Molecular dissection of competence regulated virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen capable of entering the competent state naturally and acquiring DNA from the environment for recombination. In addition to improving genome plasticity, a natural competence system directly contributes to pneumococcal virulence during pneumonia and bacteremia, independent of DNA uptake. Competence is activated when the competence stimulating peptide (CSP), encoded by the comC gene, is processed and exported through the ComAB transporter and stimulates the membrane localized two component system sensor kinase, ComD. Activated ComD autophosphorylates and activates its response regulator ComE, which in turn induces the expression of 24 “early” genes, including the comABCDE, comX1 and comX2 and ComM. Encoded by two identical redundant genes, ComX is an alternative sigma factor which activates the expression of 80 “late” genes, 16 of which are required for DNA uptake and recombination. Some of the proteins encoded by the “late” competence genes, including LytA, CbpD, and CibAB, are capable of attacking and lysing other non-competent pneumococcal cells to release DNA, in a process called allolysis or fratricide. One of these “late” genes, dprA, encodes a dual-functional protein called DNA processing protein A (DprA), which protects incoming single strand DNA for genetic transformation, as well as shutting off the activated competence system. We found that deletion of dprA resulted in prolonged and higher expression of competence genes in vitro in competent pneumococcus, and caused attenuation in mouse models of acute pneumonia and bacteremia. Also, when compared to the parental wild-type serotype 2 strain D39, ∆dprA entered competent state more easily, at lower concentrations of exogenously provided CSP. LytA, CbpD and CibAB were over-expressed in ∆dprA during competence induction in vitro. Deletion of the “combox”, the ComX binding site on the promoters of cbpD and cibAB genes abolished the attenuation ∆dprA genetic background, suggesting overexpression of the allolysis factors during competent state accounted for the virulence attenuation in ∆dprA. Additionally, deletion of the “early” competence gene comM in the ∆dprA genetic background also compensated for the virulence loss, suggesting that overexpression of ComM also contributed to virulence attenuation in ∆dprA. Lastly, because of its inability to turn off the competence regulon, overexpression of all the competence genes consumed excessive nutrients and energy that would otherwise diverted for normal cellular functions, which might also contribute to virulence attenuation. Even though the biology of pneumococcal competence development has been studied for nearly a century, the development of natural competence during pneumococcal diseases have not been examined. We generated a pneumococcal reporter strain using the wild-type serotype 2 strain D39 with the firefly luciferase gene (luc) fused to the promoter of a highly upregulated “late” competence gene ssbB. The resulting reporter strain D39 ssbB-luc was used to track the development of natural competence during pneumonia-derived sepsis (pneumonic sepsis). D39 ssbB-luc cells infecting the lungs of CD-1 mice were able to naturally enter the competent state during pneumonic sepsis. In contrast to the transient short burst of competent state in vitro (~2 hours) in liquid medium, the competent state during pneumonic sepsis was prolonged and sustained, beginning at approximately 20 hours post infection (hpi) and lasted for 12 hours to 40 hours. In mice where the competent state lasted for ~12 hours, the animals quickly progressed from acute pneumonia to sepsis, and the competence signal kept increasing until death. For mice with more prolonged course of the disease, the naturally-developed competent state might wane, but would increase again when the infection entered the sepsis state and lasted for >40 hours until the animal approached endpoint. Exogenously provided CSP had minimal impact on the development of natural competence during pneumonic sepsis. The amount of initial inoculum (1 x 106 – 5 x 107 CFU per mouse), as well as the subsequent bacterial burden in the lung after infection do not seem to be the key factors in determining the timing of natural competence development. DNA processing protein A (DprA), which is responsible for efficient competence shut off in vitro, was highly expressed during pneumonic sepsis, but failed to turn off the competent state in infected animals. Importantly, natural competence development was also detected in both serotypes 3 and 4 clinical pneumococcal strains. Lastly, during pneumonic sepsis, competent D39 bacteria appeared to transfer the competence signal by using a cell-cell contact mechanism rather than the quorum-sensing model based on freely diffusive CSP. Genetic transformation in pneumococcus involves both DNA uptake and homologous recombination. Previously, our lab has identified the small cytoplasmic RNA (scRNA) as essential for transformation. ScRNA is a component of the signal recognition particle (SRP) that forms a complex with both Ffh and FtsY, and together, delivers nascent peptide to the Sec translocon for membrane targeting or secretion. By marker-less deletion, we found that the first nucleotides 6-49 of the scRNA gene, which constitutes part of the helix IV structure, was required for genetic transformation. Considerable efforts were devoted for the deletion and analysis of both ffh and ftsY genes, because ∆ffh and ∆ftsY were unable to grow on commonly used Todd-Hewitt broth unless supplemented with free amino acids. Similar to ∆scRNA, deletion of ffh and ftsY abolished genetic transformation. Deletion of the insertase gene yidC2, which functional overlaps with SRP, did not affect transformation. Proteomic analysis revealed that three key proteins CglA, CglB, and CglC, which are required for the assembly of the indispensable a Type IV pilus-like DNA uptake apparatus, were found in reduced levels on the membrane of ∆scRNA than D39. Collectively, these results suggest that reduced efficiency in targeting and/or assembling DNA uptake apparatus impairs the genetic transformation in ∆scRNA

Research progress of Yttrium-90 microsphere selective internal radiation therapy in downstaging and conversion of hepatocellular carcinoma

The incidence and mortality of hepatocellular carcinoma (HCC) in China are among the highest in the world, imposing a heavy social burden. Liver resection and liver transplantation are the primary radical treatments for HCC, although most patients are no longer able to meet the surgical requirements at initial diagnosis. Yttrium-90 microsphere selective internal radiation therapy (90Y-SIRT) has the advantages of shrinking tumors, enlarging residual liver, regressing portal vein tumor thrombus and improving the quality of life, which can be used for conversion, downstaging and bridging therapy for HCC before surgical treatment, enabling patients regain the chance of radical treatment and reducing the postoperative recurrence rate. This review focuses on the clinical application and progress of 90Y-SIRT in this field

Molecular dissection of competence regulated virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen capable of entering the competent state naturally and acquiring DNA from the environment for recombination. In addition to improving genome plasticity, a natural competence system directly contributes to pneumococcal virulence during pneumonia and bacteremia, independent of DNA uptake. Competence is activated when the competence stimulating peptide (CSP), encoded by the comC gene, is processed and exported through the ComAB transporter and stimulates the membrane localized two component system sensor kinase, ComD. Activated ComD autophosphorylates and activates its response regulator ComE, which in turn induces the expression of 24 “early” genes, including the comABCDE, comX1 and comX2 and ComM. Encoded by two identical redundant genes, ComX is an alternative sigma factor which activates the expression of 80 “late” genes, 16 of which are required for DNA uptake and recombination. Some of the proteins encoded by the “late” competence genes, including LytA, CbpD, and CibAB, are capable of attacking and lysing other non-competent pneumococcal cells to release DNA, in a process called allolysis or fratricide. One of these “late” genes, dprA, encodes a dual-functional protein called DNA processing protein A (DprA), which protects incoming single strand DNA for genetic transformation, as well as shutting off the activated competence system. We found that deletion of dprA resulted in prolonged and higher expression of competence genes in vitro in competent pneumococcus, and caused attenuation in mouse models of acute pneumonia and bacteremia. Also, when compared to the parental wild-type serotype 2 strain D39, ∆dprA entered competent state more easily, at lower concentrations of exogenously provided CSP. LytA, CbpD and CibAB were over-expressed in ∆dprA during competence induction in vitro. Deletion of the “combox”, the ComX binding site on the promoters of cbpD and cibAB genes abolished the attenuation ∆dprA genetic background, suggesting overexpression of the allolysis factors during competent state accounted for the virulence attenuation in ∆dprA. Additionally, deletion of the “early” competence gene comM in the ∆dprA genetic background also compensated for the virulence loss, suggesting that overexpression of ComM also contributed to virulence attenuation in ∆dprA. Lastly, because of its inability to turn off the competence regulon, overexpression of all the competence genes consumed excessive nutrients and energy that would otherwise diverted for normal cellular functions, which might also contribute to virulence attenuation. Even though the biology of pneumococcal competence development has been studied for nearly a century, the development of natural competence during pneumococcal diseases have not been examined. We generated a pneumococcal reporter strain using the wild-type serotype 2 strain D39 with the firefly luciferase gene (luc) fused to the promoter of a highly upregulated “late” competence gene ssbB. The resulting reporter strain D39 ssbB-luc was used to track the development of natural competence during pneumonia-derived sepsis (pneumonic sepsis). D39 ssbB-luc cells infecting the lungs of CD-1 mice were able to naturally enter the competent state during pneumonic sepsis. In contrast to the transient short burst of competent state in vitro (~2 hours) in liquid medium, the competent state during pneumonic sepsis was prolonged and sustained, beginning at approximately 20 hours post infection (hpi) and lasted for 12 hours to 40 hours. In mice where the competent state lasted for ~12 hours, the animals quickly progressed from acute pneumonia to sepsis, and the competence signal kept increasing until death. For mice with more prolonged course of the disease, the naturally-developed competent state might wane, but would increase again when the infection entered the sepsis state and lasted for >40 hours until the animal approached endpoint. Exogenously provided CSP had minimal impact on the development of natural competence during pneumonic sepsis. The amount of initial inoculum (1 x 106 – 5 x 107 CFU per mouse), as well as the subsequent bacterial burden in the lung after infection do not seem to be the key factors in determining the timing of natural competence development. DNA processing protein A (DprA), which is responsible for efficient competence shut off in vitro, was highly expressed during pneumonic sepsis, but failed to turn off the competent state in infected animals. Importantly, natural competence development was also detected in both serotypes 3 and 4 clinical pneumococcal strains. Lastly, during pneumonic sepsis, competent D39 bacteria appeared to transfer the competence signal by using a cell-cell contact mechanism rather than the quorum-sensing model based on freely diffusive CSP. Genetic transformation in pneumococcus involves both DNA uptake and homologous recombination. Previously, our lab has identified the small cytoplasmic RNA (scRNA) as essential for transformation. ScRNA is a component of the signal recognition particle (SRP) that forms a complex with both Ffh and FtsY, and together, delivers nascent peptide to the Sec translocon for membrane targeting or secretion. By marker-less deletion, we found that the first nucleotides 6-49 of the scRNA gene, which constitutes part of the helix IV structure, was required for genetic transformation. Considerable efforts were devoted for the deletion and analysis of both ffh and ftsY genes, because ∆ffh and ∆ftsY were unable to grow on commonly used Todd-Hewitt broth unless supplemented with free amino acids. Similar to ∆scRNA, deletion of ffh and ftsY abolished genetic transformation. Deletion of the insertase gene yidC2, which functional overlaps with SRP, did not affect transformation. Proteomic analysis revealed that three key proteins CglA, CglB, and CglC, which are required for the assembly of the indispensable a Type IV pilus-like DNA uptake apparatus, were found in reduced levels on the membrane of ∆scRNA than D39. Collectively, these results suggest that reduced efficiency in targeting and/or assembling DNA uptake apparatus impairs the genetic transformation in ∆scRNA.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

Influence mechanism of surface hardness of soil samples on plasma

Abstract Laser-induced breakdown spectroscopy (LIBS) can be used to quantitatively analyze heavy metal elements in soil, but the matrix effects have always been an important problem affecting the accuracy of quantitative analysis. The influence mechanism of matrix effects on plasma needs to be further studied. In this paper, the main purpose is to analyze the influence mechanism of sample surface hardness on plasma. The trend of plasma temperature, the ratio of ion line to atomic line of Cr and Cu elements, and the calibration curve of Cr and Cu elements with the change of sample surface hardness are analyzed, respectively. The results show that the plasma temperature and the ratio of ion line to atomic line of Cr and Cu elements increase with the increase of sample surface hardness. Meanwhile, the root mean square error (RMSE) and average relative error (ARE) of Cr and Cu elements in the samples with a hardness of 98HA are lower than those of the samples with a hardness of 43HA and 70HA. For samples with a surface hardness of 98HA, the RMSE and ARE of Cr are 0.027wt% and 9.763%, and those of Cu are 0.047wt% and 15.862%, respectively. However, the stability of the training data decreases with the surface hardness increases. The relative standard deviations (RSD) of Cr and Cu elements are 19.383% and 34.383%, respectively, for samples with a surface hardness of 98HA. By studying the influence of sample surface hardness on plasma temperature and calibration curve, the influence mechanism of sample surface hardness on plasma was explored.</jats:p

DprA-Dependent Exit from the Competent State Regulates Multifaceted Streptococcus pneumoniae Virulence

Streptococcus pneumoniae (pneumococcus) causes multiple infectious diseases. The pneumococcal competence system facilitates genetic transformation, spreads antibiotic resistance, and contributes to virulence. DNA-processing protein A (DprA) regulates the exit of pneumococcus from the competent state. Previously, we have shown that DprA is important in both bacteremia and pneumonia infections. Here, we examined the mechanisms of virulence attenuation in a Δ dprA mutant. </jats:p

Quantitative analysis of Cr in soil based on variable selection coupled with multivariate regression using laser-induced breakdown spectroscopy

Compared with LASSO, ALASSO can better select the variables of soil LIBS spectra.</jats:p