Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

ABSTRACTEnterobacter cloacaecomplex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation ofampC,ampR(which encodes the regulator protein ofampC), andampG(encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression ofampCin different ways: one involving NagZ (aN-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established forPseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutiveampCoverexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of adacBdeletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistancein vivoas opposed toP. aeruginosawheredacBmutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

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Draft Genome Sequences of Two Enterobacter cloacae subsp. cloacae Strains Isolated from Australian Hematology Patients with Bacteremia

Genome Announcements ◽

10.1128/genomea.00756-17 ◽

2017 ◽

Vol 5 (33) ◽

Cited By ~ 1

Author(s):

Lex E. X. Leong ◽

David Shaw ◽

Lito Papanicolas ◽

Diana Lagana ◽

Ivan Bastian ◽

...

Keyword(s):

Gut Microbiota ◽

Draft Genome ◽

Enterobacter Cloacae ◽

Opportunistic Pathogen ◽

Healthy Individuals ◽

Genome Sequences ◽

Content Type ◽

Extended Spectrum ◽

Genes Encoding

ABSTRACT Enterobacter cloacae is a common member of the gut microbiota in healthy individuals. However, it is also an opportunistic pathogen, capable of causing bacteremia. We report the draft genomes of two Enterobacter cloacae subspecies cloacae strains isolated from hematology patients with bacteremia. Both isolates carry genes encoding extended-spectrum β-lactamases.

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Acinetobacter baumanniiOxyR Regulates the Transcriptional Response to Hydrogen Peroxide

Infection and Immunity ◽

10.1128/iai.00413-18 ◽

2018 ◽

Vol 87 (1) ◽

Cited By ~ 16

Author(s):

Lillian J. Juttukonda ◽

Erin R. Green ◽

Zachery R. Lonergan ◽

Marie C. Heffern ◽

Christopher J. Chang ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Drug Targets ◽

Transcriptional Response ◽

Transcriptional Regulator ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

World Health ◽

Content Type

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that causes diverse infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired antimicrobial-resistance mechanisms,A. baumanniiisolates are commonly multidrug resistant, and infections are notoriously difficult to treat. The World Health Organization recently highlighted carbapenem-resistantA. baumanniias a “critical priority” for the development of new antimicrobials because of the risk to human health posed by this organism. Therefore, it is important to discover the mechanisms used byA. baumanniito survive stresses encountered during infection in order to identify new drug targets. In this study, by use ofin vivoimaging, we identified hydrogen peroxide (H2O2) as a stressor produced in the lung duringA. baumanniiinfection and defined OxyR as a transcriptional regulator of the H2O2stress response. Upon exposure to H2O2,A. baumanniidifferentially transcribes several hundred genes. However, the transcriptional upregulation of genes predicted to detoxify hydrogen peroxide is abolished in anA. baumanniistrain in which the transcriptional regulatoroxyRis genetically inactivated. Moreover, inactivation ofoxyRin both antimicrobial-susceptible and multidrug-resistantA. baumanniistrains impairs growth in the presence of H2O2. OxyR is a direct regulator ofkatEandahpF1, which encode the major H2O2-degrading enzymes inA. baumannii, as confirmed through measurement of promoter binding by recombinant OxyR in electromobility shift assays. Finally, anoxyRmutant is less fit than wild-typeA. baumanniiduring infection of the murine lung. This work reveals a mechanism used by this important human pathogen to survive H2O2stress encountered during infection.

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Aberrantly high activation of a FoxM1–STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00396-0 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Jun Liu ◽

Jipeng Li ◽

Ke Wang ◽

Haiming Liu ◽

Jianyong Sun ◽

...

Keyword(s):

Cell Proliferation ◽

Poor Prognosis ◽

Soft Agar ◽

Transcriptional Level ◽

Regulation Mechanism ◽

Strong Positive Correlation ◽

Cell Viability Assay ◽

High Level

AbstractFork-head box protein M1 (FoxM1) is a transcriptional factor which plays critical roles in cancer development and progression. However, the general regulatory mechanism of FoxM1 is still limited. STMN1 is a microtubule-binding protein which can inhibit the assembly of microtubule dimer or promote depolymerization of microtubules. It was reported as a major responsive factor of paclitaxel resistance for clinical chemotherapy of tumor patients. But the function of abnormally high level of STMN1 and its regulation mechanism in cancer cells remain unclear. In this study, we used public database and tissue microarrays to analyze the expression pattern of FoxM1 and STMN1 and found a strong positive correlation between FoxM1 and STMN1 in multiple types of cancer. Lentivirus-mediated FoxM1/STMN1-knockdown cell lines were established to study the function of FoxM1/STMN1 by performing cell viability assay, plate clone formation assay, soft agar assay in vitro and xenograft mouse model in vivo. Our results showed that FoxM1 promotes cell proliferation by upregulating STMN1. Further ChIP assay showed that FoxM1 upregulates STMN1 in a transcriptional level. Prognostic analysis showed that a high level of FoxM1 and STMN1 is related to poor prognosis in solid tumors. Moreover, a high co-expression of FoxM1 and STMN1 has a more significant correlation with poor prognosis. Our findings suggest that a general FoxM1-STMN1 axis contributes to cell proliferation and tumorigenesis in hepatocellular carcinoma, gastric cancer and colorectal cancer. The combination of FoxM1 and STMN1 can be a more precise biomarker for prognostic prediction.

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Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02425-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5589-5593 ◽

Cited By ~ 25

Author(s):

Anna L. Sartor ◽

Muhammad W. Raza ◽

Shahid A. Abbasi ◽

Kathryn M. Day ◽

John D. Perry ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Epidemiology ◽

Genetic Relatedness ◽

Antibiotic Resistance Genes ◽

Enterobacter Cloacae ◽

Content Type ◽

Plasmid Replicon ◽

Genes Encoding ◽

Clonal Spread ◽

Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

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The Transporter-Mediated Cellular Uptake and Efflux of Pharmaceutical Drugs and Biotechnology Products: How and Why Phospholipid Bilayer Transport Is Negligible in Real Biomembranes

Molecules ◽

10.3390/molecules26185629 ◽

2021 ◽

Vol 26 (18) ◽

pp. 5629

Author(s):

Douglas B. Kell

Keyword(s):

Lipid Bilayers ◽

Drug Targets ◽

Drug Transport ◽

Phospholipid Bilayer ◽

Substrate Uptake ◽

Pharmaceutical Drugs ◽

Natural Evolution ◽

Lipid Ii ◽

High Concentrations

Over the years, my colleagues and I have come to realise that the likelihood of pharmaceutical drugs being able to diffuse through whatever unhindered phospholipid bilayer may exist in intact biological membranes in vivo is vanishingly low. This is because (i) most real biomembranes are mostly protein, not lipid, (ii) unlike purely lipid bilayers that can form transient aqueous channels, the high concentrations of proteins serve to stop such activity, (iii) natural evolution long ago selected against transport methods that just let any undesirable products enter a cell, (iv) transporters have now been identified for all kinds of molecules (even water) that were once thought not to require them, (v) many experiments show a massive variation in the uptake of drugs between different cells, tissues, and organisms, that cannot be explained if lipid bilayer transport is significant or if efflux were the only differentiator, and (vi) many experiments that manipulate the expression level of individual transporters as an independent variable demonstrate their role in drug and nutrient uptake (including in cytotoxicity or adverse drug reactions). This makes such transporters valuable both as a means of targeting drugs (not least anti-infectives) to selected cells or tissues and also as drug targets. The same considerations apply to the exploitation of substrate uptake and product efflux transporters in biotechnology. We are also beginning to recognise that transporters are more promiscuous, and antiporter activity is much more widespread, than had been realised, and that such processes are adaptive (i.e., were selected by natural evolution). The purpose of the present review is to summarise the above, and to rehearse and update readers on recent developments. These developments lead us to retain and indeed to strengthen our contention that for transmembrane pharmaceutical drug transport “phospholipid bilayer transport is negligible”.

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Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00363-20 ◽

2020 ◽

Vol 64 (11) ◽

Cited By ~ 2

Author(s):

Matt D. Johansen ◽

Wassim Daher ◽

Françoise Roquet-Banères ◽

Clément Raynaud ◽

Matthéo Alcaraz ◽

...

Keyword(s):

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Zebrafish Model ◽

Content Type ◽

Attractive Option ◽

Conventional Antibiotics ◽

Susceptibility Profiles ◽

Combination Regimens

ABSTRACT Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.

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Spatiotemporal Distribution of Pseudomonas aeruginosa Alkyl Quinolones under Metabolic and Competitive Stress

mSphere ◽

10.1128/msphere.00426-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Tianyuan Cao ◽

Jonathan V. Sweedler ◽

Paul W. Bohn ◽

Joshua D. Shrout

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Species ◽

Metabolic Stress ◽

Opportunistic Pathogen ◽

Cell Interaction ◽

Chemical Imaging ◽

Carbon Limitation ◽

Bacterial Strains ◽

Content Type ◽

High Level

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen important to diseases such as cystic fibrosis. P. aeruginosa has multiple quorum-sensing (QS) systems, one of which utilizes the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). Here, we use hyperspectral Raman imaging to elucidate the spatiotemporal PQS distributions that determine how P. aeruginosa regulates surface colonization and its response to both metabolic stress and competition from other bacterial strains. These chemical imaging experiments illustrate the strong link between environmental challenges, such as metabolic stress caused by nutritional limitations or the presence of another bacterial species, and PQS signaling. Metabolic stress elicits a complex response in which limited nutrients induce the bacteria to produce PQS earlier, but the bacteria may also pause PQS production entirely if the nutrient concentration is too low. Separately, coculturing P. aeruginosa in the proximity of another bacterial species, or its culture supernatant, results in earlier production of PQS. However, these differences in PQS appearance are not observed for all alkyl quinolones (AQs) measured; the spatiotemporal response of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) is highly uniform for most conditions. These insights on the spatiotemporal distributions of quinolones provide additional perspective on the behavior of P. aeruginosa in response to different environmental cues. IMPORTANCE Alkyl quinolones (AQs), including Pseudomonas quinolone signal (PQS), made by the opportunistic pathogen Pseudomonas aeruginosa have been associated with both population density and stress. The regulation of AQ production is known to be complex, and the stimuli that modulate AQ responses are not fully clear. Here, we have used hyperspectral Raman chemical imaging to examine the temporal and spatial profiles of AQs exhibited by P. aeruginosa under several potentially stressful conditions. We found that metabolic stress, effected by carbon limitation, or competition stress, effected by proximity to other species, resulted in accelerated PQS production. This competition effect did not require cell-to-cell interaction, as evidenced by the fact that the addition of supernatants from either Escherichia coli or Staphylococcus aureus led to early appearance of PQS. Lastly, the fact that these modulations were observed for PQS but not for all AQs suggests a high level of complexity in AQ regulation that remains to be discerned.

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Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00243-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 17

Author(s):

Katharina Schaufler ◽

Torsten Semmler ◽

Lothar H. Wieler ◽

Darren J. Trott ◽

Johann Pitout ◽

...

Keyword(s):

Escherichia Coli ◽

High Risk ◽

Multidrug Resistant ◽

Phenotypic Analysis ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

Niche Adaptation ◽

High Level

ABSTRACT The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n = 107) and ST10 (n = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.

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Identification of Genes That Contribute to the Pathogenesis of Invasive Pneumococcal Disease byIn VivoTranscriptomic Analysis

Infection and Immunity ◽

10.1128/iai.00295-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3268-3278 ◽

Cited By ~ 44

Author(s):

Abiodun D. Ogunniyi ◽

Layla K. Mahdi ◽

Claudia Trappetti ◽

Nadine Verhoeven ◽

Daphne Mermans ◽

...

Keyword(s):

Invasive Pneumococcal Disease ◽

Pneumococcal Disease ◽

Targeted Mutagenesis ◽

Content Type ◽

Genome Wide ◽

Blood Relative ◽

High Level ◽

Upregulated Genes

ABSTRACTStreptococcus pneumoniae(the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-widein vivotranscriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliAand ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiOand ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products ofaliA,malX, andpiuAare promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such asS. pyogenes,Haemophilus influenzaetype b, andNeisseria meningitidis.

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Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04694-14 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4669-4679 ◽

Cited By ~ 23

Author(s):

Nilmar Silvio Moretti ◽

Leonardo da Silva Augusto ◽

Tatiana Mordente Clemente ◽

Raysa Paes Pinto Antunes ◽

Nobuko Yoshida ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Drug Targets ◽

Wild Type ◽

Content Type ◽

Damage Repair ◽

Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

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