PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-9 (9)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Complete Genome Sequence of SfII, a Serotype-Converting Bacteriophage of the Highly Prevalent Shigella flexneri Serotype 2a 
Genome Announcements  2013;1(5):e00626-13.
SfII is a serotype-converting temperate bacteriophage of the highly prevalent Shigella flexneri serotype 2a. We isolated the SfII phage from a wild-type strain of S. flexneri serotype 2a. Here, we present the complete genome sequence of this phage.
doi:10.1128/genomeA.00626-13
PMCID: PMC3772137  PMID: 24029753
2.  Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri 
BMC Genomics  2013;14:677.
Background
Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.
Results
In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.
Conclusions
This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.
doi:10.1186/1471-2164-14-677
PMCID: PMC3851460  PMID: 24090466
Shigella flexneri; Bacteriophage; O-antigen modification; Serotype conversion
3.  Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri 
BMC Biochemistry  2012;13:13.
Background
Thirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions.
Results
In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines–R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52–53, GS 138–139 and SYG 274–276), phenylalanine-proline motifs (FP 78–79 and FPV 282–84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110–111, GR 269–270 and D333 found in hydrophilic loops were also found to be critical to Oac function.
Conclusions
By studying the effect of the mutations on Oac’s function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110–111, GR 269–270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.
doi:10.1186/1471-2091-13-13
PMCID: PMC3467182  PMID: 22793174
Shigella flexneri; O-acetyltransferase (Oac); Critical amino acids
4.  Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri 
BMC Biochemistry  2011;12:67.
Background
The three bacteriophage genes gtrA, gtrB and gtr(type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr(type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
Results
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
Conclusion
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
doi:10.1186/1471-2091-12-67
PMCID: PMC3259042  PMID: 22188643
5.  IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses 
During T cell–dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell–intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell–autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1+ GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21.
doi:10.1084/jem.20091738
PMCID: PMC2822609  PMID: 20142429
6.  A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c▿ † 
Journal of Bacteriology  2009;191(21):6612-6617.
The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
doi:10.1128/JB.00628-09
PMCID: PMC2795301  PMID: 19717593
7.  Complete Genomic Sequence of SfV, a Serotype-Converting Temperate Bacteriophage of Shigella flexneri 
Journal of Bacteriology  2002;184(7):1974-1987.
Bacteriophage SfV is a temperate serotype-converting phage of Shigella flexneri. SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage λ, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that orf-41 encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two S. flexneri strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has cos sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of Escherichia coli O157:H7 and Haemophilus influenzae. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the E. coli K-12 genome were noted, suggesting that these three phages have common evolutionary origins.
doi:10.1128/JB.184.7.1974-1987.2002
PMCID: PMC134923  PMID: 11889106
8.  Serotype 1a O-Antigen Modification: Molecular Characterization of the Genes Involved and Their Novel Organization in the Shigella flexneri Chromosome 
Journal of Bacteriology  1999;181(15):4711-4718.
The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.
PMCID: PMC103612  PMID: 10419979
9.  The Periplasmic Enzyme, AnsB, of Shigella flexneri Modulates Bacterial Adherence to Host Epithelial Cells 
PLoS ONE  2014;9(4):e94954.
S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.
doi:10.1371/journal.pone.0094954
PMCID: PMC3998974  PMID: 24762742

Results 1-9 (9)