O4 Antigen Research Articles

Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways, to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multi-module proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain-length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid (Kdo)-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137), and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.

The protective capacity of anti-O4 antigen antibodies against Salmonella infection is influenced by the presence or absence of the O5 antigen

Anti-O-antigen antibodies, such as anti-O4 antigen IgG, induce protective immunity against Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. S. Typhimurium belongs to the group O4, which can be classified into two serological variants, namely factor O5 antigen positive (O5+) and factor O5 antigen negative (O5−). In this study, we determined the protective immunity induced by anti-O4 antigen IgG against O5+ and O5− S. Typhimurium infection in a mouse model. Unexpectedly, anti-O4 antigen IgG induced protection against O5− of S. Typhimurium, but not against O5+ of S. Typhimurium. We suggest that the affinity of the O4 antigen with anti-O4 antigen IgG is stronger in the O5− S. Typhimurium compared to the O5+ S. Typhimurium. Although anti-O4 antigen IgG has the potential to protect against S. Typhimurium infection, the effects of anti-O4 antigen IgG in protection against Salmonella infection differ depending on the presence or absence of the O5 antigen.

Issue Cover (August 2017)

Front cover: Rat spinal cord oligodendrocytes differentiating in vitro. A pure oligodendroglial lineage cell (OLC) culture from the rat neonatal spinal cord is stained for oligodendrocyte markers, O4 antigen (red) and galactocerebroside (green) by immunocytochemistry. Nuclei are stained with DAPI (Blue). By comparing pure oligodendrocyte cultures from rat spinal cords and forebrains, we demonstrated differing intrinsic biological properties between these two OLC groups originated from distinct CNS regions. Further understanding the regional differences of oligodendrocytes will help to improve treatments for demyelination in different CNS regions and to facilitate the development of stem cell-derived OLCs for cell transplantation therapies for demyelination. Read the full article ‘Differing intrinsic biological properties between forebrain and spinal oligodendroglial lineage cells’ by M. Horiuchi, Y. Suzuki-Horiuchi, T. Akiyama, A. Itoh, D. Pleasure, E. Carstens and T. Itoh (J. Neurochem. 2017, vol. 142 (3), pp. 378–391) on doi: 10.1111/jnc.14074

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.

Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration

We demonstrate that there are significantly more p75 neurotrophin receptor- (NTR)-expressing cells in olfactory ensheathing cell (OEC) primary cultures from olfactory nerve rootlets (ONR), but a greater proportion of O4 antigen- and PSA-NCAM-expressing cells in parallel cultures from the nerve fibre layer of the olfactory bulb (OB). By co-culturing adult rat retinal ganglion cells (RGCs) with OECs derived from either ONR or OB tissue, we compared their neurite regrowth-promoting properties. In phenotypically unsorted cultures, there is greater RGC neurite regrowth on ONR OECs compared to OB OECs. Following immunoselection of ONR cells for p75 NTR, there is increased RGC neurite regrowth on the enriched population compared to the unselected cell population or the p75 NTR depleted population. When p75 NTR-enriched cells from ONR and OB cultures are compared directly, tissue source-related differences are no longer observed. Our previous work implicated a pertussis toxin (PTx)-sensitive G protein-linked signalling pathway in OEC regulation of neurite regrowth. We show that this pathway probably operates in interactions between the p75 NTR-positive and -negative cells; separated populations lose the PTx-mediated enhancement of neurite regrowth-promoting properties seen in mixed cultures. Optimum neurite regrowth is observed when both phenotypes are present in cultures from either ONR or OB, and where glial G-protein signalling is disabled by PTx before co-culture with neurons. We thus propose that p75 NTR-positive cells, whilst being the more effective neurite regrowth promoting subpopulation in isolation, cooperate with negative cells to provide optimum support for axonal regrowth.

MICROGLIA AS A STEM CELL

Microglia is considered the only cell population of mesodermal origin, which comprises 10% of the cells in brain parenchyma. Recent neural stem cell (NSC) studies demonstrate that the brain has regenerative potential. NSCs do not give rise to microglial cells, however indicating that NSCs alone cannot complete the regenetion of the brain. Although the role of microglia is not fully understood, the recent study demonstrated that rat primary microglia cell expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. In our study, in purified primary cell culture microglia cells could be differentiated into other glial and neural cells.

Microglia, a potential source of neurons, astrocytes, and oligodendrocytes.

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 days, followed by culture in the presence of 70% serum for 2 days. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labeled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes.

Leydig cells of the human testis possess astrocyte and oligodendrocyte marker molecules

It has been established, that Leydig cells of the human testis possess neuroendocrine properties and are therefore a member of the diffuse neuroendocrine (paraneuron) system. In the present study, we examined whether Leydig cells of adult (51-86 year of age) and developing (between the 15th and 36th week of gestation) human testes are immunopositive for glial cell-specific antigens such as glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), cyclic 2',3'-nucleotide-3'-phosphodiesterase (CNPase), A2B5-antigen (A2B5) and O4-antigen (O4). With the use of Western blots and dot blot analyses, respectively, GFAP, CNPase, GalC, A2B5 and O4 were found in whole testes and Leydig cell protein extracts of adult men. Corresponding immunohistochemical studies revealed presence of these antigens in the cytoplasm of Leydig cells both of adult testes and testes during prenatal development. Some differences in staining intensity of single antigens were observed probably depending on the functional and/or developmental stage of the single cells. In addition, GFAP-, GalC- and CNPase-immunopositivity was found in numerous Sertoli cells of the seminiferous tubules. Moreover, some connective tissue cells (compartmentalizing cells or Co-cells) of the intertubular space showed immunopositivity for CNPase, A2B5 and GalC. The results obtained show that Leydig cells of the human testis, in addition to their endocrine, neuronal and neuroendocrine features, possess qualities of both astrocytes and oligodendrocytes and thus show qualities of multipotential cells. Leydig cells probably differentiate to a phenotype that is characteristic for cells in the developing nervous system. Furthermore, the established immunohistochemical similarities are consistent with the assumption that foetal and postnatal Leydig cells are of common origin.

Selective Expression of Nkx-2.2 Transcription Factor in Chicken Oligodendrocyte Progenitors and Implications for the Embryonic Origin of Oligodendrocytes

Recent studies have demonstrated that oligodendrocytes originate from the ventral region of the developing spinal cord. However, the precise neuroepithelial origin of oligodendrocytes remains controversial, and the transcriptional control of oligodendrocyte lineage specification is largely unknown. Here we present evidence that oligodendrocytes in the embryonic chicken spinal cord can be generated from neuroepithelial cells that express the Nkx-2.2 homeodomain transcription factor. Nkx-2.2 expression is initially confined to a narrow stripe of neuroepithelium flanking the floor plate. Later, Nkx-2.2+ cells migrate ventrally and dorsolaterally into the surrounding gray and white matter regions where they undergo rapid proliferation. Double labeling experiments revealed that Nkx-2.2+ cells coexpress markers specific for oligodendrocyte progenitors, e.g., PDGFRα+, O4, and R-mAb antigens. In the brain, the Nkx-2.2 cells are also highly migratory and can generate oligodendrocytes. The persistent expression of the Nkx-2.2 homeodomain transcription factor in the oligodendrocyte lineage suggests its important role in the control of oligodendrocyte development.

Schwann-like macroglia in adult rat brain.

Olfactory ensheathing cells (OECs) share properties with astrocytes and Schwann cells. This study was designed to test the hypothesis that glia with properties similar to those exhibited by OECs might be present in brain areas other than the olfactory bulb. We found tanycytes and pituicytes to express a distinctive set of immunological markers in common with OECs and nonmyelinating Schwann cells, namely low-affinity neurotrophin receptor (p75NTR), O4 antigen, estrogen receptor-alpha type, and insulin-like growth factor 1 (IGF-1). The two glial types could be cultured from adult hypothalamus and neurohypophysis, respectively, using the methods developed for olfactory OECs. Both glial types displayed morphologies reminiscent of Schwann cells, in primary culture. Schwann-like central glia presented a preferred growth substrate for dorsal root ganglion neurites and, when making intimate contacts with them, manifested a myelinating phenotype. These combined properties define a type of CNS macroglia that would not fit within conventional central glia types.

Schwann cell development in embryonic mouse nerves

Previously we proposed that Schwann cell development from the neural crest is a two-step process that involves the generation of one main intermediate cell type, the Schwann cell precursor. Until now Schwann cell precursors have only been identified in the rat, and much remains to be learned about these cells and how they generate Schwann cells. Here we identify this cell in the mouse and analyze its transition to form Schwann cells in terms of timing, molecular expression, and extracellular signals and intracellular pathways involved in survival, proliferation, and differentiation. In the mouse, the transition from precursors to Schwann cells takes place 2 days earlier than in the rat, i.e., between embryo days 12/13 and 15/16, and is accompanied by the appearance of the O4 antigen and the establishment of an autocrine survival circuit. Beta neuregulins block precursor apoptosis and support Schwann cell generation in vitro, a process that is accelerated by basic fibroblast growth factor 2. The development of Schwann cells from precursors also involves a change in the intracellular survival signals utilized by neuregulins: To block precursor death neuregulins need to signal through both the mitogen-activated protein kinase and the phosphoinositide-3-kinase pathways although neuregulins support Schwann cell survival by signaling through the phosphoinositide-3-kinase pathway alone. Last, we describe the generation of precursor cultures from single 12-day-old embryos, a prerequisite for culture studies of genetically altered precursors when embryos are non-identical with respect to the transgene in question. J. Neurosci. Res. 56:334–348, 1999. © 1999 Wiley-Liss, Inc.

Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster.

The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigí, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581-7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5alpha by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster.

Monoclonal antibody 14F7, which recognizes a stage-specific immature oligodendrocyte surface molecule, inhibits oligodendrocyte differentiation mediated in co-culture with astrocytes.

Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17-26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5- cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development.

Monoclonal antibody 14F7, which recognizes a stage‐specific immature oligodendrocyte surface molecule, inhibits oligodendrocyte differentiation mediated in co‐culture with astrocytes

Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17–26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5− cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development. J. Neurosci. Res. 54:79–96, 1998. © 1998 Wiley-Liss, Inc.

Cells of the oligodendrocyte lineage proliferate following cortical stab wounds: an in vitro analysis.

We have previously shown that a cortical stab wound induces the proliferation of microglia and astrocytes in situ, but no evidence was obtained for proliferation of cells of the oligodendrocyte lineage (Amat et al., 1996). To study further the properties of cells involved in repair following brain injury, groups of adult rats received either sham operations or bilateral stab wounds. Proliferating cells were labeled in vivo 3 days later with [3H]-thymidine (Thy) and sacrificed the same day. Oligodendrocyte-enriched preparations were isolated, cultured, and analyzed. The fate and antigenic phenotype of the proliferating cells was analyzed using three-color immunofluorescence combined with autoradiography at 1, 2, 3, 5, and 10 days in vitro (DIV). Cells were immunostained for ganglioside GD3 (glial stem cells), O4 antigen (cells of the oligodendrocyte lineage), galactosyl ceramide (GC, differentiated oligodendrocytes), and GFAP (astrocytes). Thymidine-labeled O4+/GC- cells were found only in cultures from wounded animals and most of them differentiated in vitro as mature oligodendrocytes, but no Thy+/O4+/GC+ oligodendrocytes were seen at 1, 2, or 3 DIV. There was also a marked increase in the number of Thy+/GD3+ cells in the experimental cultures. In both experimental and control groups the total number of Thy+ and Thy- GD3+ cells declined with time in culture concomitant with an increase in total number of both Thy+ and Thy- GFAP+ astrocytes, and without any significant change in the Thy+ cell fraction of O4+ oligodendrocytes in the experimental cultures. Therefore most of the GD3+/O4- cells apparently differentiated as GFAP+ astrocytes, not as oligodendrocytes. We conclude that O4+/GC- oligodendrocyte precursor cells, but not differentiated oligodendrocytes, proliferate in response to brain injury. These cells proliferate slowly or not at all in normal adult animals and constitute a phenotypically and kinetically distinct group from the GD3+ glial precursors. This result is consistent with the existence within the adult CNS of a quiescent premyelinating oligodendrocyte. We propose that these immature committed oligodendrocytes are induced to proliferate at the wound site and serve as a source of new oligodendrocytes.

The O4 specific antigen moiety of lipopolysaccharide but not the K54 group 2 capsule is important for urovirulence of an extraintestinal isolate of Escherichia coli

Group 2 capsules and lipopolysaccharides are regarded as important virulence factors in extraintestinal isolates of Escherichia coli, but their specific contributions to bladder and renal infections, if any, are unknown. Proven isogenic derivatives deficient in the K54 antigen alone (CP9.137), the O4 antigen alone (CP921), or both the K54 and O4 antigens (CP923) were compared with their wild-type parent (CP9 [O4/K54JH5]) for growth in human urine in vitro and for virulence in vivo in a mouse model of ascending urinary tract infection (UTI). Growth of CP9.137 and CP921 was equivalent to that of CP9 in human urine. CP923 demonstrated a small but reproducible decrease in log-phase growth but achieved the same plateau density. In the mouse model of UTI, the isogenic mutant deficient in the 04 antigen alone (CP921) and, to a greater degree, the derivative deficient in both the K54 and O4 antigens (CP923) were significantly less virulent in nearly all parameters measured. In contrast, the K54 knockout derivative was as virulent as its parent, CP9, in causing bladder infection and nearly as virulent in causing renal infection. These results demonstrate an important role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of UTI. The possibility that the K54 antigen also plays a minor role cannot be excluded.

In vitro differentiation of embryonic stem cells into glial cells and functional neurons.

Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyte- or oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltage-dependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.

Loss of the O4 antigen moiety from the lipopolysaccharide of an extraintestinal isolate of Escherichia coli has only minor effects on serum sensitivity and virulence in vivo.

The O-specific antigen in extraintestinal isolates of Escherichia coli is believed to be an important virulence factor. To assess its role in the pathogenic process, proven isogenic derivatives with either a complete (CP921) or nearly complete (CP920) deficiency of the O4 antigen were obtained by TnphoA'1-mediated transposon mutagenesis of an O4/K54/H5 blood isolate (CP9). By utilizing a previously reported isogenic K54 capsule-deficient derivative (CP9.137), additional isogenic derivatives deficient in both the K54 capsular antigen and either all (CP923) or nearly all (CP922) of the O4 antigen were also constructed. These strains and their wild-type parent were evaluated in vitro for serum sensitivity and in vivo by intraperitoneal challenge of outbred mice. The complete or nearly complete loss of the O4 antigen (CP920 and CP921) resulted in only a minor increase in serum sensitivity. In contrast, CP9.137 had a significant increase in serum sensitivity, and CP922 and CP923 were extremely serum sensitive. When tested in vivo, the complete or nearly complete loss of the O4 antigen resulted in a small but significant increase (P < or = 0.05), not the expected decrease, in virulence compared with its wild-type parent. In contrast, CP9.137 and CP922 were significantly less virulent (P < or = 0.05). These studies do not exclude a role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of extraintestinal E. coli infection; however, they demonstrate that the O4 antigen plays only a minor role in serum resistance in vitro and that its loss does not diminish and perhaps enhances the virulence of CP9 in vivo after intraperitoneal challenge.

The construction and characterization of colanic aciddeficient mutants in an extraintestinal isolate of Escherichia coli (O4/K54/H5)

Extraintestinal strains of Escherichia coli possess a variety of virulence factors that enablethem to cause disease. These strains express a group 2 capsular polysaccharide which is important in the pathogenic process. Extraintestinal strains evaluated to date are also capable of producing the group 1 capsular polysaccharide colanic acid. The blood isolate CP9 (O4/K54/H5) constitutively produces the group 2, K54 capsule but can be induced to produce colanic acid. In this report we assess whether colanic acid contributes to the pathogenesis of this extraintestinal pathogen. CP9 and its derivatives that are deficient in their ability to produce colanic acid (TR94), the K54 group 2 capsule ± colanic acid (CP9.137, TR1374) and the 04 specific antigen ± colanic acid (CP921,CP925) were used to test whether the group 1 capsule colanic acid conferred protection against the bactericidal effects of serum and recombinant bactericidal/permeability-increasing protein (rBPI-23) in vitro. Additionally, CP9, CP9.137 and TR94 were evaluated in the rat granuloma pouch, an in vivo model for localized infection, and by intraperitoneal inoculation into mice, a systemic infection model. In summary, the inability of CP9 to produce colanic acid in the presence or absence of its K54 and O4 antigens had no effect on its ability to survive these host defenses in vitro and did not affect its virulence in these two in vivo models of infection.