Biomedical subjects

Ian R Henderson

Publications and source records attributed to Ian R Henderson.

At least 19 recordsLinked to original sources

PubMed2007

A conserved extended signal peptide region directs posttranslational protein translocation via a novel mechanism.

Members of the type V secretion family are among the most prevalent secreted proteins in Gram-negative bacteria. A subset of this family, including Pet, the prototypical member of the Enterobacteriaceae serine proteases, possess unusual signal peptides which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which the authors have named the extended signal peptide region (ESPR), demonstrate remarkable conservation. In contrast, the N2, H2 and C regions show significant variability, and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the ESPR remains obscure. Here, it is shown that proteins possessing the ESPR are translocated in a posttranslational fashion. The presence of the ESPR severely impairs inner membrane translocation. Mutational analysis suggests that the ESPR delays inner membrane translocation by adopting a particular conformation, or by interacting with a cytoplasmic or inner membrane co-factor, prior to inner membrane translocation.

Amino Acid Sequence↗

PubMed2006

Phylogenetic comparisons reveal multiple acquisitions of the toxin genes by enterotoxigenic Escherichia coli strains of different evolutionary lineages.

Escherichia coli is a diverse bacterial species which is widely distributed in the environment but also exists as a commensal and pathogen of different host species. Human intestinal pathogenic E. coli causes over 160 million cases of diarrhea and an estimated 1 million deaths per year. The majority of deaths are attributable to one pathovar of E. coli, namely, enterotoxigenic E. coli. The pathogenesis of enterotoxigenic E. coli is dependent on the production of a colonization factor to promote adhesion to the intestinal epithelium and the elaboration of heat-labile or heat-stable toxins which induce a secretory diarrhea. Despite the high morbidity and mortality associated with enterotoxigenic E. coli infection, little is known of the genetic background of this global pathogen. Here we demonstrate by multilocus sequence typing that enterotoxigenic E. coli isolates are present in all phylogenetic lineages of E. coli, indicating that acquisition of the toxin genes may be sufficient to generate an enterotoxigenic E. coli strain. In addition, screening of diarrheal isolates for the presence of additional genes previously associated with the virulence of enterotoxigenic E. coli revealed that they were not abundant. These observations have significant implications for disease epidemiology and for the design of effective vaccines.

Bacterial Toxins↗

PubMed2006

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

Arabidopsis↗

PubMed2006

Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis.

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

Arabidopsis↗

PubMed2006

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Arabidopsis↗

PubMed2006

RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in arabidopsis.

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes.

Arabidopsis↗

PubMed2006

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

DNA Methylation↗

PubMed2006

Type III secretion: what's in a name?

The term 'type III secretion' has seen widespread use. However, problems persist in nomenclature. We propose that the standard abbreviation for this kind of secretion should be 'T3S' and that 'type III secretion system' should be abbreviated to 'T3SS'. There is also a need for a new terminology to distinguish flagellar and non-flagellar type III secretion systems that reflects their common evolutionary ancestry but does not obscure their distinctive features. Finally, the use of the term 'type III secretion' to cover cytolysin-mediated translocation is to be deprecated because an authentic type III secretion system has already been described in gram-positive bacteria, namely the flagellar protein export apparatus.

Endocytosis↗

PubMed2006

Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of food and water-borne E. coli-mediated human diarrhoea worldwide. The incidence in developing countries is estimated at 650 million cases per year, resulting in 800 000 deaths, primarily in children under the age of five. ETEC is also the most common cause of diarrhoea among travellers, including the military, from industrialized nations to less developed countries. In addition, ETEC is a major pathogen of animals, being responsible for scours in cattle and neonatal and postweaning diarrhoea in pigs and resulting in significant financial losses. Studies on the pathogenesis of ETEC infections have concentrated on the plasmid-encoded heat-stable and heat-labile enterotoxins and on the plasmid-encoded antigenically variable colonization factors. Relatively little work has been carried out on chromosomally encoded virulence factors. Here, we review the known virulence factors of ETEC and highlight the future for combating this major disease.

Animals↗

PubMed2006

The unusual extended signal peptide region of the type V secretion system is phylogenetically restricted.

The plasmid encoded toxin, Pet, is a prototypical member of the serine protease autotransporters of the Enterobacteriaceae. In addition to the passenger and beta-domains typical of autotransporters, in silico predictions indicate that Pet possesses an unusually long N-terminal signal sequence. The signal sequence can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which we have termed the extended signal peptide region, demonstrate remarkable conservation. In contrast, the N2, H2 and C regions demonstrate significant variability and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the extended signal peptide region remains obscure and surprisingly it has not been proven that the extended signal peptide region is actually synthesized as part of the signal sequence. Here, we demonstrate that the extended signal peptide region is present only in Gram-negative bacterial proteins originating from the classes Beta- and Gammaproteobacteria, and more particularly only in proteins secreted via the Type V secretion pathway: autotransporters, TpsA exoproteins of the two-partner system and trimeric autotransporters. In vitro approaches demonstrate that the DNA region encoding the extended signal peptide region is transcribed and translated.

Amino Acid Sequence↗

PubMed2005

Genomic analysis of the protein secretion systems in Clostridium acetobutylicum ATCC 824.

Consistent information about protein secretion in Gram-positive bacteria is essentially restricted to the model organism Bacillus subtilis. Among genome-sequenced clostridia, Clostridium acetobutylicum has been the most extensively studied from a physiological point of view and is the organism for which the largest variety of molecular biology tools have been developed. Following in silico analyses, both secreted proteins and protein secretion systems were identified. The Tat (Twin arginine translocation; TC #2.A.64) pathway and ABC (ATP binding cassette) protein exporters (TC #3.A.1.) could not be identified, but the Sec (secretion) pathway (TC #3.A.5) appears to be used prevalently. Similarly, a flagella export apparatus (FEA; TC #3.A.6.), holins (TC #1.E.), and an ESAT-6/WXG100 (early secreted antigen target of 6 kDa/proteins with a WXG motif of approximately 100 residues) secretion system were identified. Here, we report for the first time the identification of a fimbrilin protein exporter (FPE; TC #3.A.14) and a Tad (tight adherence) export apparatus in C. acetobutylicum. This investigation highlights the potential use of this saprophytic bacterium in biotechnological and biomedical applications as well as a model organism for studying protein secretion in pathogenic Gram-positive bacteria.

Amino Acid Sequence↗

PubMed2005

Protein secretion systems in Fusobacterium nucleatum: genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis.

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.

Amino Acid Motifs↗

PubMed2005

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Alleles↗

PubMed2005

Gardening the genome: DNA methylation in Arabidopsis thaliana.

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.

Arabidopsis↗

PubMed2005

Prevalence of pathogenicity island IICFT073 genes among extraintestinal clinical isolates of Escherichia coli.

Uropathogenic Escherichia coli is the most common cause of urinary tract infection (UTI). Cystitis in women is by far the most common UTI; pyelonephritis in both sexes and prostatitis in men are more severe but are less frequent complaints. The ability of E. coli to cause UTI is associated with specific virulence determinants, some of which are encoded on pathogenicity islands (PAI). One such PAI (PAI IICFT073), of the prototypical uropathogenic E. coli strain CFT073, contains 116 open reading frames, including iron-regulated genes, carbohydrate biosynthetic genes, the serine protease autotransporter picU, a two-partner secretion system, a type I secretion system, mobility genes, and a large number of hypothetical genes. To determine the association of PAI IICFT073 with UTI, PCR was used to examine the prevalence of the five virulence-associated loci among the ECOR collection and a collection of E. coli isolated from patients with cystitis, pyelonephritis, prostatitis, or septicemia. All PAI IICFT073 loci were found to be more prevalent among the B2 phylogenetic group than any other group within the ECOR collection and among invasive prostatitis strains than were cystitis or pyelonephritis strains. These data support the theory that clinical isolates causing prostatitis are more virulent than those producing cystitis or pyelonephritis in women.

Bacterial Proteins↗

PubMed2005

Distribution of the serine protease autotransporters of the Enterobacteriaceae among extraintestinal clinical isolates of Escherichia coli.

Urinary tract infections continue to be among the most common extraintestinal diseases. Cystitis in women is by far the most common urinary tract infection; pyelonephritis in both sexes and prostatitis in men are more severe but less frequent complaints. Escherichia coli is by far the most common cause of urinary tract infection. It is believed that uropathogenic E. coli is adept at colonizing the urinary tract via the production of specific virulence factors. Recently, a novel virulence determinant, Vat, was described for the prototypical uropathogenic E. coli strain CFT073. Vat is a member of the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of the autotransporters. Previously, SPATEs have been described for all pathovars of E. coli, but until recently their presence had been noticeably absent in nonpathogenic E. coli. In this report we describe the prevalence and phylogenetic distribution of the SPATEs among uropathogenic E. coli and the ECOR collection, demonstrating an association between the presence of the SPATEs, including Vat, and uropathogenic E. coli phylogroups. In addition, we describe the distribution of SPATEs among nonpathogenic E. coli.

Enterobacteriaceae↗

PubMed2004

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli.

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.

Cloning, Molecular↗

PubMed2004

The autotransporter secretion system.

The type V secretion system includes the autotransporter family, the two-partner system and the Oca family. The autotransporter secretion process involving first the translocation of the precursor through the inner membrane and then its translocation through the outer membrane via a pore formed by a beta-barrel is reviewed.

Bacteria↗