Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin.

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy.

Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae.

Carbapenemase and extended ß-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.

Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-ß-d-Galf-(1→3)-ß-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F.

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: →4)-ß-D-Glcp-(1→3)-[[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-ß-D-Galp-(1→4)]-ß-L-Rhap-(1→. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted ß-Gal is linked to ß-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-ß-Gal to resonate in the α-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal α-Rha: →4)-ß-D-Glcp-(1→4)-[Gro-(2→P→3)]-ß-D-Galp-(1→4)-ß-L-Rhap-(1→. The immunodominant terminal α-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B.

Identification of Klebsiella capsule synthesis loci from whole genome data.

Klebsiella pneumoniae is a growing cause of healthcare-associated infections for which multi-drug resistance is a concern. Its polysaccharide capsule is a major virulence determinant and epidemiological marker. However, little is known about capsule epidemiology since serological typing is not widely accessible and many isolates are serologically non-typeable. Molecular typing techniques provide useful insights, but existing methods fail to take full advantage of the information in whole genome sequences. We investigated the diversity of the capsule synthesis loci (K-loci) among 2503 K. pneumoniae genomes. We incorporated analyses of full-length K-locus nucleotide sequences and also clustered protein-encoding sequences to identify, annotate and compare K-locus structures. We propose a standardized nomenclature for K-loci and present a curated reference database. A total of 134 distinct K-loci were identified, including 31 novel types. Comparative analyses indicated 508 unique protein-encoding gene clusters that appear to reassort via homologous recombination. Extensive intra- and inter-locus nucleotide diversity was detected among the wzi and wzc genes, indicating that current molecular typing schemes based on these genes are inadequate. As a solution, we introduce Kaptive, a novel software tool that automates the process of identifying K-loci based on full locus information extracted from whole genome sequences (https://github.com/katholt/Kaptive). This work highlights the extensive diversity of Klebsiella K-loci and the proteins that they encode. The nomenclature, reference database and novel typing method presented here will become essential resources for genomic surveillance and epidemiological investigations of this pathogen.

The diversity of Klebsiella pneumoniae surface polysaccharides.

Klebsiella pneumoniae is considered an urgent health concern due to the emergence of multi-drug-resistant strains for which vaccination offers a potential remedy. Vaccines based on surface polysaccharides are highly promising but need to address the high diversity of surface-exposed polysaccharides, synthesized as O-antigens (lipopolysaccharide, LPS) and K-antigens (capsule polysaccharide, CPS), present in K. pneumoniae. We present a comprehensive and clinically relevant study of the diversity of O- and K-antigen biosynthesis gene clusters across a global collection of over 500 K. pneumoniae whole-genome sequences and the seroepidemiology of human isolates from different infection types. Our study defines the genetic diversity of O- and K-antigen biosynthesis cluster sequences across this collection, identifying sequences for known serotypes as well as identifying novel LPS and CPS gene clusters found in circulating contemporary isolates. Serotypes O1, O2 and O3 were most prevalent in our sample set, accounting for approximately 80 % of all infections. In contrast, K serotypes showed an order of magnitude higher diversity and differ among infection types. In addition we investigated a potential association of O or K serotypes with phylogenetic lineage, infection type and the presence of known virulence genes. K1 and K2 serotypes, which are associated with hypervirulent K. pneumoniae, were associated with a higher abundance of virulence genes and more diverse O serotypes compared to other common K serotypes.

Iron Modulates Butyrate Production by a Child Gut Microbiota In Vitro.

UNLABELLED: The aim of this study was to investigate the effect of iron (Fe) availability on butyrate production in the complex bacterial ecosystem of the human gut. Hence, different Fe availabilities were mimicked in an in vitro colonic fermentation model (the polyfermenter intestinal model called PolyFermS) inoculated with immobilized gut microbiota from a child and in batch cultures of the butyrate producer Roseburia intestinalis. Shifts in the microbial community (16S rRNA sequencing and quantitative PCR), metabolic activity (high-performance liquid chromatography), and expression of genes involved in butyrate production were assessed. In the PolyFermS, moderate Fe deficiency resulted in a 1.4-fold increase in butyrate production and a 5-fold increase in butyryl-coenzyme A (CoA):acetate CoA-transferase gene expression, while very strong Fe deficiency significantly decreased butyrate concentrations and butyrate-producing bacteria compared with the results under normal Fe conditions. Batch cultures of R. intestinalis grown in a low-Fe environment preferentially produced lactate and had reduced butyrate and hydrogen production, in parallel with upregulation of the lactate dehydrogenase gene and downregulation of the pyruvate:ferredoxin-oxidoreductase gene. In contrast, under high-Fe conditions, R. intestinalis cultures showed enhanced butyrate and hydrogen production, along with increased expression of the corresponding genes, compared with the results under normal-Fe conditions. Our data reveal the strong regulatory effect of Fe on gut microbiota butyrate producers and on the concentrations of butyrate, which contributes to the maintenance of host gut health. IMPORTANCE: Fe deficiency is one of the most common nutritional deficiencies worldwide and can be corrected by Fe supplementation. In this in vitro study, we show that environmental Fe concentrations in a continuous gut fermentation model closely mimicking a child's gut microbiota strongly affect the composition of the gut microbiome and its metabolic activity, particularly butyrate production. The differential expression of genes involved in the butyrate production pathway under different Fe conditions and the enzyme cofactor role of Fe explain the observed modulation of butyrate production. Our data reveal that the level of dietary Fe reaching the colon affects the microbiome, and its essential function of providing the host with beneficial butyrate.

Complete Genome Sequence of the Probiotic Bifidobacterium thermophilum Strain RBL67.

Bifidobacterium thermophilum RBL67, an isolate from infant feces, exhibits bacteriocin-like antimicrobial activity against Listeria spp. and Salmonella spp. and protects HT29-MTX cells against Salmonella infection. Here, the complete genome sequence of the probiotic B. thermophilum strain RBL67 is presented.

Comparative genome analysis of Streptococcus infantarius subsp. infantarius CJ18, an African fermented camel milk isolate with adaptations to dairy environment.

BACKGROUND: Streptococcus infantarius subsp. infantarius (Sii) belongs to the Streptococcus bovis/Streptococcus equinus complex associated with several human and animal infections. Sii is a predominant bacterium in spontaneously fermented milk products in Africa. The genome sequence of Sii strain CJ18 was compared with that of other Streptococcus species to identify dairy adaptations including genome decay such as in Streptococcus thermophilus, traits for its competitiveness in spontaneous milk fermentation and to assess potential health risks for consumers. RESULTS: The genome of Sii CJ18 harbors several unique regions in comparison to Sii ATCC BAA-102T, among others an enlarged exo- and capsular polysaccharide operon; Streptococcus thermophilus-associated genes; a region containing metabolic and hypothetical genes mostly unique to CJ18 and the dairy isolate Streptococcus gallolyticus subsp. macedonicus; and a second oligopeptide transport operon. Dairy adaptations in CJ18 are reflected by a high percentage of pseudogenes (4.9%) representing genome decay which includes the inactivation of the lactose phosphotransferase system (lacIIABC) by multiple transposases integration. The presence of lacS and lacZ genes is the major dairy adaptation affecting lactose metabolism pathways also due to the disruption of lacIIABC.We constructed mutant strains of lacS, lacZ and lacIIABC and analyzed the resulting strains of CJ18 to confirm the redirection of lactose metabolism via LacS and LacZ.Natural competence genes are conserved in both Sii strains, but CJ18 contains a lower number of CRISPR spacers which indicates a reduced defense capability against alien DNA. No classical streptococcal virulence factors were detected in both Sii strains apart from those involved in adhesion which should be considered niche factors. Sii-specific virulence factors are not described. Several Sii-specific regions encoding uncharacterized proteins provide new leads for virulence analyses and investigation of the unclear association of dairy and clinical Sii with human diseases. CONCLUSIONS: The genome of the African dairy isolate Sii CJ18 clearly differs from the human isolate ATCC BAA-102T. CJ18 possesses a high natural competence predisposition likely explaining the enlarged genome. Metabolic adaptations to the dairy environment are evident and especially lactose uptake corresponds to S. thermophilus. Genome decay is not as advanced as in S. thermophilus (10-19%) possibly due to a shorter history in dairy fermentations.

Metabolism of oligosaccharides and starch in lactobacilli: a review.

Oligosaccharides, compounds that are composed of 2-10 monosaccharide residues, are major carbohydrate sources in habitats populated by lactobacilli. Moreover, oligosaccharide metabolism is essential for ecological fitness of lactobacilli. Disaccharide metabolism by lactobacilli is well understood; however, few data on the metabolism of higher oligosaccharides are available. Research on the ecology of intestinal microbiota as well as the commercial application of prebiotics has shifted the interest from (digestible) disaccharides to (indigestible) higher oligosaccharides. This review provides an overview on oligosaccharide metabolism in lactobacilli. Emphasis is placed on maltodextrins, isomalto-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, and raffinose-family oligosaccharides. Starch is also considered. Metabolism is discussed on the basis of metabolic studies related to oligosaccharide metabolism, information on the cellular location and substrate specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, and the presence of metabolic genes in genomes of 38 strains of lactobacilli. Metabolic pathways for disaccharide metabolism often also enable the metabolism of tri- and tetrasaccharides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. This general restriction to intracellular glycosyl hydrolases differentiates lactobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp.

Reactive oxygen species-inducible ECF σ factors of Bradyrhizobium japonicum.

Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H(2)O(2)-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H(2)O(2)-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5' ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H(2)O(2) stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.

Complete genome sequence of the African dairy isolate Streptococcus infantarius subsp. infantarius strain CJ18.

Streptococcus infantarius subsp. infantarius, a member of the Streptococcus bovis/Streptococcus equinus complex, is highly prevalent in artisanal dairy fermentations in Africa. Here the complete genome sequence of the dairy-adapted S. infantarius subsp. infantarius CJ18 strain--a strain predominant in traditionally fermented camel milk (suusac) from Kenya--is presented.