Shigella flexneri is the causative agent of dysentery, and its pathogenesis is mediated by a type III secretion system (T3SS). S. flexneri secretes effector proteins into the eukaryotic cell via the T3SS, and these proteins usurp host cellular functions to the benefit of the bacteria. OspF and OspC1 are known to be secreted by S. flexneri, but their functions are unknown. We transformed S. flexneri with a plasmid that expresses a twohemagglutinin tag (2HA) in frame with OspF or OspC1 and verified that these proteins are secreted in a T3SS-dependent manner. Immunofluorescence of HeLa cells infected with S. flexneri expressing OspF-2HA or OspC1-2HA revealed that both proteins localize in the nucleus and cytoplasm of host cells. To elucidate the function of these T3SS effectors, we constructed ⌬ospF and ⌬ospC1 deletion mutants by allelic exchange. We found that ⌬ospF and ⌬ospC1 mutants invade host cells and form plaques in confluent monolayers similar to wild-type S. flexneri. However, in the polymorphonuclear (PMN) cell migration assay, a decrease in neutrophil migration was observed for both mutants in comparison to the migration of wild-type bacteria. Moreover, infection of polarized T84 intestinal cells infected with ⌬ospF and ⌬ospC1 mutants resulted in decreased phosphorylation of extracellular signal-regulated kinase 1/2 in comparison to that of T84 cells infected with wild-type S. flexneri. To date, these are the first examples of T3SS effectors implicated in mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway activation. Ultimately, OspF and OspC1 are essential for PMN transepithelial migration, a phenotype associated with increased inflammation and bacterial access to the submucosa, which are fundamental aspects of S. flexneri pathogenesis.Shigella species are responsible for dysentery (shigellosis) in humans, which starts as an acute infection in the large intestine, which is followed by cramps, diarrhea, and fever. The infection is usually self-limiting, but it can also cause damage to the colonic mucosa, intestinal bleeding, and death if untreated. Worldwide, Shigella spp. infections are responsible for approximately 163 million cases of dysentery and 1 million deaths each year (23). The majority of infections occur in third-world countries, where contaminated food and drinking water are common (23); however, even developed nations still have multiple Shigella outbreaks every year (47). Therefore, studying the pathogenesis of these gram-negative bacteria is of utmost importance, particularly in light of emerging antibiotic resistance, the lack of an appropriate vaccine, and the potential for use of Shigella as a bioweapon (23,33,42).Following ingestion, Shigella flexneri eventually reaches the large intestine, the target site for infection, where access to the basolateral membrane is a prerequisite for the invasion of epithelial cells (see reference 48 for a review). M cells in the large intestine phagocytose and subsequently transcytose the bacteria from the lumen to t...
We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a ␥-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission. The norepinephrine transporter (NET)1 at presynaptic nerve terminals mediates the uptake of released norepinephrine, resulting in the rapid termination of synaptic transmission and thereby controlling the fine tuning of neuronal activities. Psychostimulants, including amphetamines, and tricyclic antidepressants, such as desipramine, exert their pharmacological effects by acting on NET (1, 2). Molecular cloning studies have resulted in the isolation of human NET cDNA (3), followed by several other monoamine neurotransmitter transporters from several species (4, 5).There is a line of evidence suggesting that functional expression of monoamine neurotransmitter transporters is regulated in different ways, e.g. direct or indirect protein phosphorylation, long term modification at the level of mRNA expression, translation, and processing (4 -6). Recently, gene targeting of these transporters by homologous recombination was utilized to investigate their physiological relevance in vivo. Elimination of the dopamine transporter (DAT) in knockout mice produces hyperlocomotion and indifference to cocaine and amphetamines (7).The regulatory importance of alternative RNA splicing has been suggested in many genes, including members of the ␥-aminobutyric acid (GABA)/norepinephrine transporter family. In the glycine transporter GLYT1, alternative splicing of 5Ј-noncoding and -coding sequences confers differential cellspecific expression of GLYT1 gene products (8). Two different cDNAs of bovine NET, which display different COOH termini, have been reported (9, 10). Based on the analysis of human and mouse NET genes at the exon-intron boundary for this 3Ј-region (11, 12), it is suggested that the difference may occur from alternative splicing.To gain further understanding of the importance of these transporters in monoaminergic nervous system functions, we isolated and characterized cDNAs encoding rat NET that displayed RNA splice variants at the 3Ј-region including both coding and noncoding regions. One splice form, which codes different predicted CO...
The Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) causes specific cell death in dopaminergic neurons after accumulation by the dopamine transporter (DAT). COS cells, a non-neuronal cell line insensitive to high doses of MPP+, becomes sensitive to MPP+ when transfected with the rat DAT cDNA. We analyzed the bi-directional transport of MPP+ and its toxicity in several cell lines expressing wild or mutant DATs. Cell death in COS cells expressing wild DAT by exposure to MPP+ was concentration-dependent and cocaine-reversible. Increased wild DAT expression caused higher sensitivities to the toxin in HeLa cells. Although several mutant DATs demonstrated greater transport activity than the wild-type, they displayed similar or lower sensitivity to MPP+ toxicity. Reverse transport of preloaded [3H]MPP+ through DAT was facilitated in COS cells expressing certain mutant DATs, which consistently displayed less sensitivity to MPP+ toxicity. These results suggest that re-distribution of MPP+ due to influx/efflux turnover through the transporter is a key factor in MPP+ toxicity.
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