H1 helix of colicin U causes phospholipid membrane permeation.

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

Structure and genetics of the O-antigen of Escherichia coli O169 related to the O-antigen of Shigella boydii type 6.

The O-polysaccharide (O-antigen) of Escherichia coli O169 was studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: [Formula: see text] The O-polysaccharide of E. coli O169 differs from that of Shigella boydii type 6 only in the presence of a side-chain glucose residue. A comparison of the O-antigen biosynthesis gene clusters between the galF to gnd genes in the genomes of the two bacteria revealed their close relationship. The glycosyltransferase gene responsible for the formation of the ß-D-Glcp-(1 → 6)-α-D-Galp linkage in the O-antigen was identified in the gene cluster.

Arginine-dependent acid-resistance pathway in Shigella boydii.

Ability to survive the low pH of the human stomach is considered be an important virulent determinant. It was suggested that the unique acid tolerance of Shigella boydii 18 CDPH, the strain implicated in a 1998 outbreak, may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arginine-dependent acid-resistance (ADAR) pathway. This pathway was assumed to be absent in Shigella sp. Here, we have examined occurrence and efficacy of ADAR pathway in 21 S. boydii strains obtained from the American Type Culture Collection (ATCC) along with strains of S. flexneri (n = 7), S. sonnei (n = 4), and S. dysenteriae (n = 2). The eight S. boydii strains were able to induce ADAR to survive the acid challenge at pH 2.0; additional 8 strains could tolerate acid challenge at pH 2.5 but not at pH 2.0. The remaining five S. boydii strains were not able to induce ADAR pathway and could not survive acid challenge even at pH 2.5. ADAR pathway also appears to be present in all four Shigella sp. Shigella ADAR pathway was induced when cells were grown under partial oxygen pressure while its expression in E. coli required mere fermentative growth on glucose.

The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65.

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-kappaB, to the host cell nucleus. NF-kappaB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-kappaB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-kappaB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-kappaB activation. Whereas NleE inhibited both TNFalpha and IL-1beta stimulated p65 nuclear translocation and IkappaB degradation, NleB inhibited the TNFalpha pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-kappaB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli.

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli, had lipopolysaccharide profiles quite distinct from the respective strain of E. coli. It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.

Role of RNA polymerase sigma-factor (RpoS) in induction of glutamate-dependent acid-resistance of Escherichia albertii under anaerobic conditions.

Escherichia albertii is a potential enteric food-borne pathogen with poorly defined genetic and biochemical properties. Acid resistance is perceived to be an important property of enteric pathogens, enabling them to survive passage through stomach acidity so that they may colonize the mammalian gastrointestinal tract. We analyzed glutamate-dependent acid-resistance pathway (GDAR) in five E. albertii strains that have been identified so far. We observed that the strains were unable to induce GDAR under aerobic growth conditions. Mobilization of the rpoS gene restored aerobic induction of this acid-resistance pathway, indicating that all five strains may have a dysfunctional sigma-factor. On the other hand, under anaerobic growth conditions where GDAR is induced in an RpoS-independent manner (i.e. in Shigella spp. and Escherichia coli O157:H7 strains), only three out of five E. albertii strains successfully induced GDAR. The remainder of the two strains exhibited dependence on functional RpoS even under anaerobic conditions to express GDAR, a regulatory function previously considered to be redundant. The data indicate that certain E. albertii strains may have an alternate RpoS-dependent pathway for acid-resistance under anaerobic growth conditions.

Structural and genetic characterization of Shigella boydii type 17 O antigen and confirmation of two new genes involved in the synthesis of glucolactilic acid.

Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.

Structural and molecular characterization of Shigella boydii type 16 O antigen.

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis).

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.

Structural and genetic characterization of the Shigella boydii type 18 O antigen.

Shigella strains are important human pathogens and are normally identified by their O antigens. O antigen is an essential part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria and plays a role in pathogenicity. Structural and genetic organization of the Shigella boydii type 18 O antigen was investigated. As judged by sugar and methylation analyses and NMR spectroscopy data, the O antigen has a linear pentasaccharide repeating unit (O unit), which consists of three L-rhamnose residues, and one residue each of D-galacturonic acid (D-GalA) and N-acetylgalactosamine (D-GalNAc), and the following structure of the O unit was established. -->3)-beta-L-Rhap-(1-->4)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-GalpA-(1-->3)-alpha-D-GalpNAc-(1--> The O antigen gene cluster of S. boydii type 18, which contains nine open reading frames (ORFs), was found between galF and gnd. Based on homology, all of the ORFs were identified as O antigen synthesis genes, involved in the synthesis of rhamnose, transfer of sugars, and processing of O unit. Genes specific for S. boydii type 18 were identified, which can be potentially used for the development of a PCR-based assay for the identification and detection of this strain.

Correlation between Congo red binding and contact haemolysin production in Shigella species.

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.

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.

Expression of fimbriae and hemagglutination activity in Shigella boydii.

This report describes the presence of type 1 fimbriae on Shigella boydii 5 which agglutinate guinea pig erythrocytes and feature mannose-sensitive adherence. Morphologically, the fimbriae were thin, rigid cylinders 2-5 microm in length and 35 nm in diameter, and the organella retained axial holes. This is the first study to have revealed the existence of type 1 fimbriae on S. boydii.

Binding of Shigella to rat and human intestinal mucin.

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [35S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.

Contact-haemolysin production by entero-invasive Escherichia coli and shigellae.

Entero-invasive Escherichia coli (EIEC) and shigellae were tested for contact-haemolysin (CH) with red blood cells (RBCs) of guinea-pig, rabbit, rat, mouse, monkey, man, sheep and chicken; all bacteria showed the best lysis with guinea-pig RBCs. The best culture medium for CH activity of shigellae was tryptic soy broth, and for EIEC it was casamino acid-yeast extract broth with 1 mM CaCl2. CH production by all species was best at the slightly alkaline pH which is optimal for growth; it was also dependent on the presence of a large (140-Mda) plasmid. Pre-treatment of bacteria with homologous antisera inhibited CH activity. Various treatments of bacterial cells and RBCs suggested that CH may be a protein molecule, and that a chitotriose-like moiety may serve as CH receptor. RBCs that were incubated with bacteria at 4 degrees C, or with heat-killed bacteria at 37 degrees C, were not lysed; also, isolated cell-surface components (lipopolysaccharide and outer-membrane protein) did not lyse RBCs. This suggests that metabolically active cells are required for CH activity. Production of CH by both EIEC and shigellae is consistent with a common mechanism for the virulence of these organisms.

[Isolation of Shigella boydii serotype 14 with altered biochemical characteristics in a water-borne epidemic of dysentery]. / Izolacija Shigella boydii, serotip 14 izmenjenih biohemijskih osobina u hidricnoj epidemiji dizenterije.

Two water-borne epidemics of bacillary dysentery have been described in a garrison in which the leading causative agent has been shigella boydii, serotype of 14 altered biochemical characteristics isolated for the first time in Yugoslavia. All the isolated strains of this shigella were mannitol negative and they degradated gylocose to acidity and gas already after 24 hours, and arabinose and sorbitol to acidity and gas after 48 hours. The clinical picture of the diseased was characteristic of bacillary dysentery and did not differ from clinical picture of patients in whom other serotypes of shigella were isolated.

Congo red binding and salt aggregation as indicators of virulence in Shigella species.

Smooth strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii, and Shigella sonnei which form pigmented colonies (Pcr+) on Congo red agar were virulent in the Sereny test. Smooth variants unable to bind Congo red (Pcr-) were avirulent. Measurements of dye uptake from solution showed that S. dysenteriae type 1 bound the most dye, followed in order of uptake by S. flexneri, S. boydii, and S. sonnei. Using the salt aggregation test (SAT) to determine cell surface hydrophobicity, we found the same order of species. The SAT could not, however, detect differences in surface properties between Pcr+ and Pcr- pairs of isogenic smooth strains. Enteroinvasive Escherichia coli strains used in the study showed SAT and Congo red-binding properties which were similar to those of the S. flexneri strains. A direct correlation was found between pigment-binding ability and the presence of the large 140-megadalton plasmid in S. flexneri, enteroinvasive E. coli, and S. boydii but not in S. dysenteriae type 1 or S. sonnei strains. Congo red interacted with outer membranes and outer membrane proteins of S. dysenteriae type 1 but not with lipopolysaccharides. However, rough mutants of Shigella species deficient in lipopolysaccharides bound Congo red and formed pigmented colonies, showing that dye binding as a virulence assay may be misinterpreted in such cases. There was complete correlation of the Pcr+ phenotype with virulence in the smooth strains in this study, suggesting that Congo red binding can be utilized as a quick and reliable alternative to the Sereny test.

The relation between production of cytotoxin and clinical features in shigellosis.

The relation between in vitro production of HeLa cell cytotoxin by strains of Shigella and clinical symptomatology was determined for 35 travelers from the United States who developed shigellosis in Guadalajara, Mexico. There were 25 patients with Shigella sonnei, eight with Shigella flexneri, one with Shigella boydii, and one with Shigella dysenteriae. These strains were evaluated for in vitro production of cytotoxin. The amount of cytotoxin did not correlate with the number of stools passed, the severity of abdominal pain, or the presence of nausea or vomiting. However, patients with strains of Shigella that produced more cytotoxic activity were more likely to have fever (P less than .02) and occult blood in their stools (P less than .004). The cytotoxicity produced by 30 (86%) strains could not be neutralized with rabbit antiserum to purified, formaldehyde-treated Shiga toxin from S. dysenteriae type 1 strain 60 R; the cytotoxicity of five (14%) of the strains was partially neutralized. When only nonneutralizable cytotoxin was considered, the presence of fecal leukocytes (P less than .04), as well as of occult blood (P less than .002) and fever (P less than .02), correlated with the amount of cytotoxin. The amount of nonneutralizable cytotoxin produced by shigella strains was related to the clinical findings. This cytotoxic activity was infrequently attributable to "Shiga toxin".