. putida cells extracted from the root surface of bean at 48 and 72 hr after inoculation.