Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.
The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-κB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-κB activation. We show that inhibition of NF-κB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-κB activation by preventing activation of IKKβ and consequently the degradation of the NF-κB inhibitor, IκB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-κB signaling pathways.
SummaryCyclic-di-GMP (c-di-GMP) regulates many important bacterial processes. Freely diffusible intracellular c-di-GMP is determined by the action of metabolizing enzymes that allow integration of numerous input signals. c-di-GMP specifically regulates multiple cellular processes by binding to diverse target molecules. This review highlights important questions in research into the mechanisms of c-di-GMP signalling and its role in bacterial physiology.
Caenorhabditis elegans mtf-1 encodes matefin, which has a predicted SUN domain, a coiled-coil region, an anti-erbB-2 IgG domain, and two hydrophobic regions. We show that matefin is a nuclear membrane protein that colocalizes in vivo with Ce-lamin, the single nuclear lamin protein in C. elegans, and binds Ce-lamin in vitro but does not require Ce-lamin for its localization. Matefin is detected in all embryonic cells until midembryogenesis and thereafter only in germ-line cells. Embryonic matefin is maternally deposited, and matefin is the first nuclear membrane protein known to have germ line-restricted expression. Animals homozygous for an mtf-1 deletion allele show that matefin is essential for germ line maturation and survival. However, matefin is also required for embryogenesis because mtf-1 (RNAi) embryos die around the Ϸ300-cell stage with defects in nuclear structure, DNA content, and chromatin morphology. Down-regulating matefin in mes-3 animals only slightly enhances embryonic lethality, and elimination of UNC-84, the only other SUN-domain gene in C. elegans, has no affect on mtf-1 (RNAi) animals. Thus, mtf-1 mediates a previously uncharacterized pathway(s) required for embryogenesis as well as germ line proliferation or survival. Lamins are nuclear intermediate filament proteins found in metazoan cells at the nuclear periphery and in the nucleoplasm (1). Lamins interact with most known inner nuclear membrane proteins as well as with several nucleoplasmic proteins (2). Nuclear architecture, cell cycle progression, DNA replication, and RNA transcription and splicing all depend on lamins (3, 4). Consistent with such roles, many of these laminbinding proteins also bind transcription repressors and chromatin proteins.To understand nuclear lamins and lamin-associated protein functions in vivo we turned to Caenorhabditis elegans, studying its single lamin protein, Ce-lamin (5), and three inner nuclear membrane proteins, Ce-emerin, Ce-MAN1 (5, 6), and UNC-84 (7). UNC-84 contains an Ϸ120-residue SUN (Sad1p-UNC-84 homology) domain (8) with an unknown function. The SUN domain is also found in four human proteins, two of which localize at the nuclear envelope (9). UNC-84 is expressed in most C. elegans cells, and it depends on Ce-lamin for its nuclearenvelope localization (7). However, mutations in unc-84 cause nuclear migration or nuclear anchoring defects in only a subset of cells, leading to uncoordinated movement (8). At least two nuclear-envelope proteins, UNC-83 and ANC-1, require the SUN domain of UNC-84 for their nuclear-envelope localization and ability to regulate nuclear position (10). To explain their nuclear-envelope anchoring, a ''bridging model'' was proposed in which the transmembrane domains of UNC-83 and ANC-1 cross the outer nuclear membrane (ONM), and their luminal domains interact with the luminal domain of UNC-84 embedded in the inner nuclear membrane (7, 10).Based on the hypothesis that the SUN domain defined a new family of nuclear-envelope proteins, we searched the C. elegans genome for ot...
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