Shielding Surfaces from Viruses and Bacteria with a Multiscale Coating.

The spread of viral and bacterial pathogens mediated by contact with surfaces is a leading cause of infection worldwide. COVID-19 and the continuous rise of deaths associated with antibiotic-resistant bacteria highlight the need to impede surface-mediated transmission. A sprayable coating with an intrinsic ability to resist the uptake of bacteria and viruses from surfaces and droplets, such as those generated by sneezing or coughing, is reported. The coating also provides an effective microbicidal functionality against bacteria, providing a dual barrier against pathogen uptake and transmission. This antimicrobial functionality is fully preserved following scratching and other induced damage to its surface or 9 days of submersion in a highly concentrated suspension of bacteria. The coatings also register an 11-fold decrease in viral contamination compared to the noncoated surfaces.

Functional Role of YnfA, an Efflux Transporter in Resistance to Antimicrobial Agents in Shigella flexneri.

Shigella flexneri has become a significant public health concern accounting for the majority of shigellosis cases worldwide. Even though a multitude of efforts is being made into the development of a vaccine to prevent infections, the absence of a licensed global vaccine compels us to enormously depend on antibiotics as the major treatment option. The extensive-unregulated use of antibiotics for treatment along with natural selection in bacteria has led to the rising of multidrug-resistance Shigella strains. Out of the various mechanisms employed by bacteria to gain resistance, efflux transporters are considered to be one of the principal contributors to antimicrobial resistance. The small multidrug-resistance family consists of unique small proteins that act as efflux pumps and are involved in extruding various antimicrobial compounds. The present study aims to demonstrate the role of an efflux transporter YnfA belonging to the SMR family and its functional involvement in promoting antimicrobial resistance in S. flexneri. Employing various genetic, computational, and biochemical techniques, we show how disrupting the YnfA transporter, renders the mutant Shigella strain more susceptible to some antimicrobial compounds tested in this study, and significantly affects the overall transport activity of the bacteria against ethidium bromide and acriflavine when compared with the wild-type Shigella strain. We also assessed how mutating some of the conserved amino acid residues of YnfA alters the resistance profile and efflux activity of the mutant YnfA transporter. This study provides a functional understanding of an uncharacterized SMR transporter YnfA of Shigella.

YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri.

The intracellular pathogen Shigella flexneri, which is the causative agent of bacillary dysentery, significantly influences the worldwide implication of diarrheal infections, consequentially causing about 1.1 million deaths each year. Due to a nonavailability of an authorized vaccine and the upsurge of multidrug resistance amongst Shigella strains, there has been a huge demand for further genetic analyses which could help in the advancement of new/improved drugs, and finding vaccine candidates against the pathogen. The present study aims to illustrate the role of the yfiB gene in Shigella virulence, part of the periplasmic YfiBNR tripartite signalling system. This system is involved in the regulation of cyclic-di-GMP levels inside the bacterial cells, a vital messenger molecule impacting varied cellular processes such as biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms such as adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect the native c-di-GMP levels. We found that this subsequently causes a negative effect on the biofilm formation, bacterial invasion, host-surface attachment, and the overall virulence of Shigella. This study also carried out a structural and functional assessment of the YfiB protein and determined critical amino acid residues, essential for proper functioning of this signalling system. The present work improves our understanding of the in vivo persistence and survival of Shigella, brings light to the c-di-GMP led regulation of Shigella virulence, and provides a prospective new target to design anti-infection drugs and vaccines against S. flexneri and other bacterial pathogens.

Identification of critical residues of O-antigen-modifying O-acetyltransferase B (OacB) of Shigella flexneri.

BACKGROUND: Shigellosis is an acute gastrointestinal disease caused primarily by the bacterium Shigella flexneri. Upon ingestion, S. flexneri initiates a serotype-specific immune response that targets the O-antigen of the pathogen's lipopolysaccharide. O-antigen subunits are modified by the addition of chemical moieties, which give rise to new serotypes of S. flexneri. Nineteen different serotypes of S. flexneri have been recognized. A recently identified O-antigen-modifying enzyme, O-acetyltransferase B (OacB), which adds an acetyl residue at either position 3 or 4 of RhamnoseIII (3/4-O-acetylation) in serotypes 1a, 1b, 2a, 5a, 7a, Y, and 6 and position 6 of N- acetylglucosamine (6-O-acetylation) in serotypes 2a, 3a, Y and Yv of the O-antigen subunits. Critical residues in other proteins involved in O-antigen modifications such as glucosyltransferases (Gtrs) and acetyltransferase (Oac) of S. flexneri have been identified, whereas identification of important amino acids in OacB function is yet to be determined. RESULTS: Hydrophobicity analysis showed that OacB is a transmembrane protein with 11 transmembrane segments, 12 loops, and periplasmic N- and cytoplasmic C- termini. Bioinformatics analyses revealed that OacB contains acetyltransferase-3 domain and several conserved residues. Using site-directed mutagenesis, selected amino acids were mutated to alanine to elucidate their role in the mechanism of action of OacB. Seven amino acids R47, H58, F98, W71, R116, R119, and S146 were found critical for the OacB function. CONCLUSION: In the absence of a three-dimensional structure of the serotype converting enzyme, O-acetyltransferase B (OacB), a clear role of important residues in the mechanism of action is precluded. Therefore, in this study, using site-directed mutagenesis, seven residues critical to the function of OacB were identified. The lack of agglutination of cell expressing mutant OacB in the presence of the antiserum indicated the functional role of the corresponding residues. Hence, this study provides significant information about key residues in OacB which might be involved in forming the catalytic sites of this O-antigen modifying enzyme of S. flexneri.

Newly Emerged Serotype 1c of Shigella flexneri: Multiple Origins and Changing Drug Resistance Landscape.

Bacillary dysentery caused by Shigella flexneri is a major cause of under-five mortality in developing countries, where a novel S. flexneri serotype 1c has become very common since the 1980s. However, the origin and diversification of serotype 1c remain poorly understood. To understand the evolution of serotype 1c and their antimicrobial resistance, we sequenced and analyzed the whole-genome of 85 clinical isolates from the United Kingdom, Egypt, Bangladesh, Vietnam, and Japan belonging to serotype 1c and related serotypes of 1a, 1b and Y/Yv. We identified up to three distinct O-antigen modifying genes in S. flexneri 1c strains, which were acquired from three different bacteriophages. Our analysis shows that S. flexneri 1c strains have originated from serotype 1a and serotype 1b strains after the acquisition of bacteriophage-encoding gtrIc operon. The maximum-likelihood phylogenetic analysis using core genes suggests two distinct S. flexneri 1c lineages, one specific to Bangladesh, which originated from ancestral serotype 1a strains and the other from the United Kingdom, Egypt, and Vietnam originated from ancestral serotype 1b strains. We also identified 63 isolates containing multiple drug-resistant genes in them conferring resistance against streptomycin, sulfonamide, quinolone, trimethoprim, tetracycline, chloramphenicol, and beta-lactamase. Furthermore, antibiotic susceptibility assays showed 83 (97.6%) isolates as either complete or intermediate resistance to the WHO-recommended first- and second-line drugs. This changing drug resistance pattern demonstrates the urgent need for drug resistance surveillance and renewed treatment guidelines.

Functional Annotation and Curation of Hypothetical Proteins Present in A Newly Emerged Serotype 1c of Shigella flexneri: Emphasis on Selecting Targets for Virulence and Vaccine Design Studies.

Shigella flexneri is the principal cause of bacillary dysentery, contributing significantly to the global burden of diarrheal disease. The appearance and increase in the multi-drug resistance among Shigella strains, necessitates further genetic studies and development of improved/new drugs against the pathogen. The presence of an abundance of hypothetical proteins in the genome and how little is known about them, make them interesting genetic targets. The present study aims to carry out characterization of the hypothetical proteins present in the genome of a newly emerged serotype of S. flexneri (strain Y394), toward their novel regulatory functions using various bioinformatics databases/tools. Analysis of the genome sequence rendered 4170 proteins, out of which 721 proteins were annotated as hypothetical proteins (HPs) with no known function. The amino acid sequences of these HPs were evaluated using a combination of latest bioinformatics tools based on homology search against functionally identified proteins. Functional domains were considered as the basis to infer the biological functions of HPs in this case and the annotation helped in assigning various classes to the proteins such as signal transducers, lipoproteins, enzymes, membrane proteins, transporters, virulence, and binding proteins. This study contributes to a better understanding of growth, survival, and disease mechanism at molecular level and provides potential new targets for designing drugs against Shigella infection.

Transcriptional response of Caenorhabditis elegans when exposed to Shigella flexneri.

In recent years, researchers have begun to use Caenorhabditis elegans as a potential animal model to study Shigella pathogenesis. This study aims to further develop this model using RNA-sequencing to understand which pathways/cellular characteristics are affected and potentially cause death in Shigella-exposed worms. We identified 1631 differentially expressed genes in Shigella-exposed worms (6 h exposure). A number of these genes encode proteins involved in fatty-acid ß-oxidation (FAO), antioxidant defense and autophagy. The down-regulation of acyl-CoA dehydrogenases would impede FAO, reducing the overall energy to combat Shigella in the worm's intestinal tract. This is potentially coupled with the production of reactive oxygen species (ROS) that may not be fully quenched by antioxidant defense proteins, leading to damaged cellular organelles in the worm's intestinal cells. These cells may undergo autophagy to remove the mounting damage, but may eventually undergo cell death.

Plasmids of Shigella flexneri serotype 1c strain Y394 provide advantages to bacteria in the host.

BACKGROUND: Shigella flexneri has an extremely complex genome with a significant number of virulence traits acquired by mobile genetic elements including bacteriophages and plasmids. S. flexneri serotype 1c is an emerging etiological agent of bacillary dysentery in developing countries. In this study, the complete nucleotide sequence of two plasmids of S. flexneri serotype 1c strain Y394 was determined and analysed. RESULTS: The plasmid pINV-Y394 is an invasive or virulence plasmid of size 221,293 bp composed of a large number of insertion sequences (IS), virulence genes, regulatory and maintenance genes. Three hundred and twenty-eight open reading frames (ORFs) were identified in pINV-Y394, of which about a half (159 ORFs) were identified as IS elements. Ninety-seven ORFs were related to characterized genes (majority of which are associated with virulence and their regulons), and 72 ORFs were uncharacterized or hypothetical genes. The second plasmid pNV-Y394 is of size 10,866 bp and encodes genes conferring resistance against multiple antibiotics of clinical importance. The multidrug resistance gene cassette consists of tetracycline resistance gene tetA, streptomycin resistance gene strA-strB and sulfonamide-resistant dihydropteroate synthase gene sul2. CONCLUSIONS: These two plasmids together play a key role in the fitness of Y394 in the host environment. The findings from this study indicate that the pathogenic S. flexneri is a highly niche adaptive pathogen which is able to co-evolve with its host and respond to the selection pressure in its environment.

Genome Analysis of Shigella flexneri Serotype 3b Strain SFL1520 Reveals Significant Horizontal Gene Acquisitions Including a Multidrug Resistance Cassette.

Shigella flexneri is a major etiological agent of shigellosis in developing countries, primarily occurring in children under 5 years of age. We have sequenced, for the first time, the complete genome of S. flexneri serotype 3b (strain SFL1520). We used a hybrid sequencing method--both long-read MinION Flow (Oxford Nanopore Technologies) and short-read MiSeq (Illumina) sequencing to generate a high-quality reference genome. The SFL1520 chromosome was found to be ∼4.58 Mb long, with 4,729 coding sequences. Despite sharing a substantial number of genes with other publicly available S. flexneri genomes (2,803), the SFL1520 strain contains 1,926 accessory genes. The phage-related genes accounted for 8% of the SFL1520 genome, including remnants of the Sf6 bacteriophage with an intact O-acetyltransferase gene specific to serotype 3b. The SFL1520 chromosome was also found to contain a multiple-antibiotic resistance cassette conferring resistance to ampicillin, chloramphenicol, streptomycin, and tetracycline, which was potentially acquired from a plasmid via transposases. The phylogenetic analysis based on core genes showed a high level of similarity of SFL1520 with other S. flexneri serotypes; however, there were marked differences in the accessory genes of SFL1520. In particular, a large number of unique genes were identified in SFL1520 suggesting significant horizontal gene acquisition in a relatively short time period. The major virulence traits of SFL1520 (such as serotype conversion and antimicrobial resistance) were associated with horizontal gene acquisitions highlighting the role of horizontal gene transfer in S. flexneri diversity and evolution.

Bacteriophages are the major drivers of Shigella flexneri serotype 1c genome plasticity: a complete genome analysis.

BACKGROUND: Shigella flexneri is the primary cause of bacillary dysentery in the developing countries. S. flexneri serotype 1c is a novel serotype, which is found to be endemic in many developing countries, but little is known about its genomic architecture and virulence signatures. We have sequenced for the first time, the complete genome of S. flexneri serotype 1c strain Y394, to provide insights into its diversity and evolution. RESULTS: We generated a high-quality reference genome of S. flexneri serotype 1c using the hybrid methods of long-read single-molecule real-time (SMRT) sequencing technology and short-read MiSeq (Illumina) sequencing technology. The Y394 chromosome is 4.58 Mb in size and shares the basic genomic features with other S. flexneri complete genomes. However, it possesses unique and highly modified O-antigen structure comprising of three distinct O-antigen modifying gene clusters that potentially came from three different bacteriophages. It also possesses a large number of hypothetical unique genes compared to other S. flexneri genomes. CONCLUSIONS: Despite a high level of structural and functional similarities of Y394 genome with other S. flexneri genomes, there are marked differences in the pathogenic islands. The diversity in the pathogenic islands suggests that these bacterial pathogens are well adapted to respond to the selection pressures during their evolution, which might contribute to the differences in their virulence potential.

Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage.

BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition. RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype. CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage, SfMu, of Shigella flexneri.

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri.

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain.

BACKGROUND: Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of RhaIII, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome. RESULTS: In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a. CONCLUSIONS: This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.

Shigella flexneri infection in Caenorhabditis elegans: cytopathological examination and identification of host responses.

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.

The periplasmic enzyme, AnsB, of Shigella flexneri modulates bacterial adherence to host epithelial cells.

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.

Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri.

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.

Complete Genome Sequence of SfII, a Serotype-Converting Bacteriophage of the Highly Prevalent Shigella flexneri Serotype 2a.

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.

Topological investigation of glucosyltransferase V in Shigella flexneri using the substituted cysteine accessibility method.

Modification of the lipopolysaccharide O-antigen of Shigella converts the serotype, which is significant as acquired immune responses are serotype specific. Glucosyltransferases (Gtrs) modify the O-antigen by the addition of glucosyl-groups; however the precise mechanism of O-antigen modification is not fully understood. This study aims to substantiate inferences made on the GtrV topological structure using the substituted cysteine accessibility method (SCAM). Twenty-one amino acid residues were tested to clarify three features of GtrV: the extramembrane regions, a proposed reentrant loop, and a membrane border region. Overall, the results agreed with a previous topology proposed for GtrV. The topology of GtrV consists of 11 extramembrane regions with a cytoplasmic N-terminus, periplasmic C-terminus and 9 transmembrane (TM) helices. The existence of a reentrant loop between TM helices IV and V was verified, and the cytoplasmic membrane border region of TM helix II was examined in depth.

Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri.

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.

Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri.

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.