The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism–mutualism continuum.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?

Painful peripheral neuropathies are precipitated by nerve injury from disease or trauma. All such injuries will be accompanied by an inflammatory reaction, a neuritis, that will mobilize the immune system. The role of the inflammation itself is difficult to determine in the presence of structural damage to the nerve. A method has been devised to produce a focal neuritis in the rat sciatic nerve that involves no more than trivial structural damage to the nerve. This experimental focal neuritis produces neuropathic pain sensations (heat- and mechano-hyperalgesia, and cold- and mechano-allodynia) in the ipsilateral hind paw. The abnormal pain sensations begin in 1–2 days and last for 4–6 days, with a subsequent return to normal. These results suggest that there is a neuroimmune interaction that occurs at the outset of nerve injury (and perhaps episodically over time in slow developing conditions like diabetic neuropathy) that produces neuropathic pain. The short duration of the phenomena suggest that they may prime the system for more slowly developing mechanisms of abnormal pain (e.g., ectopic discharge in axotomized primary afferent neurons) that underlie the chronic phase of painful neuropathy.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

Highly plastic chromosomal organization in Salmonella typhi.

Gene order in the chromosomes of Escherichia coli K-12 and Salmonella typhimurium LT2, and in many other species of Salmonella, is strongly conserved, even though the genera diverged about 160 million years ago. However, partial digestion of chromosomal DNA of Salmonella typhi, the causal organism of typhoid fever, with the endonuclease I-CeuI followed by separation of the DNA fragments by pulsed-field gel electrophoresis showed that the chromosomes of independent wild-type isolates of S. typhi are rearranged due to homologous recombination between the seven rrn genes that code for ribosomal RNA. The order of genes within the I-CeuI fragments is largely conserved, but the order of the fragments on the chromosome is rearranged. Twenty-one different orders of the I-CeuI fragments were detected among the 127 wild-type strains we examined. Duplications and deletions were not found, but transpositions and inversions were common. Transpositions of I-CeuI fragments into sites that do not change their distance from the origin of replication (oriC) are frequently detected among the wild-type strains, but transpositions that move the fragments much further from oriC were rare. This supports the gene dosage hypothesis that genes at different distances from oriC have different gene dosages and, hence, different gene expression, and that during evolution genes become adapted to their specific location; thus, cells with changes in gene location due to transpositions may be less fit. Therefore, gene dosage may be one of the forces that conserves gene order, although its effects seem less strong in S. typhi than in other enteric bacteria. However, both the gene dosage and the genomic balance hypotheses, the latter of which states that the origin (oriC) and terminus (TER) of replication must be separated by 180 degrees C, need further investigation.

A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine.

Cells of the exocrine pancreas produce digestive enzymes potentially harmful to the intestinal mucosa. Dopamine has been reported to protect against mucosal injury. In looking for the source of dopamine in the small intestine, we found that the duodenal juice contains high levels of dopamine and that the pancreas itself has a high dopamine [and dihydroxyphenylalanine (dopa)] content that does not change significantly after chemical sympathectomy. Furthermore, we were able to demonstrate tyrosine hydroxylase (TH) activity in control pancreas as well as in pancreas from rats after chemical sympathectomy. Immunostaining and in situ hybridization histochemistry confirmed both the presence of TH, dopamine, and the dopamine transporter, and the mRNAs encoding TH and dopamine transporter, and the presence of both types of vesicular monoamine transporters in the exocrine cells of the pancreas. Since there are no catecholaminergic enteric ganglia in the pancreas, the above results indicate that pancreatic cells have all the characteristics of dopamine-producing cells. We suggest that the pancreas is an important source of nonneuronal dopamine in the body, and that this dopamine has a role in protecting the intestinal mucosa and suggests that dopamine D1b receptor agonists might be used to help mucosal healing in the gastrointestinal tract.

Endothelin-B receptor is expressed by neural crest cells in the avian embryo.

Disruptions of the genes encoding endothelin 3 (EDN3) and its receptor endothelin-B receptor (EDNRB) in the mouse result in defects of two neural crest (NC)-derived lineages, the melanocytes, and the enteric nervous system. To assess the mechanisms through which the EDN3/EDNRB signaling pathway can selectively act on these NC derivatives, we have studied the spatiotemporal expression pattern of the EDNRB gene in the avian embryo, a model in which NC development has been extensively studied. For this purpose, we have cloned the quail homologue of the mammalian EDNRB cDNA. EDNRB transcripts are present in NC cells before and during their emigration from the neural tube at all levels of the neuraxis. At later developmental stages, the receptor remains abundantly expressed in the peripheral nervous system including the enteric nervous system. In a previous study, we have shown that EDN3 enhances dramatically the proliferation of NC cells when they are at the pluripotent stage. We propose that the selective effect of EDN3 or EDNRB gene inactivation is due to the fact that both melanocytes and enteric nervous system precursors have to colonize large embryonic areas (skin and bowel) from a relatively small population of precursors that have to expand considerably in number. It is therefore understandable that a deficit in one of the growth-promoting pathways of NC cells has more deleterious effects on long-range migrating cells than on the NC derivatives which develop close to the neural primordium like the sensory and sympathetic ganglia.

TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis.

Expression of the Bacillus subtilis nrgAB operon is derepressed during nitrogen-limited growth. We have identified a gene, tnrA, that is required for the activation of nrgAB expression under these growth conditions. Analysis of the DNA sequence of the tnrA gene revealed that it encodes a protein with sequence similarity to GlnR, the repressor of the B. subtilis glutamine synthetase operon. The tnrA mutant has a pleiotropic phenotype. Compared with wild-type cells, the tnrA mutant is impaired in its ability to utilize allantoin, gamma-aminobutyrate, isoleucine, nitrate, urea, and valine as nitrogen sources. During nitrogen-limited growth, transcription of the nrgAB, nasB, gabP, and ure genes is significantly reduced in the tnrA mutant compared with the levels seen in wild-type cells. In contrast, the level of glnRA expression is 4-fold higher in the, tnrA mutant than in wild-type cells during nitrogen restriction. The phenotype of the tnrA mutant indicates that a global nitrogen regulatory system is present in B. subtilis and that this system is distinct from the Ntr regulatory system found in enteric bacteria.

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.

Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium.

In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate. To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis. Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH). Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase). By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+. When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S. typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool. When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool. Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate. Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.

Similarities and dissimilarities of phage genomes.

Genomic similarities and contrasts are investigated in a collection of 23 bacteriophages, including phages with temperate, lytic, and parasitic life histories, with varied sequence organizations and with different hosts and with different morphologies. Comparisons use relative abundances of di-, tri-, and tetranucleotides from entire genomes. We highlight several specific findings. (i) As previously shown for cellular genomes, each viral genome has a distinctive signature of short oligonucleotide abundances that pervade the entire genome and distinguish it from other genomes. (ii) The enteric temperate double-stranded (ds) phages, like enterobacteria, exhibit significantly high relative abundances of GpC = GC and significantly low values of TA, but no such extremes exist in ds lytic phages. (iii) The tetranucleotide CTAG is of statistically low relative abundance in most phages. (iv) The DAM methylase site GATC is of statistically low relative abundance in most phages, but not in P1. This difference may relate to controls on replication (e.g., actions of the host SeqA gene product) and to MutH cleavage potential of the Escherichia coli DAM mismatch repair system. (v) The enteric temperate dsDNA phages form a coherent group: they are relatively close to each other and to their bacteria] hosts in average differences of dinucleotide relative abundance values. By contrast, the lytic dsDNA phages do not form a coherent group. This difference may come about because the temperate phages acquire more sequence characteristics of the host because they use the host replication and repair machinery, whereas the analyzed lytic phages are replicated by their own machinery. (vi) The nonenteric temperate phages with mycoplasmal and mycobacterial hosts are relatively close to their respective hosts and relatively distant from any of the enteric hosts and from the other phages. (vii) The single-stranded RNA phages have dinucleotide relative abundance values closest to those for random sequences, presumably attributable to the mutation rates of RNA phages being much greater than those of DNA phages.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.