Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) is a gamma-2 herpesvirus believed to be the etiologic agent responsible for KS. The pathogenesis of this potentially lifethreatening neoplasm is complex and unclear, and it is currently unknown how KSHV causes KS. Id (named for inhibitor of DNA binding or inhibitor of differentiation) proteins were identified in 1990 and found to be naturally occurring dominant-negative inhibitors of basic helix-loop-helix transcription factors. Id-1, the most well-studied member of this family, has since been shown to play a key role in several biological systems including cellular differentiation, cell cycle regulation, and tumorigenesis. In this report, we demonstrate that Id-1 is expressed at high levels in KS tumor cells both in vitro and in vivo but is expressed at relatively modest levels in endothelial cells (ECs), the likely precursor of the KS tumor cell. Infection of precursor cells with KSHV may be responsible for this enhanced expression, as KSHV infection induced Id-1 27-fold in ECs under our experimental conditions. Furthermore, we demonstrate that the KSHV-encoded latency-associated nuclear antigen (LANA) protein appears to be involved. Expression of LANA in ECs resulted in Id-1 induction that was almost identical to the induction seen with KSHV-infected ECs. These results demonstrate the expression of Id-1 in KS tumor cells and indicate the KSHV LANA protein may be, at least in part, responsible. This may be an important mechanism by which KSHV allows KS tumor cells to escape normal cell cycle regulation and enhances their proliferation.Id proteins are a family of four helix-loop-helix (HLH) proteins (Id-1 to Id-4) initially recognized as growth factor-inducible genes that inhibit cellular differentiation (5, 36). These proteins lack the basic amino acid sequence characteristic of basic HLH (bHLH) transcription factors that is necessary for DNA binding and target gene transcription (5). Instead, Id proteins bind to bHLH transcription factors and form nonfunctional heterodimers, thereby acting as naturally occurring dominant-negative inhibitors (5).Although Id proteins have traditionally been viewed as negative regulators of cell differentiation, recent studies indicate they have wider biological roles, including cell cycle regulation, embryonic development, cell death, and tumorigenesis (36). Several of these functions may be related to recently described interactions of Id proteins with certain non-HLH proteins, including ETS transcription factors, pRb, the pocket proteins p107 and p130, MIDA1, and Pax proteins (22, 51). With respect to normal cell cycle regulation, Id genes are induced in the G 1 phase and expression is down regulated in quiescent, senescent, or terminally differentiated cells. These proteins can promote cell cycle progression through several distinct mechanisms. Id proteins prevent expression of the cyclin-dependent kinase inhibitor (CDKI) p21 by blocking bHLH transcription factor activity (41). Id-2 and I...

Immunoglobulin (Ig)-binding proteins are employed by a variety of organisms to evade the immune system. We now report for the first time that meningococcal strains from several capsular groups exhibit Ig-binding activity that is dependent on human serum factors. A protein mediating Ig binding was identified as T and B cell stimulating protein B (TspB) by immunoprecipitation and by mass spectroscopic analysis of tryptic peptides. Recombinant TspB and derivatives verified Ig binding, with a preference for human IgG2 Fc, and localized the IgG-binding region to a highly conserved subdomain of TspB. Antiserum produced in mice against the conserved subdomain, detected the presence of TspB on the cell surface by flow cytometry when bacteria were grown in the presence of human serum. By fluorescence microscopy, we observed formation of an extracellular matrix having characteristics of a biofilm containing TspB, human IgG, DNA, and large aggregates of bacteria. TspB is encoded by gene ORF6 in prophage DNA, which others have shown is associated with invasive meningococcal strains. Knocking out ORF6 genes eliminated IgG binding and formation of large bacterial aggregates in biofilm. Reintroduction of a wild-type ORF6 gene by phage transduction restored the phenotype. The results show that TspB mediated IgG binding and aggregate/biofilm formation triggered by factors in human serum. As has been observed for other Ig-binding proteins, the activities mediated by TspB may provide protection against immune responses, which is in accordance with the association of prophage DNA carrying ORF6 with invasive meningococcal strains.

The ability of the human bacterial pathogen Neisseria meningitidis to cause invasive disease depends on survival in the bloodstream via mechanisms to suppress complement activation. In this study, we show that prophage genes coding for T and B cell stimulating protein B (TspB), which is an immunoglobulin-binding protein, are essential for survival of N. meningitidis group B strain H44/76 in normal human serum (NHS). H44/76 carries three genes coding for TspB. Mutants having all tspB genes inactivated did not survive in >5% NHS or IgG-depleted NHS. TspB appeared to inhibit IgM-mediated activation of the classical complement pathway, since survival of the tspB triple knockout was the same as that of the parent strain or a complemented mutant when the classical pathway was inactivated by depleting NHS of C1q and was increased in IgM-depleted NHS. A mutant solely carrying tspB gene nmbh4476_0681 was as resistant as the parent strain, while mutants carrying only nmbh4476_0598 or nmbh4476_1698 were killed in >5% NHS. The phenotype associated with TspB is formation of a matrix containing TspB, IgG, and DNA that envelopes aggregates of bacteria. Recombinant proteins corresponding to particular subdomains of TspB were found to have human IgG Fc␥-and/or DNA-binding activity, but only TspB derivatives containing both domains formed large, biofilm-like aggregates when combined with purified IgG and DNA. Recognizing the role of TspB in serum resistance may lead to a better understanding of why strains that carry tspB genes are associated with invasive meningococcal disease. Invasive Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. The reasons for why some N. meningitidis strains cause disease and others do not are not well understood. With the exception of periodic epidemics occurring mainly in sub-Saharan Africa, disease caused by pathogenic N. meningitidis is relatively rare. However, asymptomatic carriage is comparatively common, ranging from ϳ5% to Ͼ80% depending on the population studied (1, 2). Host factors associated with increased risk of disease include complement deficiencies, carriage state, genetics, social behavior, and geographic location (reviewed in reference 3). Strains causing invasive disease, on the other hand, appear to be limited to those from a few, so-called hypervirulent lineages (2). However, not many specific characteristics of N. meningitidis strains have been identified that can be linked directly to disease. In an epidemiological study comparing disease-causing isolates with carriage isolates by microarray analysis during an outbreak of meningococcal disease in the Czech Republic, Bille et al. found a statistically significant association of the presence of prophage DNA with isolates that caused disease (4). However, the reasons for the link between the prophage DNA and invasive disease were not determined. Recently, we showed that the prophage gene ORF6 codes for an IgG-binding protein specific for the Fc portion of a human IgG2 paraprotein (5). The protein,...

Under conditions of nitrogen stress, leguminous plants form symbioses with soil bacteria called rhizobia. This partnership results in the development of structures called root nodules, in which differentiated endosymbiotic bacteria reduce molecular dinitrogen for the host. The establishment of rhizobium-legume symbioses requires the bacterial synthesis of oligosaccharides, exopolysaccharides, and capsular polysaccharides. Previous studies suggested that the 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular polysaccharide produced by strain Sinorhizobium meliloti Rm1021 contributes to symbiosis with Medicago sativa under some conditions. However, a conclusive symbiotic role for this polysaccharide could not be determined due to a lack of mutants affecting its synthesis. In this study, we have further characterized the synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We showed that mutants lacking the enigmatic rkp-1 gene cluster fail to display the Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of unusually high M r . In addition, we have demonstrated that mutations in kdsB2, smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric capsule. Our studies also suggest a role for the capsular polysaccharide in symbiosis. Previous reports have shown that the overexpression of rkpZ from strain Rm41 allows for the symbiosis of exoY mutants of Rm1021 that are unable to produce the exopolysaccharide succinoglycan. Our results demonstrate that mutations in the rkp-1 cluster prevent this phenotypic suppression of exoY mutants, although mutations in kdsB2, smb20804, and smb20805 have no effect.