Slipped-strand mispairing within a polycytidine tract in transcriptional regulator mga leads to M protein phase variation and Mga length polymorphism in Group A Streptococcus.

The M protein, a major virulence factor of Group A Streptococcus (GAS), is regulated by the multigene regulator Mga. An unexplained phenomena frequently occurring with in vitro genetic manipulation or culturing of M1T1 GAS strains is the loss of M protein production. This study was aimed at elucidating the basis for the loss of M protein production. The majority of M protein-negative (M-) variants had one C deletion at a tract of 8 cytidines starting at base 1,571 of the M1 mga gene, which is designated as c.1571C[8]. The C deletion led to a c.1571C[7] mga variant that has an open reading frame shift and encodes a Mga-M protein fusion protein. Transformation with a plasmid containing wild-type mga restored the production of the M protein in the c.1571C[7] mga variant. Isolates producing M protein (M+) were recovered following growth of the c.1571C[7] M protein-negative variant subcutaneously in mice. The majority of the recovered isolates with reestablished M protein production had reverted back from c.1571C[7] to c.1571C[8] tract and some M+ isolates lost another C in the c.1571C[7] tract, leading to a c.1571C[6] variant that encodes a functional Mga with 13 extra amino acid residues at the C-terminus compared with wild-type Mga. The nonfunctional c.1571C[7] and functional c.1571C[6] variants are present in M1, M12, M14, and M23 strains in NCBI genome databases, and a G-to-A nonsense mutation at base 1,657 of M12 c.1574C[7] mga leads to a functional c.1574C[7]/1657A mga variant and is common in clinical M12 isolates. The numbers of the C repeats in this polycytidine tract and the polymorphism at base 1,657 lead to polymorphism in the size of Mga among clinical isolates. These findings demonstrate the slipped-strand mispairing within the c.1574C[8] tract of mga as a reversible switch controlling M protein production phase variation in multiple GAS common M types.

The sagA/pel locus does not regulate the expression of the M protein of the M1T1 lineage of group A Streptococcus.

Altered expression of Group A Streptococcus (GAS) virulence factors, including the M protein, can result as a consequence of spontaneous genetic changes that occur during laboratory and animal passage. Occurrence of such secondary mutations during targeted gene deletion could confound the interpretation of effects attributable to the function of the gene being investigated. Contradicting reports on whether the sagA/pel locus regulates the M protein-encoding emm might be due to inconsistent occurrence of mutations unrelated with sagA. This study examined the possibility that altered emm expression observed in association with sagA/pel deletion mutants is artifactual. sagA deletion mutants (MGAS2221ΔsagA) of M1T1 isolate MGAS2221 obtained using liquid broth for GAS growth during the deletion process had diminished emm transcription and no detectable M protein production. In contrast, a ΔsagA mutant of another closely genetically related M1T1 isolate had normal emm expression. The sagB gene does not regulate emm; however, one of three MGAS2221ΔsagB mutants had diminished emm expression. The emm regulator mga was downregulated in these M protein expression-negative strains. These results argue that sagA deletion does not directly cause the downregulation of emm expression. Indeed, two MGAS2221ΔsagA mutants obtained using agar plates for GAS growth during the deletion process both had normal emm expression. We conclude that the sagA/pel locus does not regulate emm expression in the M1T1 lineage and provide a protocol for targeted gene deletion that we find less prone to the generation of mutants exhibiting downregulation in emm expression.

Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors.

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 µM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

Pleomorphic adenoma gene-like 2 regulates expression of the p53 family member, p73, and induces cell cycle block and apoptosis in human promonocytic U937 cells.

The proto-oncogene, pleomorphic adenoma gene-like 2 (PLAGL2), is implicated in a variety of cancers including acute myeloid leukemia (AML), malignant glioma, colon cancer, and lung adenocarcinoma. There is additional evidence that PLAGL2 can function as a tumor suppressor by initiating cell cycle arrest and apoptosis. Interestingly, PLAGL2 has also been implicated in human myelodysplastic syndrome, a disease that is characterized by ineffective hematopoiesis and can lead to fatal cytopenias (low blood counts) as a result of increased apoptosis in the marrow, or, in about one-third of cases, can progress to AML. To gain a better understanding of the actions of PLAGL2 in human myeloid cells, we generated a stable PLAGL2-inducible cell line, using human promonocytic U937 cells. PLAGL2 expression inhibited cell proliferation which correlated with an accumulation of cells in G1, apoptotic DNA-laddering, an increase in caspase 3, 8, and 9 activity, and a loss of mitochondrial transmembrane potential. There was significant increase in the p53 homologue, p73, with PLAGL2 expression, and consistent with mechanisms of p73-regulated cell cycle control and apoptosis, there was increased expression of known p73 target genes p21, DR5, TRAIL, and Bax. PLAGL2-induced cell cycle block was abolished in the presence of p73 siRNA. Together, these data support a role for PLAGL2 in cell cycle regulation and apoptosis via activation of p73.

Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex.

The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

Differential regulation of iron- and manganese-specific MtsABC and heme-specific HtsABC transporters by the metalloregulator MtsR of group A Streptococcus.

The genome of the human pathogen group A Streptococcus (GAS) encodes the transporters MtsABC, FtsABCD, and HtsABC to take up ferric and manganese ions, ferric ferrichrome, and heme, respectively. The GAS genome also encodes two metalloregulators PerR and MtsR. To understand the regulation of the expression of these transporters, the mtsR and perR deletion mutants of a GAS serotype M1 strain were generated, and the effects of the deletions and Fe(3+), Mn(2+), and Zn(2+) on the expression of mtsA, htsA, and ftsB were examined. Mn(2+) dramatically depresses mtsA transcription and levels of the MtsA protein but does not downregulate the expression of htsA and ftsB. Fe(3+) decreases the expression of mtsA and htsA but has no effect on ftsB expression. Zn(2+) has no effect on the expression of all three genes. The deletion of mtsR abolishes the Mn(2+)- and Fe(3+)-induced depression of mtsA expression and the Fe(3+)-dependent decrease in htsA expression. The deletion of mtsR does not significantly alter GAS virulence in a mouse model of subcutaneous infection. The deletion of perR does not affect the expression of the genes in response to the metal ions. MtsR binds to the mts promoter region in the presence of Mn(2+) or Fe(2+). The results indicate that MtsR differentially regulates the expression of mtsABC and htsABC.

Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR.

The regulator VicR of the two-component regulatory system VicRK is essential in several Gram-positive bacteria. However, the authors were able to generate an unconditional vicR insertional mutant of group A Streptococcus. This mutant grew well in rich media but not in non-immune human blood and serum, had attenuated virulence, and was unstable in mice. Complementation of the mutant with vicR expressed in trans restored its phenotype to wild-type. A vicK deletion mutant had a phenotype similar to that of the vicR mutant. Phagocytosis and killing of the vicR mutant were normal, suggesting that VicRK does not regulate processes involved in evasion of host defence. Microarray analysis showed that vicR inactivation down-regulated the transcription of 13 genes, including putative cell wall hydrolase gene pcsB and spy1058-1060, which encode a putative phosphotransferase system enzyme II for carbohydrate transport, and upregulated the expression of five genes, including spy0183 and spy0184, which encode an osmoprotectant transporter OpuA. Consistent with microarray analysis, the vicR mutant took up more of the osmoprotectants betaine and proline and was sensitive to osmotic stress, indicating that vicR inactivation induced osmotic stress and increased susceptibility to osmotic pressure. Additionally, a spy1060 deletion mutant also displayed attenuated virulence. These results suggest that VicRK regulates processes involved in cell wall metabolism, nutrient uptake, and osmotic protection.

ABC transporter FtsABCD of Streptococcus pyogenes mediates uptake of ferric ferrichrome.

BACKGROUND: The Streptococcus pyogenes or Group A Streptococcus (GAS) genome encodes three ABC transporters, namely, FtsABCD, MtsABC, and HtsABC, which share homology with iron transporters. MtsABC and HtsABC are believed to take up ferric (Fe3+) and manganese ions and heme, respectively, while the specificity of FtsABCD is unknown. RESULTS: Recombinant FtsB, the lipoprotein component of FtsABCD, was found to bind Fe3+ ferrichrome in a 1:1 stoichiometry. To investigate whether FtsABCD transports Fe3+ ferrichrome, GAS isogenic strains defective in lipoprotein gene ftsB and permease gene ftsC were generated, and the effects of the mutations on uptake of Fe3+ ferrichrome were examined using radioactive 55Fe3+ ferrichrome. FtsB was produced in the wild-type strain but not in the ftsB mutant, confirming the ftsB inactivation. While wild-type GAS took up 3.6 x 10(4) Fe3+ ferrichrome molecules per bacterium per min at room temperature, the ftsB and ftsC mutants did not have a detectable rate of Fe3+ ferrichrome uptake. The inactivation of ftsB or ftsC also decreased 55Fe3+ ferrichrome uptake by >90% under growth conditions in the case of limited uptake time. Complementation of the ftsB mutant with a plasmid carrying the ftsB gene restored FtsB production and 55Fe3+ ferrichrome association at higher levels compared with the parent strain. The inactivation of mtsA and htsA and Fe-restricted conditions enhanced the production of FtsB and Fe3+ ferrichrome uptake. CONCLUSION: The FtsB protein bound Fe3+ ferrichrome, and inactivation of ftsB or ftsC, but not htsA or mtsA, diminished Fe3+ ferrichrome uptake, indicating that FtsABCD, but not HtsABC and MtsABC, is the transporter that takes up Fe3+ ferrichrome in GAS. Fe acquisition systems are virulence factors in many bacterial pathogens and are attractive vaccine candidates. The elucidation of the FtsABCD specificity advances the understanding of Fe acquisition processes in GAS and may help evaluating the GAS Fe acquisition systems as vaccine candidates.