Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

An impedimetric aptasensor for Shigella dysenteriae using a gold nanoparticle-modified glassy carbon electrode.

This work describes an aptasensor for the foodborne pathogen Shigella dysenteriae (S. dysenteriae). A glassy carbon electrode (GCE) was modified with gold nanoparticles (AuNPs) by electrodeposition. Then, thiolated aptamer for S. dysenteriae detection was self-assembled on the surface of the modified GCE, and any free residual AuNPs were blocked with 6-mercapto-1-hexanol. The size, morphology, and distribution of the AuNPs were characterized by field emission scanning electron microscopy. Detection of S. dysenteriae was performed measurement of the charge transfer resistance (Rct) before and after addition of S. dysenteriae using hexacyanoferrate as an electrochemical probe. The interaction between the aptamer and outer-membrane proteins of S. dysenteriae lead to an increase in the Rct of the sensor. The assay has a linear dynamic range that extends from 101 to 106 CFU.mL-1 and a limit of detection of 100 CFU.mL-1. It can differentiate between alive S. dysenteriae and other pathogens. Dead S. dysenteriae cells do not have any effect on selectivity. Unpasteurized and pasteurized skim milk and some water samples were spiked with S. dysenteriae and then successfully examined by this method. The results were validated by real-time PCR. The method is fast, low-cost, highly sensitive, and specific. Hence, it represents a valuable tool in food quality control. Graphical abstract Schematic presentation of a label free impedimetric aptasensor for Shigella dysenteriae using a glassy carbon electrode modified with gold nanoparticles (AuNPs) and 6-mercapto-1-hexanol (MCH). The limit of detection of this aptasensor is as low as 1 CFU.mL-1 for target bacteria.

Diversity of Class 1 Integron Gene Cassette Rearrangements Selected under Antibiotic Pressure.

UNLABELLED: Integrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Gene cassettes are expressed via a Pc promoter and can be excised from or integrated into the integron by integrase IntI. Although the mechanisms of gene cassette integration and excision are well known, the kinetics and modes of gene cassette shuffling leading to new gene cassette arrays remain puzzling. It has been proposed that under antibiotic selective pressure, IntI-mediated rearrangements can generate integron variants in which a weakly expressed gene cassette moves closer to Pc, thus leading to higher-level resistance. To test this hypothesis, we used an integron with four gene cassettes, intI1-aac(6')-Ib-dfrA15-aadA1-catB9, and applied selective pressure with chloramphenicol, resistance to which is encoded by catB9. Experiments were performed with three different Pc variants corresponding to three IntI1 variants. All three integrases, even when not overexpressed, were able to bring catB9 closer to Pc via excision of the dfrA15 and aadA1 gene cassettes, allowing their host bacteria to adapt to antibiotic pressure and to grow at high chloramphenicol concentrations. Integrase IntI1(R32_H39), reported to have the highest recombination activity, was able, when overexpressed, to trigger multiple gene cassette rearrangements. Although we observed a wide variety of rearrangements with catB9 moving closer to Pc and leading to higher chloramphenicol resistance, "cut-and-paste" relocalization of catB9 to the first position was not detected. Our results suggest that gene cassette rearrangements via excision are probably less cost-effective than excision and integration of a distal gene cassette closer to Pc. IMPORTANCE: Integrons are bacterial genetic elements able to capture and express gene cassettes. Gene cassettes are expressed via a Pc promoter; the closer they are to Pc, the more strongly they are expressed. Gene cassettes can be excised from or integrated into the integron by integrase IntI. The kinetics and modes of gene cassette shuffling, leading to new gene cassette arrays remain puzzling. We used an integron with 4 antibiotic resistance gene cassettes and applied selective pressure with the antibiotic for which resistance was encoded by cassette 4. All IntI variants were able to bring cassette 4 closer to Pc. Rearrangements occur via excision of the previous gene cassettes instead of cut-and-paste relocalization of the fourth gene cassette.

Shiga toxin induces membrane reorganization and formation of long range lipid order.

Lateral variation of the in-plane orientation of lipids in a bilayer is referred to as texture. The influence of the protein Shiga toxin on orientational membrane texture was studied in phosphatidylcholine lipid bilayers using polarization two-photon fluorescence microscopy and atomic force microscopy. A content of 1% of glycosphingolipid globotriaosylceramide (Gb3) receptor lipids in a bilayer was used to bind the Shiga toxin B-subunit to the surface of gel domains. Binding of the Shiga toxin B-subunit to lipids led to the modulation of orientational membrane texture in gel domains and induced membrane reordering. When Shiga toxin was added above the lipid chain melting temperature, the toxin interaction with the membrane induced rearrangement and clustering of Gb3 lipids that resulted in the long range order and alignment of lipids in gel domains. The toxin induced redistribution of Gb3 lipids inside gel domains is governed by the temperature at which Shiga toxin was added to the membrane: above or below the phase transition. The temperature is thus one of the critical factors controlling lipid organization and texture in the presence of Shiga toxin. Lipid chain ordering imposed by Shiga toxin binding can be another factor driving the reconstruction of lipid organization and crystallization of lipids inside gel domains.

Proteomes of pathogenic Escherichia coli/Shigella group surveyed in their host environments.

Proteomic studies on Shigella dysenteriae, Shigella flexneri, enterohemorrhagic Escherichia coli and uropathogenic E. coli (UPEC) are reviewed. UPEC causes infections in the urogenital tract, whereas the other species colonize and, to varying degrees, invade the intestinal tract. Type III secretion systems used to breach the mucosal barrier by the intestinal pathogens revealed distinct expression patterns in different host environments. Dynamic adaptations to changes in nutrient availability and oxygen were observed, including increased reliance on anaerobic respiration and mixed acid fermentation in vivo. Utilization of carbon and nitrogen resources by the bacteria varied considerably depending on the host model investigated. Shigellae and UPEC adapted to metal ion sequestration in the mammalian host by enhancing expression of various receptors and transporters for iron and zinc. This appears to reflect the preferred intracellular life stage of Shigella spp. and responses of UPEC to high levels of lipocalin and lactotransferrin in the urinary tract.

Identification and characterization of a novel lytic peptidoglycan transglycosylase (MltC) in Shigella dysenteriae.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.

AGAP2 regulates retrograde transport between early endosomes and the TGN.

The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are approximately 20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN.

Shiga toxin B subunits induce VWF secretion by human endothelial cells and thrombotic microangiopathy in ADAMTS13-deficient mice.

Diarrhea-associated hemolytic uremic syndrome (D+HUS) is the most common cause of acute renal failure among children. Renal damage in D+HUS is caused by Shiga toxin (Stx), which is elaborated by Shigella dysenteriae and certain strains of Escherichia coli, in North America principally E coli O157:H7. Recent studies demonstrate that Stx also induces von Willebrand factor (VWF) secretion by human endothelial cells and causes thrombotic thrombocytopenic purpura, a disease with similarities to D+HUS, in Adamts13(-/-) mice. Stx occurs in 2 variants, Stx1 and Stx2, each of which is composed of 1 catalytically active A subunit that is responsible for cytotoxicity, and 5 identical B subunits that mediate binding to cell-surface globo-triaosylceramide. We now report that B subunits from Stx1 or Stx2 can stimulate the acute secretion of VWF in the absence of the cytotoxic A subunit. This rapid effect requires binding and clustering of globotriaosylceramide, and depends on plasma membrane cholesterol and caveolin-1 but not clathrin. Furthermore, similar to Stx2 holotoxin, the isolated Stx2B subunits induce thrombotic microangiopathy in Adamts13(-/-) mice. These results demonstrate the existence of a novel Stx B-induced lipid raft-dependent signaling pathway in endothelial cells that may be responsible for some of the biological effects attributed previously to the cytotoxic Stx A subunit.

Differential response of the human renal proximal tubular epithelial cell line HK-2 to Shiga toxin types 1 and 2.

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb(3) and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

GroEL dependency affects codon usage--support for a critical role of misfolding in gene evolution.

It has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding, yet evidence for this remains largely circumstantial. In contrast, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. We propose that putative error limitation in cis can be elucidated by examining the interaction between codon usage and chaperoning processes. Using Escherichia coli as a model system, we find that codon optimality covaries with dependency on the chaperonin GroEL. Sporadic but not obligate substrates of GroEL exhibit higher average codon adaptation and are conspicuously enriched for optimal codons at structurally sensitive sites. Further, codon optimality of sporadic clients is more conserved in the E. coli clone Shigella dysenteriae. We suggest that highly expressed genes cannot routinely use GroEL for error control so that codon usage has evolved to provide complementary error limitation. These findings provide independent evidence for a role of misfolding in shaping gene evolution and highlight the need to co-characterize adaptations in cis and trans to unravel the workings of integrated molecular systems.

Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator.

Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human beta-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.

Evaluation of the Anti-Shigellosis Activity of Dietary Isothiocyanates in Galleria mellonella Larvae.

Cruciferous vegetables, widely present in daily diets, are a rich source of organosulfur compounds with proven health benefits, especially chemopreventive or antioxidative effects. Isothiocyanate derivatives (ITCs) exhibit a broad spectrum of biological and pharmacological activity and recently, their antibacterial properties have been of particular importance. Here, we have focused on the anti-shigellosis activity of sulforaphane (SFN) and phenethyl ITC (PEITC). The genus Shigella causes gastroenteritis in humans, which constitutes a threat to public health. Production of a potent Stx toxin by S. dysenteriae type 1 results not only in more severe symptoms but also in serious sequela, including the hemolytic uremic syndrome. Here, we present evidence that two aliphatic and aromatic ITCs derivatives, SFN and PEITC, have an effective antibacterial potency against S. dysenteriae, also negatively regulating the stx gene expression. The molecular mechanism of this effect involves induction of the global stress-induced stringent response. ITCs also inhibit bacterial virulence against the Vero and HeLa cells. We present evidence for the therapeutic effect of sulforaphane and phenethyl ITC against a S. dysenteriae infection in the Galleria mellonella larvae model. Thus, our results indicate that isothiocyanates can be effectively used to combat dangerous bacterial infections.

Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: heme binding by an induced fit mechanism.

Shigella dysentriae and other Gram-negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 A resolution the 3D structure of the TonB-dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C-terminal domain that folds into a 22-stranded transmembrane beta-barrel, which is filled by the N-terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 A apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA-Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N-terminal TonB-box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB-box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB-box.

The Shigella dysenteriae serotype 1 proteome, profiled in the host intestinal environment, reveals major metabolic modifications and increased expression of invasive proteins.

Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the type III secretion system effectors OspC2 and IpaB, were detected as antigens in Western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.

Priming of CD4+ T cells with porin of Shigella dysenteriae activates the cells toward type 1 polarization.

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive T(h) cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4(+) T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-gamma and had no effect on IL-4 expression. In parallel to the predominant release of the T(h)1 cytokine IFN-gamma, up-regulation of CCR5 on the immune CD4(+) T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1 alpha and regulated on activation normal T cell expressed and secreted showed T(h)1 bias. The porin-primed CD4(+) T cells expressed the mRNA for T-box expressed in T cells, a T(h)1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector T(h)1 cells. The immune CD4(+) T cells proliferated and released IL-2 and IFN-gamma profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4(+) T cells of T(h)1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity.

Functional characterization of the Shigella dysenteriae heme ABC transporter.

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.

Differential expression of ompC and ompF in multidrug-resistant Shigella dysenteriae and Shigella flexneri by aqueous extract of Aegle marmelos, altering its susceptibility toward beta-lactam antibiotics.

Steadily increasing resistance among Shigella to beta-lactams, aminoglycosides, and tetracycline has compromised the utility of these commonly used antimicrobial agents. Also, undesirable side effects of certain antibiotics have triggered immense interest in search of alternative therapies using medicinal plants. One such medicinal plant used since ancient times to cure diarrhea is Aegle marmelos. The present study exemplifies the susceptibility of beta-lactam-resistant Shigella dysenteriae and Shigella flexneri toward beta-lactam antibiotics, when grown in the presence of aqueous extract of A. marmelos (AEAM), by altering porin channels. This was demonstrated by antibiotic sensitivity test using disc diffusion method and MIC test. Susceptibility toward beta-lactam antibiotic is associated with changes in outer membrane porins OmpC (approximately 42 kDa) and OmpF (approximately 38 kDa) and cytosolic proteins of approximately 26 kDa, OmpR, a transcriptional regulator. Expression of ompF is increased in S. dysenteriae and S. flexneri grown in the presence of AEAM due to down-regulation of ompR, which is conformed by reverse transcriptase polymerase chain reaction. In conclusion, AEAM influences susceptibility of beta-lactam-resistant Shigella toward beta-lactam antibiotics by altering porin channels. Hence, AEAM along with beta-lactam can be used for treatment of multidrug-resistant Shigella.

Biochemical characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc biosynthetic pathways in Shigella dysenteriae type 7 and Escherichia coli O7.

O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-d-glucose (d-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-d-glucose (d-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-d-Qui4N and dTDP-d-Qui4NAc (the nucleotide-activated precursors of d-Qui4NGlyAc and d-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-d-Qui4N is synthesized from alpha-d-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-d-Qui4N into dTDP-d-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioA(D7)) and E. coli O7 (VioA(O7)) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-d-Qui4N and dTDP-d-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.

Close relation of the O-polysaccharide structure of Escherichia coli O168 and revised structure of the O-polysaccharide of Shigella dysenteriae type 4.

The O-polysaccharide was isolated from the lipopolysaccharide of Escherichia coli O168 and studied by chemical analyses and Smith degradation along with (1)H and (13)C NMR spectroscopies. The following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text] where 6-O-acetylation of GlcNAc is partial. Reinvestigation of the O-polysaccharide of Shigella dysenteriae type 4 established earlier showed it to have the same structure except for that the lateral Fuc residue is nonstoichiometrically O-acetylated at each position.

Transcriptional and posttranscriptional regulation of Shigella shuT in response to host-associated iron availability and temperature.

Like most bacteria, Shigella must maintain a precise balance between the necessity and toxicity of iron; a balance that is achieved, at least in part, by regulating the production of bacterial iron acquisition systems in response to specific environmental signals. Using the Shigella heme utilization (Shu) system, S. dysenteriae is able to acquire iron from heme, a potentially rich source of nutritional iron within the otherwise iron-limited environment of the human host. Investigations presented within reveal two distinct molecular mechanisms underlying previously uncharacterized transcriptional and translational regulation of shuT, a gene encoding the periplasmic-binding component of the Shu system. While shuT transcription is regulated in response to iron availability via a process dependent upon the global regulator Fur and a Fur-binding site located immediately downstream of the promoter, shuT translation is regulated in response to environmental temperature via the activity of an RNA thermometer located within the 5' untranslated region of the gene. Such complex regulation likely increases the fitness of S. dysenteriae by ensuring maximal ShuT production when the pathogen is within the iron-limited and relatively warm environment of the infected host, the only environment in which heme will be encountered as a potential source of essential iron.