scholarly journals Fur regulation of the capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 419-429 ◽  
Author(s):  
Ching-Ting Lin ◽  
Chien-Chen Wu ◽  
Yu-Sheng Chen ◽  
Yi-Chyi Lai ◽  
Chia Chi ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Iron Acquisition ◽  
Regulatory Gene ◽  
Dependent Manner ◽  
Ferric Uptake Regulator ◽  
Mobility Shift ◽  
Polysaccharide Biosynthesis ◽  
Qrt Pcr ◽  
Electrophoretic Mobility Shift Assays

The ferric uptake regulator Fur has been reported to repress the expression of rmpA, a regulatory gene for the mucoid phenotype, leading to decreased capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae CG43. Here, quantitative real-time PCR (qRT-PCR) analyses and electrophoretic mobility shift assays showed that Fur also repressed the expression of the CPS regulatory genes rmpA2 and rcsA. Interestingly, deletion of rmpA or rcsA but not rmpA2 from the Δfur strain was able to suppress the deletion effect of Fur. The availability of extracellular iron affected the amount of CPS, suggesting that Fur regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of siderophores was observed in the Δfur strain, suggesting that uptake of extracellular iron in K. pneumoniae is regulated by Fur. Fur titration assays and qRT-PCR analyses demonstrated that at least six of the eight putative iron-acquisition systems, identified by a blast search in the contig database of K. pneumoniae CG43, were directly repressed by Fur. We conclude that Fur has a dual role in the regulation of CPS biosynthesis and iron acquisition in K. pneumoniae.

scholarly journals IscR Regulation of Capsular Polysaccharide Biosynthesis and Iron-Acquisition Systems in Klebsiella pneumoniae CG43

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107812 ◽  
Author(s):  
Chien-Chen Wu ◽  
Chien-Kuo Wang ◽  
Yu-Ching Chen ◽  
Tien-Huang Lin ◽  
Tzyy-Rong Jinn ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Iron Acquisition ◽  
Polysaccharide Biosynthesis

scholarly journals Role of the small RNA RyhB in the Fur regulon in mediating the capsular polysaccharide biosynthesis and iron acquisition systems in Klebsiella pneumoniae

2012 ◽  
Vol 12 (1) ◽  
pp. 148 ◽  
Author(s):  
Su-Hua Huang ◽  
Chien-Kuo Wang ◽  
Hwei-Ling Peng ◽  
Chien-Chen Wu ◽  
Ying-Tsong Chen ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Small Rna ◽  
Capsular Polysaccharide ◽  
Iron Acquisition ◽  
Polysaccharide Biosynthesis

scholarly journals RmpA Regulation of Capsular Polysaccharide Biosynthesis in Klebsiella pneumoniae CG43

2010 ◽  
Vol 192 (12) ◽  
pp. 3144-3158 ◽  
Author(s):  
H. Y. Cheng ◽  
Y. S. Chen ◽  
C. Y. Wu ◽  
H. Y. Chang ◽  
Y. C. Lai ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Promoter Activity ◽  
Deletion Mutant ◽  
Capsular Polysaccharide ◽  
Activity Measurement ◽  
Virulence Plasmid ◽  
Dependent Manner ◽  
Multicopy Plasmid ◽  
Regulatory Activity ◽  
Mobility Shift

ABSTRACT Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.

scholarly journals Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4170-4183 ◽  
Author(s):  
Hung-Yu Shu ◽  
Chang-Phone Fung ◽  
Yen-Ming Liu ◽  
Keh-Ming Wu ◽  
Ying-Tsong Chen ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Sequence Data ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Clinical Isolates ◽  
Polysaccharide Biosynthesis ◽  
Bacterial Surface ◽  
Polysaccharide Synthesis ◽  
Hospital Acquired

Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5′ end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3′ end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.

The Platelet Specific Chemokine Platelet Factor 4 Induces Expression of E-Selectin in an NF-κ B Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3908-3908
Author(s):  
Bruce S. Sachais ◽  
Peihong Ma ◽  
Ann H. Rux ◽  
Guangyao Yu
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Atherosclerotic Lesion ◽  
Surface Expression ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Platelet Factor ◽  
Umbilical Vein Endothelial Cells ◽  
Electrophoretic Mobility Shift Assays

Abstract The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4) is a platelet specific chemokine released upon platelet activation. PF4 has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin mRNA is time and dose dependent, and requires the presence of cell surface glycosaminoglycans. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. Activation of NF-κB is critical for PF4 induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. In summary, our data demonstrate that PF4 can increase expression of E-selectin by endothelial cells by activation of NF-κB. PF4 induction of endothelial E-selectin expression represents another mechanism by which platelets may participate in atherosclerotic lesion progression. These data also suggest that PF4 may participate in the proinflammatory functions of activated platelets.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

scholarly journals The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

2008 ◽  
Vol 190 (12) ◽  
pp. 4351-4359 ◽  
Author(s):  
Thomas J. Goss
Keyword(s):  
Klebsiella Pneumoniae ◽  
Upstream Region ◽  
Mobility Shift ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Sensitive Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

scholarly journals The Two-Component Signaling System VraSRssIs Critical for Multidrug Resistance and Full Virulence inStreptococcus suisSerotype 2

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Xiaojun Zhong ◽  
Yue Zhang ◽  
Yinchu Zhu ◽  
Wenyang Dong ◽  
Jiale Ma ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Capsular Polysaccharide ◽  
Gene Clusters ◽  
Mobility Shift ◽  
Content Type ◽  
Signaling Systems ◽  
Two Component ◽  
Human Infections ◽  
Host Blood ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTStreptococcus suishas received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located inS. suisserotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that theyvqFSSgene, located upstream ofvraSRSS, shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSSand VraSRSSconstitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSSinactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSSexpression observed in host blood, strongly suggested that VraSRSSis an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSSstrains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSSindirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused byvraSRSSdeletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS. Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.

scholarly journals Surface Antigen Exposure by Bismuth Dimercaprol Suppression of Klebsiella pneumoniae Capsular Polysaccharide

1999 ◽  
Vol 67 (2) ◽  
pp. 664-669 ◽  
Author(s):  
Philip Domenico ◽  
J. M. Tomas ◽  
S. Merino ◽  
X. Rubires ◽  
Burke A. Cunha
Keyword(s):  
Membrane Proteins ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Outer Membrane Proteins ◽  
Defined Medium ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
O Antigen ◽  
Bacterial Capsule

ABSTRACT The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi3+repressed Klebsiella pneumoniae capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K− cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K− cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of K. pneumoniae after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated K. pneumoniae. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.

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