Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.     

Shigella Strains Are Not Clones of Escherichia coli but Sister Species in the Genus Escherichia

Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.     

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters in Shigella

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.The Shigella outer membrane protein IcsA is unusual in that it is secreted at the bacterial old pole (9, 13, 24). The secreted protein forms a cap at the old pole (Fig. ​(Fig.1A),1A), where during the infection of host cells, it interacts with cellular actin cytoskeletal proteins to induce the formation of propulsive actin tails (6, 43, 70). Actin tail formation is essential to the spread of Shigella spp. through cell monolayers and mammalian tissues (6, 43, 47) and is critical for Shigella virulence (15, 60). IcsA is a member of the autotransporter family of secreted proteins in gram-negative bacteria. Approximately 700 autotransporter proteins are predicted to be encoded within bacterial genomes that had been annotated as of 2003 (54). All autotransporter proteins for which the site of secretion has been determined are, like IcsA, secreted at the bacterial old pole (35).Open in a separate windowFIG. 1.Design of screen for proteins that, like IcsA, localize to potential division sites independent of FtsZ. (A) Localization of IcsA on the surface of S. flexneri. Immunofluorescence using antibody to IcsA. (B) Localization of IcsA_507-620-GFP (expressed from pBAD24-icsA_507-620::gfp) to poles of single cells of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the permissive temperature (30°C). (C) Localization of IcsA_507-620-GFP to potential cell division sites of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the nonpermissive temperature (42°C). (D) Diagram of the strategy used to identify proteins of E. coli that localize to potential cell division sites independent of FtsZ, displaying a localization pattern similar to that shown for IcsA in panel C. Image from DnaK-GFP localization (expressed from leaky promoter on pCA24N-dnaK, without induction) in screen well; incomplete overlay of GFP with phase-contrast microscopy is due to the movement of cells between capturing the two images, as cells were imaged live. Size bars = 2 μm (A and B) and 5 μm (C and D). Images are representative. O/N, overnight.Several other secreted bacterial proteins are also localized to one or both cell poles; these include the Listeria monocytogenes actin assembly protein ActA (39), components of the chemotaxis apparatus in Escherichia coli and Caulobacter crescentus (1, 46, 66), the Legionella pneumophila and Agrobacterium tumifaciens type IV secretion systems (14, 40), Pseudomonas aeruginosa type IV pili (8), protein components of the cell cycle regulatory pathways in C. crescentus (reviewed in reference 72), the DNA transfer apparatus in Bacillus subtilis and Streptomyces spp. (26, 28), and polar flagella in Vibrio cholerae, Campylobacter spp., Helicobacter spp., C. crescentus, and other gram-negative bacteria. In L. monocytogenes, the polarity of ActA is established after ActA secretion and likely depends on differential growth rates of the cell wall along the length of the bacterium (56). In C. crescentus, TipN serves as a polar developmental landmark (31, 42), and RcdA provides temporal and spatial specificity in the regulated proteolysis of key factors involved in polar asymmetry (50). Beyond these studies, relatively little is known about the molecular mechanisms that mediate the proper localization of polar bacterial proteins.Chemical or genetic blockade of cell division leads to the formation of filamentous cells without septa. In cells that have been filamented by either blocking FtsI or depleting functional FtsZ, a cytoplasmic derivative of IcsA localizes at or near potential cell division sites (36), which represent the sites of future cell poles. IcsA also localizes to potential division sites independent of nucleoid occlusion (36), together indicating that the positional information directing IcsA polarity is independent of these cell division proteins and chromosome positioning. The molecules that are required for the localization of IcsA to the cell pole have not been identified.One model of IcsA localization to the pole is that freely diffusing cytoplasmic IcsA recognizes and binds a protein receptor that is present at poles and future poles. Although icsA is present only in Shigella spp., upon heterologous expression, IcsA localizes to the poles of other Enterobacteriaceae (13, 58, 59), indicating that if targeting occurs via binding to a polar receptor, the receptor is likely conserved among members of this family. In addition, since IcsA localizes independently of FtsZ and FtsI, the localization of a putative polar receptor to the pole must also be independent of these cell division proteins. To find proteins that might serve as a polar receptor for IcsA, we first conducted a genome-wide screen designed to identify the subset of E. coli proteins that localize to poles and to potential cell division sites independently of functional FtsZ. For each conserved protein that displayed this localization pattern, we then tested whether it played a role in the polar localization of IcsA. We found that, under the conditions of our screen, a green fluorescent protein (GFP) fusion to the cytoplasmic chaperone DnaK localizes to cell poles and potential cell division sites. Although DnaK is not a polar receptor for IcsA, we demonstrated that it was required for the localization of IcsA to the pole in the bacterial cytoplasm in E. coli and for the secretion of both IcsA and a second Shigella autotransporter, SepA, in native Shigella flexneri, consistent with a critical role of DnaK in the chaperoning of IcsA and SepA, and perhaps autotransporter proteins more generally, in the bacterial cytoplasm.     

Rapid Concentration and Molecular Enrichment Approach for Sensitive Detection of Escherichia coli and Shigella Species in Potable Water Samples

In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment]), that we coupled to an Escherichia coli/Shigella-specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli/Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non-E. coli/Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii, which was detected by the CRENAME procedure combined with the E. coli/Shigella-specific rtPCR assay (CRENAME + E. coli rtPCR). DNA from all 90 E. coli/Shigella strains tested was amplified by the CRENAME + E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME + E. coli rtPCR method detected 1.8 E. coli/Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli/Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli/Shigella-specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin In the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coii K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K-12 and a virulence locus for provoking keratocon-junctivitis in the eyes of guinea-pigs, named kcpA is located near purE in S. fiexnerl, and the two loci are involved in VirG expression, the KcpA～ mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompr gene from E. coli K-12, giving rise to a KcpA phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Molecular and Functional Profiling of the Polyamine Content in Enteroinvasive E. coli?: Looking into the Gap between Commensal E. coli and Harmful Shigella

Polyamines are small molecules associated with a wide variety of physiological functions. Bacterial pathogens have developed subtle strategies to exploit polyamines or manipulate polyamine-related processes to optimize fitness within the host. During the transition from its innocuous E. coli ancestor, Shigella, the aetiological agent of bacillary dysentery, has undergone drastic genomic rearrangements affecting the polyamine profile. A pathoadaptation process involving the speG gene and the cad operon has led to spermidine accumulation and loss of cadaverine. While a higher spermidine content promotes the survival of Shigella within infected macrophages, the lack of cadaverine boosts the pathogenic potential of the bacterium in host tissues. Enteroinvasive E. coli (EIEC) display the same pathogenicity process as Shigella, but have a higher infectious dose and a higher metabolic activity. Pathoadaption events affecting the cad locus have occurred also in EIEC, silencing cadaverine production. Since EIEC are commonly regarded as evolutionary intermediates between E. coli and Shigella, we investigated on their polyamine profile in order to better understand which changes have occurred along the path to pathogenicity. By functional and molecular analyses carried out in EIEC strains belonging to different serotypes, we show that speG has been silenced in one strain only, favouring resistance to oxidative stress conditions and survival within macrophages. At the same time, we observe that the content of spermidine and putrescine, a relevant intermediate in the synthesis of spermidine, is higher in all strains as compared to E. coli. This may represent an evolutionary response to the lack of cadaverine. Indeed, restoring cadaverine synthesis decreases the expression of the speC gene, whose product affects putrescine production. In the light of these results, we discuss the possible impact of pathoadaptation events on the evolutionary emergence of a polyamine profile favouring to the pathogenic lifestyle of Shigella and EIEC.     

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Structural relationships between genetically closely related O-antigens of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using ¹H and ¹³C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.     

Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.     

Changing Trends in the Prevalence of Shigella Species: Emergence of Multi-Drug Resistant Shigella sonnei Biotype g in Bangladesh

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.     

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis

Shigella sonnei is a bacterial pathogen and causative agent of bacillary dysentery. It deploys a type III secretion system to inject effector proteins into host epithelial cells and macrophages, an essential step for tissue invasion and immune evasion. Although the arsenal of bacterial effectors and their cellular targets have been studied extensively, little is known about the prerequisites for deployment of type III secreted proteins during infection. Here, we describe a novel S. sonnei adhesin, SSO1327 which is a multivalent adhesion molecule (MAM) required for invasion of epithelial cells and macrophages and for infection in vivo. The S. sonnei MAM mediates intimate attachment to host cells, which is required for efficient translocation of type III effectors into host cells. SSO1327 is non‐redundant to IcsA; its activity is independent of type III secretion. In contrast to the up‐regulation of IcsA‐dependent and independent attachment and invasion by deoxycholate in Shigella flexneri, deoxycholate negatively regulates IcsA and MAM in S. sonnei resulting in reduction in attachment and invasion and virulence attenuation in vivo. A strain deficient for SSO1327 is avirulent in vivo, but still elicits a host immune response.     

Gene decay in Shigella as an incipient stage of host-adaptation

Background

Many facultative bacterial pathogens have undergone extensive gene decay processes, possibly due to lack of selection pressure during evolutionary conversion from free-living to intracellular lifestyle. Shigella, the causative agents of human shigellosis, have arisen from different E. coli-like ancestors independently by convergent paths. As these bacteria all have lost large numbers of genes by mutation or deletion, they can be used as ideal models for systematically studying the process of gene function loss in different bacteria living under similar selection pressures.

Methodologies/Principal Findings

We compared the sequenced Shigella genomes and re-defined decayed genes (pseudogenes plus deleted genes) in these bacteria. Altogether, 85 genes are commonly decayed in the five analyzed Shigella strains and 1456 genes are decayed in at least one Shigella strain. Genes coding for carbon utilization, cell motility, transporter or membrane proteins are prone to be inactivated. Decayed genes tend to concentrate in certain operons rather than distribute averagely across the whole genome. Genes in the decayed operon accumulated more non-synonymous mutations than the rest genes and meanwhile have lower expression levels.

Conclusions

Different Shigella lineages underwent convergent gene decay processes, and inactivation of one gene would lead to a lesser selection pressure for the other genes in the same operon. The pool of superfluous genes for Shigella may contain at least two thousand genes and the gene decay processes may still continue in Shigella until a minimum genome harboring only essential genes is reached.