Characterization of BseMII, a new type IV restriction–modification system, which recognizes the pentanucleotide sequence 5′-CTCAG(N)10/8↓

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5′-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3′-protruding end. Magnesium ions and S-adenosyl-l-methionine (AdoMet) are required for cleavage. S-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5′-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-l-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Comparison of the early stages of forced unfolding for fibronectin type III modules

The structural changes accompanying stretch-induced early unfolding events were investigated for the four type III fibronectin (FN-III) modules, FN-III7, FN-III8, FN-III9, and FN-III10 by using steered molecular dynamics. Simulations revealed that two main energy barriers, I and II, have to be overcome to initiate unraveling of FN-III's tertiary structure. In crossing the first barrier, the two opposing β-sheets of FN-III are rotated against each other such that the β-strands of both β-sheets align parallel to the force vector (aligned state). All further events in the unfolding pathway proceed from this intermediate state. A second energy barrier has to be overcome to break the first major cluster of hydrogen bonds between adjacent β-strands. Simulations revealed that the height of barrier I varied significantly among the four modules studied, being largest for FN-III7 and lowest for FN-III10, whereas the height of barrier II showed little variation. Key residues affecting the mechanical stability of FN-III modules were identified. These results suggest that FN-III modules can be prestretched into an intermediate state with only minor changes to their tertiary structures. FN-III10, for example, extends 12 Å from the native “twisted” to the intermediate aligned state, and an additional 10 Å from the aligned state to further unfolding where the first β-strand is peeled away. The implications of the existence of intermediate states regarding the elasticity of fibrillar fibers and the stretch-induced exposure of cryptic sites are discussed.

Molecular and cell biology aspects of plague

A 70-kb virulence plasmid (sometimes called pYV) enables Yersinia spp. to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. The Ysc apparatus forms a channel composed of 29 proteins. Of these, 10 have counterparts in almost every type III system. Secretion of some Yops requires the assistance, in the bacterial cytosol, of small individual chaperones called the Syc proteins. These chaperones act as bodyguards or secretion pilots for their partner Yop. Yop proteins fall into two categories. Some are intracellular effectors, whereas the others are “translocators” needed to deliver the effectors across the eukaryotic plasma membrane, into eukaryotic cells. The translocators (YopB, YopD, LcrV) form a pore of 16–23 Å in the eukaryotic cell plasma membrane. The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT. YopH is a powerful phosphotyrosine phosphatase playing an antiphagocytic role by dephosphorylating several focal adhesion proteins. YopE and YopT contribute to antiphagocytic effects by inactivating GTPases controlling cytoskeleton dynamics. YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription factor NF-κB. It also induces rapid apoptosis of macrophages. Less is known about the role of the phosphoserine kinase YopO/YpkA and YopM.

Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module.

Fibronectin type III modules contain approximately 90 residues and are an extremely common building block of animal proteins. Despite containing a complex all-beta-sheet topology and eight prolines, the refolding of the 10th type III module of human fibronectin has been found to be very rapid, with native core packing, amide hydrogen bonding, and backbone conformation all recovered within 1 s at 5 degrees C. These observations indicate that this domain can overcome many structural characteristics often thought to slow the folding process.

Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.

Genetic heterogeneity in cystinuria: the SLC3A1 gene is linked to type I but not to type III cystinuria.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1

Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1β. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Identification of a pathogenicity island required for Salmonella survival in host cells.

We have identified a region unique to the Salmonella typhimurium chromosome that is essential for virulence in mice. This region harbors at least three genes: two (spiA and spiB) encode products that are similar to proteins found in type III secretion systems, and a third (spiR) encodes a putative regulator. A strain with a mutation in spiA was unable to survive within macrophages but displayed wild-type levels of epithelial cell invasion. The culture supernatants of the spi mutants lacked a modified form of flagellin, which was present in the supernatant of the wild-type strain. This suggests that the Spi secretory apparatus exports a protease, or a protein that can alter the activity of a secreted protease. The "pathogenicity island" harboring the spi genes may encode the virulence determinants that set Salmonella apart from other enteric pathogens.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3′ end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10−8 M) and leukotriene B4 (LTB4, 10−8 M). l-NAME (but not d-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by l-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.