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IgA nephropathy is a kidney disease characterized by deposition of immune complexes containing abnormally O -glycosylated IgA1 in the glomeruli. Specifically, some O -glycans are missing galactose that is normally β1,3-linked to N -acetylgalactosamine of the core 1 glycans. These galactose-deficient IgA1 glycoforms are produced by IgA1-secreting cells due to a dysregulated expression and activity of several glycosyltransferases. Galactose-deficient IgA1 in the circulation of patients with IgA nephropathy is bound by IgG autoantibodies and the resultant immune complexes can contain additional proteins, such as complement C3. These complexes, if not removed from the circulation, can enter the glomerular mesangium, activate the resident mesangial cells, and induce glomerular injury. In this review, we briefly summarize clinical and pathological features of IgA nephropathy, review normal and aberrant IgA1 O -glycosylation pathways, and discuss the origins and potential significance of natural anti-glycan antibodies, namely those recognizing N -acetylgalactosamine. We also discuss the features of autoantibodies specific for galactose-deficient IgA1 and the characteristics of pathogenic immune complexes containing IgA1 and IgG. In IgA nephropathy, kidneys are injured by IgA1-containing immune complexes as innocent bystanders. Most patients with IgA nephropathy progress to kidney failure and require dialysis or transplantation. Moreover, most patients after transplantation experience a recurrent disease. Thus, a better understanding of the pathogenetic mechanisms is needed to develop new disease-specific treatments. [ABSTRACT FROM AUTHOR]

A spontaneous mutant of Streptococcus pneumoniae strain D39 exhibiting elevated [beta]-galactosidase activity was identified. We determined that the [beta]-galactosidase activity was due to BgaA, a surface protein in S. pneumoniae, and that the expression of bgaA was regulated. Transcription analyses demonstrated expression of bgaA in the constitutive [beta]-galactosidase ([BgaA.sup.C]) mutant, but not in the parent. [beta]-Galactosidase expression was induced in the parent under specific growth conditions; however, the levels did not reach those of the [BgaA.sup.C] mutant. We localized the mutation resulting in the [BgaA.sup.C] phenotype to a region upstream of bgaA and in the promoter of a phosphoenolpyruvate-dependent phosphotransferase system (PTS) operon. The mutation was in a catabolite-responsive element (cre) and affected the binding of CcpA (catabolite control protein A), a key regulator of many carbon metabolism genes. The pts operon and bgaA were cotranscribed, and their transcription was regulated by CcpA. Deletion of ccpA altered [beta]-galactosidase activity, leading to a sevenfold increase in the parent but a fivefold decrease in the [BgaA.sup.C] mutant. The resulting [beta]-galactosidase activities were the same in the two strains, suggesting the presence of a second repressor. The presence of glucose in the growth medium resulted in pts-bgaA repression by both CcpA and the second repressor, with the latter being important in responding to the glucose concentration. Expression of [beta]-galactosidase is important for S. pneumoniae adherence during colonization of the nasopharynx, a site normally devoid of glucose. CcpA and environmental glucose concentrations thus appear to play important roles in the regulation of a niche-specific virulence factor.

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions, cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested. doi:10.1128/JB.01938-3381

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [[sup.14]C]Glc from UDP-[[sup.14]C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 [micro]M UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.

The type 3 synthase from Streptococcus pneumoniae is a processive [beta]-glycosyitransferase that assembles the type 3 polysaccharide [3)-[beta],-D-GlcUA-(1[arrow right]4)-[beta]-D-Glc.(1[arrow right] by a multieatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nueleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-([sup.14]C]]Glc or UDP-[[sup.14]C]GlcUA, we found that phospholipase D digestion of the Gle-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Gle followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not eovalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D[tilde]P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rxl, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D[tilde]P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rxl background, is important in the control of capsule synthesis.

The authors review the presentations carried out by the researches at the Sixth International Conference on Streptococcal, Lactococcal and Enterococcal Genetics convened in 2002 in Asheville, N.C. The topics of interest include the role of streptococcal, lactococcal and enterococcal species in bacterial genetics development and the discovery and analysis of yet-to-be-cultured microbes and microbes colonizing oral cavity, respiratory tract and intestinal system.

Results demonstrate that a reduced type 3 spontaneous mutant is involved in the capsule production. Data further point out that most of the phosphoglucomutase activity responsible for type 3 capsule production is derived from the cellular phosphoglucomutase.

The mutant strain JY2190 of Streptococcus pneumoniae, which does not require choline and its analogs, was isolated and characterized. There is little alteration in the lipoteichoic acid and wall teichoic acids from the mutant strains and the cell wall composition was essentially unaltered save for the reduction of the phosphate content. The mutant cells did not express autolysin and had impaired cell separation during cytokinesis.

Pneumococcal surface protein (PspA) of Streptococcus pneumoniae attaches to the bacterial cell surface through choline-mediated interactions between the protein's C-terminus and membrane-related lipoteichoic acid. Choline concentrations in choline-deficient regions causes five deletions in the C-terminus units initiating protein generation from the surface. The attachment technique effects rapid isolation of immunogenic PspA from the bacteria.

Mutant strains of Streptococcus pneumoniae were generated which had altered pneumococcal surface proteins (PspA). With these mutants, the pspA locus was defined and the protein further characterized. Based on the results and previous sequence data, the functions of the four domains within PspA were elucidated. The alpha-helical N-terminal domain is the antigenic determinant of the protein. A proline rich domain spans the cell wall, and a repeat region is involved in cell attachment with either the membrane or membrane components. The pspA gene was expressed and secreted in Escherichia coli.

The gene coding for pneumococcal surface protein (PspA) in Streptococcus pneumoniae was sequenced. Sequence analysis showed that the deduced protein possessed four structural domains. The N-terminal half corresponded to an alpha-helical domain, followed by a proline-rich domain. Succeeding this was a repeat domain consisting of 10 conserved 20-amino-acid repeats. The fourth domain was a hydrophobic region too short to serve as a membrane anchor. PspA may be attached to the cell by a unique mechanism involving the repeat domain. This domain showed significant homology to sequences in proteins from S. mutans, S. downei and Clostridium difficile.