A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Pan-Genome Reverse Vaccinology Approach for the Design of Multi-Epitope Vaccine Construct against Escherichia albertii.

Escherichia albertii is characterized as an emerging pathogen, causing enteric infections. It is responsible for high mortality rate, especially in children, elderly, and immunocompromised people. To the best of our knowledge, no vaccine exists to curb this pathogen. Therefore, in current study, we aimed to identify potential vaccine candidates and design chimeric vaccine models against Escherichia albertii from the analysis of publicly available data of 95 strains, using a reverse vaccinology approach. Outer-membrane proteins (n = 4) were identified from core genome as vaccine candidates. Eventually, outer membrane Fimbrial usher (FimD) protein was selected as a promiscuous vaccine candidate and utilized to construct a potential vaccine model. It resulted in three epitopes, leading to the design of twelve vaccine constructs. Amongst these, V6 construct was found to be highly immunogenic, non-toxic, non-allergenic, antigenic, and most stable. This was utilized for molecular docking and simulation studies against six HLA and two TLR complexes. This construct can therefore be used for pan-therapy against different strains of E. albertii and needs to be tested in vitro and in vivo.

Effect of trimethylamine N-oxide on inflammation and the gut microbiota in Helicobacter pylori-infected mice.

Diet is one of the factors contributing to symptom of Helicobacter pylori (H. pylori) infection. Trimethylamine N-oxide (TMAO), a diet-related microbial metabolite, is associated with inflammatory and metabolic diseases. The aim of this study is to investigate the effects of TMAO intake on inflammation and gut microbiota composition in H. pylori-infected mice via 16S rRNA sequencing and biochemical analyses. The in vitro experiments showed that TMAO not only increased the expression of growth- and metabolism-associated genes and the urease activity of H. pylori, but increased the production of virulence factors. Moreover, TMAO intake increased the production of inflammatory markers and reduced the richness and diversity of the gut microbiota in H. pylori-infected mice. Further analysis showed that TMAO increased the relative abundance of Escherichia_Shigella in H. pylori-infected mice, which had positive correlation with the levels of LPS, CRP, and CXCL1. Collectively, our results suggest that TMAO may aggravate H. pylori-induced inflammation by increasing the viability and virulence of H. pylori and may aggravate inflammation in association with the gut microbiota in H. pylori-infected mice. This study may provide a novel insight into the mechanism for the effect of diet-derived metabolites such as TMAO on H. pylori-induced disease development.

Protection against shigellosis caused by Shigella dysenteriae serotype 4 in guinea pigs using Escherichia albertii DM104 as a live vaccine candidate strain.

Recently, we reported the induction of protective immunity by environmental Escherichia albertii strain DM104 against Shigella dysenteriae in guinea pig model. In this study, we assessed three different immunization routes, such as intranasal, oral, and intrarectal routes, and revealed differences in immune responses by measuring both the serum IgG and mucosal IgA antibody titers. Protective efficacy of different routes of immunization was also determined by challenging immunized guinea pigs against live S. dysenteriae. It was found that intranasal immunization showed promising results in terms of antibody response and protective efficacy. All these results reconfirm our previous findings and additionally point out that the intranasal immunization of the environmental E. albertii strain DM104 in guinea pig model can be a better live vaccine candidate against shigellosis.

An environmental Escherichia albertii strain, DM104, induces protective immunity to Shigella dysenteriae in guinea pig eye model.

The environmental Escherichia albertii strain DM104, which cross-reacts serologically with Shigella dysenteriae was assessed for pathogenic properties, immunogenicity, and protective efficacy in different animal models to evaluate it as a vaccine candidate against S. dysenteriae, which causes the severe disease, shigellosis. The DM104 isolate was found to be non-invasive and did not produce any entero- or cyto-toxins. The strain also showed negative results in the mouse lethal activity assay. The non-pathogenic DM104 strain gave, however, a high protective efficacy as an ocularly administered vaccine in the guinea pig eye model against S. dysenteriae type 4 challenge. It also induced a high titer of serum IgG against S. dysenteriae type 4 whole cell lysate and lipopolysaccharide. Taken together, all these results indicate a good potential for the use of the DM104 as a live vaccine candidate against shigellosis.

Serological cross-reaction between O-antigens of Shigella dysenteriae type 4 and an environmental Escherichia albertii isolate.

An environmental freshwater bacterial isolate, DM104, appearing as Shigella-like colonies on selective agar plates was found to show strong and specific serological cross-reactivity with Shigella dysenteriae type 4. Biochemical identification according to the analytical profile index, molecular serotyping by restriction of the amplified O-antigen gene cluster (rfb-RFLP), together with phylogenetic analysis of the 16S rRNA gene and multi-locus sequence analysis, identified the isolate as Escherichia albertii. rfb-RFLP of DM104, revealed a profile different from that of S. dysenteriae type 4. However, western blot analysis of extracted lipopolysaccharides demonstrated strong cross-reactivity with S. dysenteriae type 4 using specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. dysenteriae type 4. The observed O-antigen cross-reaction between an E. albertii isolate and S. dysenteriae extends our knowledge of the extent of O-antigen cross-reaction within the Escherichia/Shigella group of organisms, and offers the possibility of using DM104 and similar cross-reacting strains as shigellosis vaccine candidates.

Evidence of interspecies O antigen gene cluster transfer between Shigella boydii 15 and Escherichia fergusonii.

An environmental bacterial isolate, Iso10, previously found to show serological cross-reactivity with type-specific Shigella boydii 15 antisera was subjected to further molecular and serological analyses that revealed interspecies transfer of the O antigen gene cluster. Western blot analysis of Iso10 cell surface extracts and purified lipopolysaccharides demonstrated strong cross-reactivity with S. boydii 15-specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. boydii 15. Biochemical and phylogenetic analyses identified the Iso10 isolate as Escherichia fergusonii. O antigen gene cluster analyses of Iso10, carried out by restriction fragment length analysis of the amplified ~10-kb O antigen-encoding gene cluster, revealed a profile highly similar to that of S. boydii 15, confirming the presence of the S. boydii 15 somatic antigen in Iso10. To the best of our knowledge, this is the first report of interspecies transfer of O antigen-encoding genes between S. boydii and E. fergusonii, and it has implications for our understanding of the role of lateral gene transfer in the emergence of novel Shigella serotypes.

Specific IgG activity against diarrheagenic bacteria in bovine immune milk and effect of pH on its antigen-binding activity upon heating.

Bovine colostrum and milk antibodies of calving and lactating cows immunized with a multivalent vaccine consisting of whole cells of three different species of pathogenic bacteria including four strains of enterotoxigenic Escherischia coli, five strains of enteropathogenic Esch. coli, three strains of enteroinvasive Esch. coli, two strains of Samonella typhi, and one strain each of Shigellia dysenteriae, Sh. sonnei and Sh. flexneri were generated, respectively. A significantly elevated activity and titre of specific IgG from bovine immune colostrum were seen for only 5 days after calving of immunized cows, however, the levels of specific IgG could be obtained continuously from the milk of immunized lactating cows until the 11th week of the entire experiment period. Subsequently, we observed that the high specific IgG activity in immune milk was relatively stable under pH 5.0-7.0 at 37 degrees C. Of importance, we identified that the specific IgG preserved its biological function for high antigen-binding activity at pH 5.5-6.5 for 30 min of heat treatment at 70 degrees C and for 350 s at 72 degrees C. Our findings suggest that the specific IgG from milk antibodies of immunized lactating cows may be used as an abundant source of hyper-immune products for prevention of multibacteria-induced diarrhea, however, the effect of pH on its antigen-binding activity upon heating should be carefully considered and designed.

Caspar, a suppressor of antibacterial immunity in Drosophila.

Drosophila has a primitive yet highly effective innate immune system. Although the infection-dependent activation mechanisms of the Drosophila immune system are well understood, its inhibitory regulation remains elusive. To find novel suppressors of the immune system, we performed a genetic screening for Drosophila mutants with hyperactivated immune responses and isolated a loss-of-function mutant of caspar whose product is homologous to Fas-associating factor 1 in mammals. Interestingly, caspar mutant flies showed increased antibacterial immune responses including increased resistance to bacterial infection and a constitutive expression of diptericin, a representative antibacterial peptide gene. Conversely, ectopic expression of caspar strongly suppressed the infection-dependent gene expression of diptericin, which allowed bacterial outgrowth. Consistent with these physiological phenotypes, Caspar negatively regulated the immune deficiency (Imd)-mediated immune responses by blocking nuclear translocation of Relish, an NF-kappaB transcription factor. In addition, we further demonstrated that Dredd-dependent cleavage of Relish, a prerequisite event for the nuclear entry of Relish, is the target of the Caspar-mediated suppression of the Imd pathway. Remarkably, Caspar was highly specific for the Imd pathway and did not affect the Toll pathway, which is crucial for antifungal immunity. Collectively, our elucidation of an inhibitory mechanism of the Imd pathway by Caspar will provide a valuable insight into understanding complex regulatory mechanisms of the innate immune systems in both Drosophila and mammals.

LTA and LPS mediated activation of protein kinases in the regulation of inflammatory cytokines expression in macrophages.

BACKGROUND: Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), the toxicants from bacteria, are potent inducers of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1). Although LTA is much less reported than that on LPS, LTA is regarded as the gram-positive equivalent to LPS in some aspects. We investigated the LTA-induced signal transduction and biological effects, as well as to compare the effect of LTA with that of LPS. METHODS: Kinase assay, ELISA and RT-PCR were performed to delineate LTA and LPS signaling as well as to determine the secretion and RNA expression of TNF and IL-1. RESULTS: Src, Lyn and MAPKs are involved in LTA and LPS signaling in murine macrophages. Additionally, blockades of PKC, PI3K and p38, respectively, caused significant inhibition of both LTA- and LPS-induced proIL-1/IL-1 and TNF expression. ERK inactivation moderately reduced LTA- and LPS-induced proIL-1/IL-1, but considerably reduced TNF expression. Inhibition of JNK engendered super-induction of IL-1 secretion, but diminished TNF secretion. Strikingly, both IL-1 and TNF protein induction were declined by overexpression of dominant negative form of JNK. CONCLUSIONS: The results clarify the similarity and difference between LTA- and LPS-mediated signal transduction and induction of inflammatory cytokines in macrophages.

Escherichia coli O157 somatic antigen is present in an isolate of E. fergusonii.

A bacterium that tested positive with antibodies specific for Escherichia coli O157 was isolated from beef during routine screening procedures. The bacterium was identified as E. fergusonii by biochemical testing and partial sequencing of 16S rRNA. The isolate was tested for the presence of genes encoding Shiga toxins, the E. coli attaching and effacing factor, enterohemolysin, and the O157 O antigen. The isolate tested negative for Shiga toxins and other E. coli O157 virulence markers but was found to harbor the genes encoding the O157 antigen. These results suggest genetic transfer of the O antigen gene cluster between E. coli O157:H7 and E. fergusonii.

[Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a].

Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.

Colicins produced by the Escherichia fergusonii strains closely resemble colicins encoded by Escherichia coli.

Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.

Analysis of enterohemorrhagic Escherichia coli serotype O157:H7 by flow cytometry using monoclonal antibodies.

To develop a rapid and specific method to detect and/or identify enterohemorrhagic Escherichia coli O157:H7, two mouse monoclonal antibodies (MAbs) were prepared. Specificities of these two MAbs (1D9 and 3E8) were determined by flow cytometry method (FCM). MAbs 3E8 and 1D9 were found to react with E. coli O157:H7, Citrobacter freundii and Salmonella group N (O:30), but not with Escherichia hermannii. With a mixture containing strains of E. coli O157:H7 and E. coli O6:H1, two different peaks appeared in FCM with MAbs, whereas a single peak appeared with polyclonal rabbit antiserum. From these findings, FCM with MAb is suggested to be a rapid, specific, and useful method to detect and identify strain(s) of E. coli O157:H7 in food ingredients.

Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia.

The antigenic relatedness of Escherichia coli O157 and four sorbitol-negative species of the genus Escherichia was examined. Isolates of Escherichia hermannii, E. fergusonii, E. vulneris, and E. blattae were tested in the tube agglutination assay by using polyclonal antisera and in the slide agglutination assay by using latex reagents. Only four isolates (17%) of E. hermannii exhibited serological cross-reactivity.

Escherichia hermannii (ATCC 33651) polysaccharide-protein conjugates: comparison of two conjugation methods for the induction of humoral responses in mice.

Escherichia hermannii (ATCC 33651) LPS O-polysaccharide was covalently linked to a carrier (bovine serum albumin) to form conjugates either directly or with a spacer arm (adipic acid dihydrazide). The immunogenicity of both conjugates at three different doses was tested in mice. Antibodies to the conjugate were produced and were shown to react with free lipopolysaccharide. The directly-coupled conjugate was found to be more immunogenic than the indirect one (i.e. lower dose necessary for a similar response). The antibody response elicited by the directly coupled conjugate (1 microgram/animal) began at 21 days and was sustained for at least 4 months. The mouse model described here may be applicable to the testing of other conjugates composed of bacterial cell wall polysaccharides and LPS O-chains.

Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus.

Clinical isolates of Escherichia hermannii which showed serological cross-reaction with polyclonal antisera to the O-polysaccharide portion of the lipopolysaccharide of E. coli O157 strains and with antisera to the O antigens of Brucella abortus and B. melitensis were found by chemical and nuclear magnetic resonance analyses to have lipopolysaccharide O chains composed of linear polymers containing 1,2- and 1,3-linked 4-acetamido-4,6-dideoxy-alpha-D-mannopyranosyl (alpha-D-Rhap4NAc) residues. Two O-antigen structures were identified; each had an unbranched pentasaccharide repeating unit, and one was composed of three 1,2- and two 1,3-linked alpha-D-Rhap4NAc residues and the other had two 1,2- and three 1,3-linked alpha-D-Rhap4NAc residues. The above-described cross-serological reactivities, which have led to false-positive identifications, are related to the common occurrence of epitopes involving the presence of N-acyl derivatives of 4-amino-4,6-dideoxy-D-mannopyranosyl residues in the O-polysaccharide portions of the respective lipopolysaccharides of the organisms. Strains of E. hermannii which did not show serological cross-reactions with E. coli O157 and Brucella antisera were found to have unique lipopolysaccharide O chains devoid of D-Rhap4NAc residues, demonstrating the existence of serotypes of E. hermannii that are distinct on the basis of their lipopolysaccharide components.

Immunological relationship among glyceraldehyde-3-phosphate dehydrogenases in the genera Enterobacter and Escherichia.

The comparative immunological study of glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) among Enterobacteriaceae carried out with an anti-Enterobacter cloacae G-3-PDH serum pointed out the large heterogeneity of the genera Enterobacter and Escherichia. The use of two-dimensional maps integrating our new data and previously acquired quantitative data confirmed these results.

[Development of Escherichia diagnostic O-test systems]. / Razrabotka diagnosticheskikh ésherikhioznykh O-test-sistem.

The principles of the rational development of new diagnostic preparations, Escherichia agglutinating O-test systems consisting of polyvalent reagents (reapols), were worked out, and two O-test systems, "small" and "large", were made up on the basis of these reagents (for the identification of 22 and 151 Escherichia serovars, respectively). The agglutinogenicity of Escherichia O-antigens was studied and found to be poorly pronounced in 40% of cases. The practical scheme for the identification of Escherichia O-antigens (key to the O-test system) has been made; the scheme permitted the determination of 84.8% of Escherichia O-test strains, only 29.1% of the strains showing due positive reactions. Ways for improving these preparations are presented.