The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Role of Pseudomonas aeruginosa mifSR Two-Component System in Antibiotic Resistance and Biofilm Formation

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments. It is one of the leading causes of morbidity and mortality in cystic fibrosis patients, and one of the main sources of nosocomial infections in the United States. One of the most prominent features of this pathogen is its wide resistance to antibiotics. P. aeruginosa employs a variety of mechanisms including efflux pumps and the expression of B-lactamases to overcome antibiotic treatment. Two chromosomally encoded lactamases, ampC and poxB, have been identified in P. aeruginosa. Sequence analyses have shown the presence of a two-component system (TCS) called MifSR (MifS-Sensor and MifR-Response Regulator), immediately upstream of the poxAB operon. It is hypothesized that the MifSR TCS is involved in B-lactam resistance via the regulation of poxB. Recently, the response regulator MifR has been reported to play a crucial role in biofilm formation, a major characteristic of chronic infections and increased antibiotic resistance. In this study, mifR and mifSR deletion mutants were constructed, and compared to the wild type parent strain PAOl for differences in growth and B-lactam sensitivity. Results obtained thus far indicate that mifR and mifSR are not essential for growth, and do not confer B-lactam resistance under the conditions tested. This study is significant because biofilm formation and antibiotic resistance are two hallmarks of P. aeruginosa infections, and finding a link between these two may lead to the development of improved treatment strategies.

Antimicrobial effect of Artemisia species on Pseudomonas aeruginosa

Antibiotic resistance has emerged as a severe problem in hospital-acquired infectious disease. The Gram-negative bacterium Pseudomonas aeruginosa is found to cause secondary infection in immune-compromised patients. Unfortunately, it is resistant to virtually all β-lactam antibiotics such as penicillin, cephalosporin and others. Researchers are seeking for new compounds to treat several antibiotic-resistant bacterial strains. Artemisia plant extracts are commonly used for their therapeutic properties by natives throughout dry regions of North and South America. Here, they are administered as an alternative medicine for stomach problems and other complex health issues. In this study, the antimicrobial effects of plant extracts from several Artemisia species as well as compounds dehydroleucodine and dehydroparishin-B (sesquiterpenes derived specifically from A. douglasiana) were used as treatments against the pathogenicity effects of P. aeruginosa. Results showed that both compounds effectively inhibit the secretion of LasB elastase, biofilm formation and type III secretion, but fail to control LasA protease. This is a significant observation because these virulent factors are crucial in establishing P.aeruginosa infection. The results from this study signify a plausible role for future alternative therapy in the biomedical field, which recommends DhL and DhP can be studied as key compounds against bacterial infections of Pseudomonas aeruginosa.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

Background In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link. Results Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP) and PA4393 (ampG). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP andampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampPoperon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampGoperon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampPand ampG dependent pathway, and at high concentrations where although both ampP and ampGplay a role, ampG may be of greater importance. Both ampP and ampG are required for maximum induction. Similar to ampC, ampP expression is inducible in an ampR-dependent manner. Importantly, ampP expression is autoregulated and ampP also regulates expression of ampG. Both AmpG and AmpP have topologies consistent with functions in transport. Together, these data suggest that the mechanism of β-lactam resistance of P. aeruginosa is distinct from well characterized systems in Enterobacteriaceae and involves a highly complicated interaction between these putative permeases and known Amp proteins.

Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa,

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

Screening for AmpR-Specific Inhibitors to Combat P. aeruginosa Infections

Pseudomonas aeruginosa, a Gram-negative bacterium, an opportunistic pathogen that infects individuals suffering from reduced immunity or damaged tissue. The treatment of these infections has become a major problem due to its increasing antibiotic resistance. Many multi-drug resistant isolates of P. aeruginosa can thwart most antibiotic classes including ?- lactams, fluoroquinolones, and aminoglycosides. Its ability to combat ?-lactams is in part due to expression of AmpC, a major chromosomally encoded ?-lactamase. The expression of ampC is positively regulated by AmpR. Besides antibiotic resistance, AmpR is an important regulator of various factors that are required for establishing acute and chronic infections. Loss of ampR makes P. aeruginosa susceptible to ?-lactams and less virulent than the wild type. We hypothesize that AmpR is a potential therapeutic target. In the absence of new drugs in the pipeline, the aim of this study is to find an AmpR-specific inhibitor to assist and improve the use of currently available ?- lactam treatment. A small-molecule library from Torrey Pines Institute will be used in this study. Two reporter systems, lux and lacZ, fused to a PampC promotor will be used to assess AmpR activity. Positive hits will be those that inhibit 50% PampC activity in the presence of sub inhibitory concentration of imipenem, a ?- lactam. The top positive hits will be screened for their ability to cause human cell-cytotoxicity. The non-cytotoxic hits will be assessed for their ability to affect P. aeruginosa virulence and antibiotic resistance using various in vitro assays. Determination of potential AmpR inhibitors will prove to be useful in fighting off infections and may save countless patients suffering from these infections.

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Characterization of the poxAB operon encoding a class D carbapenemase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.^