Immuno-informatics driven proteome-wide investigation revealed novel peptide-based vaccine targets against emerging multiple drug resistant Providencia stuartii.

The bacterium Providencia stuartii, is associated with urinary tract infections and is the most common cause of purple urine bag syndrome. The increasing multi-drug resistance pattern shown by the pathogen and lack of licensed vaccines make treatment of infections caused by P. stuartii challenging. As vaccinology data against the pathogen is scarce, an in silico proteome based Reverse Vaccinology (RV) protocol, in combination with subtractive proteomics is introduced in this work to screen potential vaccine candidates against P. stuartii. The analysis identified three potential vaccine candidates for designing broad-spectrum and strain-specific peptide vaccines: FimD4, FimD6, and FimD8. These proteins are essential for pathogen survival, localized in the outer membrane, virulent, and antigenic in nature. Immunoproteomic tools mapped surface exposed and non-allergenic 9mer B-cell derived T-cell antigenic epitopes for the proteins. The epitopes also show stable and rich interactions with the most predominant HLA allele (DRB1*0101) in the human population. Metabolic pathway annotation of the proteins indicated that fimbrial biogenesis outer membrane usher protein (FimD6) is the most suitable candidate for vaccine design, due to its involvement in several significant pathways. These pathways include: the bacterial secretion system, two-component system, ß-lactam resistance, and cationic antimicrobial peptide pathways. The predicted epitopes may provide a basis for designing a peptide-based vaccine against P. stuartii.

The complex contributions of genetics and nutrition to immunity in Drosophila melanogaster.

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and "nutritional immunology" has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional "immune system" that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.

Structural, serological, and genetic characterization of the O-antigen of Providencia alcalifaciens O40.

The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia. In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-ß-D-Quip3NFo-(1→3)-α-D-Galp-(1→3)-ß-D-GlcpA-(1→3)-ß-D-GalpNAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico. In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-D-Qui3NFo, UDP-D-Gal, UDP-D-GlcA, and UDP-D-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb, and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called K(LPS).

Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45.

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: →4)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpNAc3(Rlac-L-Ala)-(1→. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60.

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as (1)H and (13)C NMR spectroscopy, including 2D ROESY and (1)H,(13)C HMBC experiments in D(2)O and a ROESY experiment in a 9:1 H(2)O-D(2)O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure: The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.

Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria.

Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.

The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides.

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O57 containing an amide of D-galacturonic acid with L-alanine.

The O-polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O57:H29. Studies by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments, showed that the polysaccharide contains an amide of D-galacturonic acid with L-alanine and has the following pentasaccharide repeating unit: [formula: see text]

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51. Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: [carbohydrate structure: see text]

Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose.

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.

Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens O23.

INTRODUCTION: The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide--OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp. MATERIALS AND METHODS: LPS and alkali-treated LPS of P. alcalifaciens O23 and serologically related P. rustigianii O14, P. mirabilis O13 and P. myxofaciens as well as O-antiserum against P. alcalifaciens O23 were used. Serological characterization of P. alcalifaciens O23 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot. RESULTS AND CONCLUSIONS: The OPS of P. alcalifaciens, O23, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-L-lysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, O23, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifiaciens O23 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens O23 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of O23-specific antibodies.

Structure and cross-reactivity of the O-antigen of Providencia stuartii O18 containing 3-acetamido-3,6-dideoxy-D-glucose.

The O-polysaccharide (O-antigen) of Providencia stuartii O18 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose. Anti-P. stuartii O18 serum cross-reacted with the O-antigen of Proteus genomospecies 4, which could be accounted for the marked structural similarities of the main chain.

Providencia alcalifaciens strains translocate from the gastrointestinal tract and are resistant to lytic activity of serum complement.

The ability of Providencia alcalifaciens strains, isolated from patients with diarrhoeal disease, to translocate from the gastrointestinal tract and their resistance to serum complement lytic activity were investigated and compared with previously characterized differential invasive capabilities in HeLa cells. Translocation ability to several extraintestinal sites and resistance to lysis by human serum complement were observed in both highly invasive and non-invasive strains. These characteristics have not been previously described in P. alcalifaciens and their potential role in causing disseminated infections should therefore be considered.

Invasion of HEp-2 and other eukaryotic cell lines by Providenciae: further evidence supporting the role of Providencia alcalifaciens in bacterial gastroenteritis.

Wild-type strains of Providencia species were evaluated for their ability to invade HEp-2 monolayers based upon microscopic and semi-quantitative assays. Of 14 P. alcalifaciens strains tested, 3 (17%) were found to be highly invasive, 4 (22%) moderately invasive, and the remaining 61% weakly or noninvasive. HEp-2 invasion results were confirmed by thin-section electron microscopy. Invasive capabilities of P. alcalifaciens were greater at higher MOIs (100 to 1000) than at lower inocula (<10 MOI). No strain of P. stuartii or P. rettgeri tested invaded HEp-2 cells. Quantitative assays of Triton X-100-lysed, HEp-2-invaded cells indicated that between 0.001% and 0. 013% of the initial bacterial inoculum was gentamicin resistant. Further testing of select strains on various cell lines indicated the efficiency of invasion was Vero > Y1 > INT-407 > HEp-2. Two isolates recovered from a father and son with prolonged diarrhea after returning from Mexico were found to be identical on the basis of biotype, serotype, and genotype. These results provide additional evidence that some P. alcalifaciens strains cause gastroenteritis.

Structure of the O-specific polysaccharide of the bacterium Providencia alcalifaciens O23 containing a novel component: an amide of D-glucuronic acid with N(epsilon)-(1-carboxyethyl)lysine.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O23 and found to contain D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and N epsilon-(1-carboxyethyl)-N alpha-(D-glucuronoyl)lysine. On the basis of full and partial acid hydrolyses, selective solvolysis with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H, 13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), the following structure of the liner tetrasaccharide repeating unit of the polysaccharide was established [structure: see text]

Bactericidal activity of normal human serum against Morganella, Proteus, and Providencia strains.

The susceptibility od Proteus, Providencia, and Morganella strains to bactericidal action of human serum was examined. The percentage of survival was determined after one and three hours incubation with 50% human serum. The susceptible strains were treated by serum preparations with blocked classical or alternative complement activation pathways as well as with lysozyme removed. Following mechanisms of the bactericidal action of serum were found: complement activated by the classical or alternative pathway with participation of lysozyme, complement activated simultaneously via both pathways-while the participation of lysozyme was necessary for killing some strains and superfluos for others and complement activated only via the classical pathway without lysozyme.

[Susceptibility of Proteus, Morganella and Providencia strains isolated from patients with urinary tract infections on bactericidal activity of normal human serum]. / Podatnosc szczepów Proteus, Morganella i Providencia izolowanych od chorych z zakazeniami ukladu moczowego na bakteriobójcze dzialanie normalnej surowicy ludzkiej.

Usefulness of the test determining bactericidal activity of normal human serum was investigated with 50 strains of Proteus, Morganella and Providencia isolated from patients with urinary tract infections (UTI) and with 50 strains isolated from feces. It was found that strains from UTI were more frequently resistant to the action of normal human serum (50% resistant) in comparison with strains isolated from feces (30% resistant). Strains of Proteus belonging to four species were differing greatly in their susceptibility to normal human serum. They can be ranked as followings: P. mirabilis (49% of resistant strains), P. vulgaris (62%), P. morganii (72%) and P. rettgeri (100%). In studies on interaction subinhibitory concentrations of cefotaxime and normal human serum in bactericidal reaction, a synergism was found only with some strains.

Asymptomatic bacteriuria, urinary antibody, and survival in the institutionalized elderly.

OBJECTIVE: To compare clinical status of elderly institutionalized subjects with asymptomatic bacteriuria and normal urinary antibody to those with elevated urinary antibody to the major outer membrane of Escherichia coli. DESIGN: Retrospective review. SETTING: Long term facility for the elderly. PARTICIPANTS: Convenience sample of 63 elderly subjects, 26% of those resident in the institution, aged 78.8 +/- 8.4 years with urine specimens collected and stored in 1987. MAIN OUTCOME MEASURES: Differences in clinical or functional status, demographic features, and outcome during 3-years follow-up between bacteriuric subjects with normal and elevated urine antibody. RESULTS: Thirteen subjects had no bacteriuria, and 12 had infrequent, intermittent bacteriuria; 38 (60%) had persistent bacteriuria, including four with frequent, intermittent infections. In the persistently bacteriuric group, 18 (47%) had persistently elevated urine antibody. There was no significant differences between bacteriuric residents with normal vs elevated urine antibody in clinical or functional status, age, duration of residence, or infecting organisms. However, 11 of 20 in the cohort with normal urine antibody were alive at 3 years compared to 3 of 18 with elevated urine antibody (P = 0.014). CONCLUSIONS: Elderly institutionalized subjects with persistent bacteriuria and elevated urine antibody have decreased survival compared to those with normal urine antibody. No differences in underlying illness or clinical course to explain this survival difference were identified.