AIM: To compare efficacy of combined lamivudine (LAM) and adefovir dipivoxil (ADV) therapy with that of entecavir (ETV) monotherapy for hepatitis B virus (HBV)-related decompensated liver cirrhosis.
METHODS: A total of 120 naïve patients with HBV-related decompensated cirrhosis participated in this study. Sixty patients were treated with combined LAM and ADV therapy (LAM + ADV group), while the other 60 were treated with ETV monotherapy (ETV group) for two years. Tests for liver and kidney function, alpha-fetoprotein, HBV serum markers, HBV DNA load, prothrombin time (PT), and ultrasonography or computed tomography scan of the liver were performed every 1 to 3 mo. Repeated measure ANOVA and the χ2 test were performed to compare the efficacy, side effects, and the cumulative survival rates at 48 and 96 wk.
RESULTS: Forty-five patients in each group were observed for 96 wk. No significant differences in HBV DNA negative rates and alanine aminotransferase (ALT) normalization rates at weeks 48 (χ2 = 2.12 and 2.88) and 96 (χ2 = 3.21 and 3.24) between the two groups were observed. Hepatitis B e antigen seroconversion rate in the LAM + ADV group at week 96 was significantly higher in the ETV group (43.5% vs 36.4%, χ2 = 4.09, P < 0.05). Viral breakthrough occurred in 2 cases (4.4%) by week 48 and in 3 cases (6.7%) by week 96 in the LAM + ADV group, and no viral mutation was detected. In the ETV group, viral breakthrough occurred in 1 case (2.2%) at the end of week 96. An increase in albumin (F = 18.9 and 17.3), decrease in total bilirubin and in ALT (F = 16.5, 17.1 and 23.7, 24.8), reduced PT (F = 22.7 and 24.5), and improved Child-Turcotte-Pugh and the model for end-stage liver disease scores (F = 18.5, 17.8, and 24.2, 23.8) were observed in both groups. The cumulative rates of mortality and liver transplantation were 16.7% (10/60) and 18.3% (11/60) in the LAM + ADV and ETV groups, respectively.
CONCLUSION: Both LAM + ADV combination therapy and ETV monotherapy can effectively inhibit HBV replication, improve liver function, and decrease mortality.
Chronic hepatitis B; Decompensated liver cirrhosis; Lamivudine; Adefovir dipivoxil; Combination therapy; Entecavir
Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.
Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2–GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.
protein phosphorylation; signal transduction; fluorescence imaging; protein degradation; photobody
High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.
A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions.
Streptococcus gordonii is an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designated lac and gal, were identified in the S. gordonii DL1 genome, which encode genes of the tagatose pathway (lacABCD) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EIILac transports lactose and galactose, whereas EIIGal transports galactose. The expression of the gene for EIIGal was markedly upregulated in cells growing on galactose. Using promoter-cat fusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and the gal cluster was also shown to be sensitive to repression by CcpA. The deletion of lacT caused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EIILac, but had little effect on galactose utilization. S. gordonii maintained a selective advantage over Streptococcus mutans in a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, although S. mutans could persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems of S. gordonii are subject to complex regulation and that a high-affinity galactose PTS may be advantageous when S. gordonii is competing against the caries pathogen S. mutans in oral biofilms.
We detected Bartonella quintana in 48.6% of captive rhesus macaques from an animal facility in Beijing, China. Prevalence of infection increased over the period of observation. Our findings suggest that macaques may serve as reservoir hosts for B. quintana and that Pedicinus obtusus lice might act as efficient vectors.
Bartonella quintana; rhesus macaques; reservoir host; lice; transmission; China; vector-borne infections; Bartonella
The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.
Bombina maxima; Fibroblast; Immortalization; Skin; Vimentin
The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.
Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF.
Developing an effective vaccine against HIV infection remains an urgent goal. We used a DNA prime/fowlpox virus boost regimen to immunize Chinese rhesus macaques. The animals were challenged intramuscularly with pathogenic molecularly cloned SHIV-KB9. Immunogenicity and protective efficacy of vaccines were investigated by measuring IFN-γ levels, monitoring HIV-specific binding antibodies, examining viral load, and analyzing CD4/CD8 ratio. Results show that, upon challenge, the vaccine group can induce a strong immune response in the body, represented by increased expression of IFN-γ, slow and steady elevated antibody production, reduced peak value of acute viral load, and increase in the average CD4/CD8 ratio. The current research suggests that rapid reaction speed, appropriate response strength, and long-lasting immune response time may be key protection factors for AIDS vaccine. The present study contributes significantly to AIDS vaccine and preclinical research.
Commensal oral streptococci play critical roles in oral biofilm formation and promote dental health by competing with, and antagonizing the growth of, pathogenic organisms, such as Streptococcus mutans. Efficient utilization of the spectrum of carbohydrates in the oral cavity by commensal streptococci is essential for their persistence, and yet very little is known about the regulation of carbohydrate catabolism by these organisms. Carbohydrate catabolite repression (CCR) in the abundant oral commensal Streptococcus gordonii strain DL-1 was investigated using the exo-β-d-fructosidase gene (fruA) and a fructose/mannose sugar:phosphotransferase (PTS) enzyme II operon (levDEFG) as model systems. Functional studies confirmed the predicted roles of FruA and LevD in S. gordonii. ManL, the AB domain of a fructose/mannose-type enzyme II PTS permease, contributed to utilization of glucose, mannose, galactose, and fructose and exerted primary control over CCR of the fruA and levD operons. Unlike in S. mutans, ManL-dependent CCR was not sugar specific, and galactose was very effective at eliciting CCR in S. gordonii. Inactivation of the apparent ccpA homologue of S. gordonii actually enhanced CCR of fruA and levD, an effect likely due to its demonstrated role in repression of manL expression. Thus, there are some similarities and fundamental differences in CCR control mechanisms between the oral pathogen S. mutans and the oral commensal S. gordonii that may eventually be exploited to enhance the competitiveness of health-associated commensals in oral biofilms.
Carbohydrate catabolite repression (CCR) in Streptococcus mutans does not require CcpA and is exerted through a network of phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) permeases. To probe the molecular mechanisms of CCR in S. mutans, the effects of various ptsH (HPr) and hprK (HPr kinase/phosphatase) mutations on growth and CCR were evaluated. An hprKV265F mutation, which enhanced phosphorylation of HPr at Ser46, inhibited growth on multiple PTS sugars. A ptsHS46A mutation reversed the effects of hprKV265F in most cases. A strain carrying a ptsHS46D mutation, which mimics HPr(Ser-P), presented with more severe growth defects than the hprKV265F mutant. The hprKV265F mutant displayed reduced expression of the fruA and levD operons, a phenotype reversible by the introduction of the ptsHS46A mutation. The effects of the hprKV265F mutation on fruA and levD expression were independent of CcpA, but dependent on ManL (IIABMan) and, to a lesser extent, on the FruI (IIABCFru), in a sugar-specific manner. The hprKV265F mutation inhibited growth on cellobiose and lactose, but only the transcription of the cel operon was decreased. Thus, in S. mutans, serine-phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization through modulation of sugar transport activity and the transcription of catabolic operons.
Catabolite repression; HPr; Carbohydrate transport; Biofilm; Virulence
rabies; antibody; titer; dog; survey; viruses; immunization; China; letter
Transcription of the genes for a fructan hydrolase (fruA) and a
fructose/mannose sugar:phosphotransferase permease (levDEFG) in
Streptococcus mutans is activated by a four-component
regulatory system consisting of a histidine kinase (LevS), a response regulator
(LevR) and two carbohydrate-binding proteins (LevQT). The expression of the
fruA and levD operons was at baseline in a
levQ mutant and substantially decreased in a
levT null mutant, with lower expression with the cognate
inducers fructose or mannose, but slightly higher expression in glucose or
galactose. A strain expressing levQ with two point mutations
(E170A/F292S) did not require inducers to activate gene expression and displayed
altered levD expression when growing on various carbohydrates,
including cellobiose. Linker-scanning (LS) mutagenesis was used to generate
three libraries of mutants of levQ, levS and
levT that displayed various levels of altered substrate
specificity and of fruA/levD gene expression. The data support
that LevQ and LevT are intimately involved in the sensing of carbohydrate
signals, and that LevQ appears to be required for the integrity of the signal
transduction complex, apparently by interacting with the sensor kinase LevS.
Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a PlacA-cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EIIMan and EIILac, respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIABMan, led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.
Bacteria adhere to a surface and, through cell division and coordinated expression of gene products, to develop into a structurally-complex population of adherent cells. This process, known as biofilm formation, requires that intrinsic and extrinsic signals are transduced into appropriate gene expression patterns as biofilms mature. Mutational analysis has begun to reveal the complexity of systems used by Streptococcus mutans to ensure proper biofilm formation. These studies have revealed new and unique targets for the design of broadly-effective anti-caries strategies.
Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of Streptococcus mutans. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated aguR genes that were utilized to complement an aguR deletion mutant of S. mutans. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the agu operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.
The ability of Streptococcus mutans to catabolize cellobiose, a β-linked glucoside generated during the hydrolysis of cellulose, is shown to be regulated by a transcriptional regulator, CelR, which is encoded by an operon with a phospho-β-glucosidase (CelA) and a cellobiose-specific sugar phosphotransferase system (PTS) permease (EIICel). The roles of CelR, EIICel components, and certain fructose/mannose-PTS permeases in the transcriptional regulation of the cel locus were analyzed. The results revealed that (i) the celA and celB (EIIBCel) gene promoters require CelR for transcriptional activation in response to cellobiose, but read-through from the celA promoter contributes to expression of the EIICel genes; (ii) the EIICel subunits were required for growth on cellobiose and for transcriptional activation of the cel genes; (iii) CcpA plays little direct role in catabolite repression of the cel regulon, but loss of specific PTS permeases alleviated repression of cel genes in the presence of preferred carbohydrates; and (iv) glucose could induce transcription of the cel regulon when transported by EIICel. CelR derivatives containing amino acid substitutions for five conserved histidine residues in two PTS regulatory domains and an EIIA-like domain also provided important insights regarding the function of this regulator. Based on these data, a model for the involvement of PTS permeases and the general PTS proteins enzyme I and HPr was developed that reveals a critical role for the PTS in CcpA-independent catabolite repression and induction of cel gene expression in S. mutans.
Streptococcus mutans is particularly well-adapted for high-affinity, high-capacity catabolism of multiple carbohydrate sources. S. mutans EIILev, a fructose/mannose permease encoded by the levDEFG genes, and fruA, which encodes a hydrolase that releases fructose from fructan polymers, are transcriptionally regulated by the LevQRST four-component signal transduction system. Here, we demonstrate that (1) levDEFGX are co-transcribed and the levE/F intergenic region is required for optimal expression of levFGX; (2) D-mannose is a potent inducer of the levD and fruA operons; (3) CcpA regulates levD expression in a carbohydrate-specific manner; (4) deletion of the genes for the fructose/mannose-EII enzymes of S. mutans (manL, fruI, and levD) enhances levD expression; (5) repression of the LevQRST regulon by EII enzymes depends on the presence of their substrates and requires LevR, but not LevQST; and (6) CcpA inhibits expression of the manL and frul genes to indirectly control the LevQRST regulon. Further, the manL, ccpA, frul/fruCD and levD gene products differentially exert control over the cellobiose and lactose operons. Collectively, the results reveal the existence of a global regulatory network in S. mutans that governs the utilization of non-preferred carbohydrates in response to the availability and source of multiple preferred carbohydrates.
Sugar:phosphotransferase system; β-D-fructosidase; Catabolite repression; CcpA; Gene regulation
CcpA globally regulates transcription in response to carbohydrate availability in many gram-positive bacteria, but its role in Streptococcus mutans remains enigmatic. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrated that CcpA plays a direct role in carbon catabolite repression (CCR). Subsequently, the expression of 170 genes was shown to be differently expressed (≥2-fold) in glucose-grown wild-type (UA159) and CcpA-deficient (TW1) strains (P ≤ 0.001). However, there were differences in expression of only 96 genes between UA159 and TW1 when cells were cultivated with the poorly repressing substrate galactose. Interestingly, 90 genes were expressed differently in wild-type S. mutans when glucose- and galactose-grown cells were compared, but the expression of 515 genes was altered in the CcpA-deficient strain in a similar comparison. Overall, our results supported the hypothesis that CcpA has a major role in CCR and regulation of gene expression but revealed that in S. mutans there is a substantial CcpA-independent network that regulates gene expression in response to the carbohydrate source. Based on the genetic studies, biochemical and physiological experiments demonstrated that loss of CcpA impacts the ability of S. mutans to transport and grow on selected sugars. Also, the CcpA-deficient strain displayed an enhanced capacity to produce acid from intracellular stores of polysaccharides, could grow faster at pH 5.5, and could acidify the environment more rapidly and to a greater extent than the parental strain. Thus, CcpA directly modulates the pathogenic potential of S. mutans through global control of gene expression.
The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while a pcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.
Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes.
TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest.
TBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation.
The arginine deiminase system (ADS) is responsible for the production of ornithine, CO2, ammonia, and ATP from arginine. The ADS of the oral bacterium Streptococcus gordonii plays major roles in physiologic homeostasis, acid tolerance, and oral biofilm ecology. To further our understanding of the transcriptional regulation of the ADS (arc) operon, the binding of the ArcR transcriptional activator, which governs expression of the ADS in response to arginine, was investigated by DNase I protection and gel mobility shift assays. An ArcR binding sequence was found that was 27 bp in length and had little sequence similarity to binding sites of other arginine metabolism regulators. The presence of arginine at physiologically relevant concentrations enhanced the binding of ArcR to its target. Using cat fusions, various deletion and substitution mutations within the putative ArcR footprint were shown to cause dramatic reductions in expression from the arcA promoter in vivo, confirming that the 27-bp sequence is required for optimal expression and induction of the ADS by arginine. Mutation of two putative catabolite response elements (CREs) within the arc promoter region showed that both CREs contribute to catabolite repression. A thorough understanding of the regulation of the ADS in S. gordonii and related organisms is needed to develop ways to exploit arginine catabolism for the control of oral diseases. Identification of the ArcR and CcpA binding sites lays the foundation for a more complete understanding of the complex interactions of multiple regulatory proteins with elements in the arc promoter region.
Pseudomonas aeruginosa harbors a chromosomal aminoglycoside phosphotransferase gene, aph(3′)-IIb, which confers P. aeruginosa resistance to several important aminoglycoside antibiotics, including kanamycin A and B, neomycin B and C, butirosin, and seldomycin F5. The aph(3′)-IIb gene has been found to be regulated by an AraC-type transcriptional regulator (HpaA) encoded by a gene located upstream of the aph(3′)-IIb gene. In the presence of 4-hydroxyphenylacetic acid (4-HPA), HpaA activates the expression of aph(3′)-IIb as well as that of the hpa regulon which encodes metabolic enzymes for the utilization of 4-HPA. hpaA and aph(3′)-IIb form an operon, and in response to the presence of 4-HPA, the wild-type P. aeruginosa strain PAK (but not its hpaA mutant strain) displays increased resistance to neomycin. A survey of 39 clinical and 19 environmental isolates of P. aeruginosa demonstrated in all of them the presence of an hpaA-aph gene cluster, while 56 out of the 58 isolates are able to utilize the 4-HPA as a sole carbon source, suggesting a feature common to P. aeruginosa strains. Interestingly, a larger portion of clinical isolates than environmental isolates showed 4-HPA-induced resistance to neomycin. The aph(3′)-IIb gene product is likely to function as a metabolic enzyme which has a cross-reactivity with aminoglycosides. These findings provide new insight into the possible mechanism of P. aeruginosa antibiotic resistance.