Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance

ABSTRACT CTX-M β-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum β-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using bla CTX-M-1, bla CTX-M-3, and bla CTX-M-10 genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other β-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.

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Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00050-10 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3484-3488 ◽

Cited By ~ 46

Author(s):

José-Manuel Rodríguez-Martínez ◽

Patrice Nordmann ◽

Esthel Ronco ◽

Laurent Poirel

Keyword(s):

Acinetobacter Baumannii ◽

Clinical Isolate ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Extended Spectrum ◽

Class C ◽

High Level ◽

Level Resistance

ABSTRACT An AmpC-type β-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C β-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.

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Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

Infection and Immunity ◽

10.1128/iai.01138-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 187-193 ◽

Cited By ~ 12

Author(s):

Renu Verma ◽

Thaís Cabrera Galvão Rojas ◽

Renato Pariz Maluta ◽

Janaína Luisa Leite ◽

Livia Pilatti Mendes da Silva ◽

...

Keyword(s):

Escherichia Coli ◽

Soft Agar ◽

Pleiotropic Effect ◽

Biological Characteristics ◽

Wild Type ◽

Content Type ◽

Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

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C1-Inactivator as a Determining Factor in Contact Activation of Fibrinolysis

10.1055/s-0039-1689476 ◽

1975 ◽

Author(s):

C. Kluft

Keyword(s):

Fibrinolytic Activity ◽

Slow Rate ◽

Contact Activation ◽

Activation Rate ◽

Activity Generation ◽

High Level ◽

Fletcher Factor ◽

Determining Factor

The rate of contact activation of fibrinolysis is considered to reflect the activation rate of proactivator and Hageman factor. This study was undertaken to determine the role of Cl-inactivator in this process.Contact activation of fibrinolysis was performed according to Ogston et al. (1969), J. Clin. Invest. 48, 1786-1801. The rate of activity generation was measured in plasma with various levels of Cl-inactivator and appeared to be dependent on that level; i.e., a high level of Cl-inactivator corresponds with a slow rate of activity generation.It has recently been demonstrated that the fibrinolytic activity of euglobulin fractions is strongly inhibited by Cl-inactivator also present in this fraction. The activity generation of contact activation is found to be accompanied by a gradual decrease in functional Cl-inactivator in the euglobulin fraction. The fibrinolytic activity is set free by this disappearance of inhibition.It is concluded that the rate of contact activation of fibrinolysis must be interpreted in terms of the inactivation of Cl-inactivator rather than of the activation of proenzymes. All enzymes capable of inactivating Cl-inactivator can contribute to the process of contact activation of fibrinolysis. This mechanism might account for the observed defects in fibrinolysis in vitro in Fletcher Factor deficient patients.

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ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

10.1055/s-0038-1643843 ◽

1987 ◽

Author(s):

G A Vehar ◽

K M Tate ◽

D L Higgins ◽

W E Holmes ◽

H L Heyneker

Keyword(s):

Cho Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Single Chain ◽

Serine Protease Family ◽

The One ◽

Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

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TEM-158 (CMT-9), a New Member of the CMT-Type Extended-Spectrum β-Lactamases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00614-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4181-4183 ◽

Cited By ~ 8

Author(s):

Frédéric Robin ◽

Julien Delmas ◽

Amélie Brebion ◽

Damien Dubois ◽

Jean-Michel Constantin ◽

...

Keyword(s):

Hydrolytic Activity ◽

Detection Methods ◽

Extended Spectrum ◽

Accurate Detection ◽

High Level

ABSTRACT TEM-158 was found to include the substitutions previously observed for TEM-12 and TEM-35. This enzyme presented hydrolytic activity against ceftazidime and a high level of resistance against clavulanate, which can alter its detection. Its discovery highlights the need for accurate detection methods.

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Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

Journal of Virology ◽

10.1128/jvi.75.23.11284-11291.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11284-11291 ◽

Cited By ~ 161

Author(s):

David A. Einfeld ◽

Rosanna Schroeder ◽

Peter W. Roelvink ◽

Alena Lizonova ◽

C. Richter King ◽

...

Keyword(s):

Adenovirus Type ◽

Wild Type ◽

Base Modification ◽

Model Following ◽

Vector Distribution ◽

High Level ◽

Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

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Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein fromEscherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

Journal of Bacteriology ◽

10.1128/jb.180.18.4799-4803.1998 ◽

1998 ◽

Vol 180 (18) ◽

pp. 4799-4803 ◽

Cited By ~ 55

Author(s):

Frédérique Pompeo ◽

Jean van Heijenoort ◽

Dominique Mengin-Lecreulx

Keyword(s):

Active Site ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Acetyl Coa ◽

Mutant Proteins ◽

Acetyltransferase Activity ◽

Phosphate Acetyltransferase ◽

High Level

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

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Susceptibility trends of zoliflodacin against multidrug-resistant Neisseria gonorrhoeae clinical isolates in Nanjing, China (2014-2018)

10.1101/2020.05.04.078055 ◽

2020 ◽

Author(s):

Wenjing Le ◽

Xiaohong Su ◽

Xiangdi Lou ◽

Xuechun Li ◽

Xiangdong Gong ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Dna Sequencing ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Drug Resistant ◽

Extended Spectrum ◽

High Level ◽

Level Resistance

ABSTRACTPreviously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 gonococcal isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC and parE genes were determined by PCR and DNA sequencing. The MIC of zoliflodacin for N. gonorrhoeae ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 μg/ml); 21.2% (209/986) were resistant to azithromycin (≥1 μg/ml), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 143 isolates with higher zoliflodacin MICs (0.125-0.25 mg/L), all had quinolone resistance associated double or triple mutations in gyrA; 139/143 (97.2%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin has excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

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Forskolin enhances the antitumor effect of oncolytic measles virus by promoting Rab27a dependent vesicular transport system in the lung cancer A549 cells in vitro

10.21203/rs.3.rs-119567/v1 ◽

2020 ◽

Author(s):

Mao Xia ◽

Yongquan Xia ◽

Xuejing Xu ◽

Gang Meng ◽

Hong Yan ◽

...

Keyword(s):

Tumor Cells ◽

Transport System ◽

Measles Virus ◽

Antitumor Effect ◽

Vesicular Transport ◽

A549 Cells ◽

Over Expression ◽

Combined Strategy

Abstract Background: Measles vaccine strain viruses (MV-Edm) are an ideal platform for developing safe and effective oncolytic vectors. However, despite the promising pre-clinical data, understanding of determinants of efficacy and, thus, the interplay of the oncolytic virus with particular agents remains limited.Methods: We investigated the potency of forskolin enhancing the antitumor effect of oncolytic measles virus by promoting Rab27a dependent vesicular transport system. Cells were infected with MV-Edm and the vesicles were observed by TEM. The oncolytic effects of MV-Edm/Forskolin were investigated in vitro. Results: Here we demonstrate that the MV-Edm infection and spread in tumor, which are indispensable processes for the viral oncolysis, depend on the vesicular transport system of tumor cells. On the contrary, the tumor cells display a responsive mechanism to restrain the MV-Edm spread by down-regulating the expression of Rab27a, which is a key member of the vesicle transport system. Over-expression of Rab27a promotes the oncolytic efficacy of MV-Edm towards A549 tumor cells. Finally, we find a Rab27a agonist Forskolin, is capable of promoting the oncolytic effect of MV-Edm in vitro. Conclusions: Our study reveals the important role of vesicle transporter Rab27a in the whole program of MV-Edm mediated oncolysis. We also provide a combined strategy of Forskolin and MV-Edm, which may exert a synergistic anti-tumor effect, for clinical treatment for patients with tumor.

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The Association of Aberrant Expression of FGF1 and mTOR-S6K1 in Colorectal Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.706838 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tinghui Duan ◽

Diyuan Zhou ◽

Yizhou Yao ◽

Xinyu Shao

Keyword(s):

Colorectal Cancer ◽

Malignant Neoplasms ◽

Aberrant Expression ◽

Protein Levels ◽

And Migration ◽

High Level ◽

Proliferation And Migration

Colorectal cancer (CRC) is one of the most frequent malignant neoplasms worldwide, and the effect of treatments is limited. Fibroblast growth factor 1 (FGF1) has been involved in a wide variety of several malignant diseases and takes part in the tumorigenesis of CRC. However, the function and mechanism of FGF1 in CRC remains elusive. In this study, the results indicated that FGF1 is elevated in CRC tissues and linked with poor prognosis (P < 0.001). In subgroup analysis of FGF1 in CRC, regardless of any clinic-factors except gender, high level FGF1 expression was associated with markedly shorter survival (P < 0.05). In addition, the expression of p-S6K1 and FGF1 was not associated in normal tissue (P = 0.781), but their expression was closely related in tumor tissue (P = 0.010). The oncogenic role of FGF1 was determined using in vitro and in vivo functional assays. FGF1 depletion inhibited the proliferation and migration of CRC cells in vitro and vivo. FGF1 was also significantly correlated with mTOR-S6K1 pathway on the gene and protein levels (P < 0.05). In conclusion, FGF1 acts as a tumor activator in CRC, and against FGF1 may provide a new visual field on treating CRC, especially for mTORC1-targeted resistant patients.

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