1,721,040 research outputs found
Cell-based fluorescent screen to identify inhibitors of bacterial translation initiation
No full textA strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of proper assays aimed at the identification of molecules interfering with specific cell pathways and potentially applicable to the high throughput analysis of large chemical library. Here, I describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria vs. mammals. The effect of the compounds under analysis is assayed in living cells, thus allowing evaluating their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity and high mechanism specificity
Antisense RNA-dependent transcription termination sites that modulate lysogenic development of satellite phage P4
No full textIn the lysogenic state, bacteriophage P4 prevents the expression of its own replication genes, which are encoded in the left operon, through premature transcription termination. The phage factor responsible for efficient termination is a small, untranslated RNA (Cl RNA), which acts as an antisense RNA and controls transcription termination by pairing with two complementary sequences (seqA and seqC) located within the leader region of the left operon. A Rho-dependent termination site, t(imm), was previously shown to be involved in the control of P4 replication gene expression. In the present study, by making use of phage ΦR73 as a cloning vector and of suppressor tRNA(Gly) as a reporter gene, we characterized two additional terminators, t1 and t4. Although transcription termination at neither site requires the Rho factor, only t1 has the typical structure of a Rho-independent terminator. t1 is located between the P(LE) promoter and the cl gene, whereas t4 is located between cl and t(imm). Efficient termination at t1 requires the Cl RNA and the seqA target sequence; in vitro, the Cl RNA enhanced termination at t1 in the absence of any bacterial factor. A P4 mutant, in which the t1 terminator has been deleted, can still lysogenize both Rho+ and Rho- strains and exhibits increased expression of Cl RNA. These data indicate that t1 and the Rho-dependent t(imm) terminators are not essential for lysogeny. t1 is involved in Cl RNA autoregulation, whereas t4 appears to be the main terminator necessary to prevent expression of the lytic genes in the lysogenic state
RNase III-independent autogenous regulation of Escherichia coli polynucleotide phosphorylase via translational repression
Full text linkThe complex post-transcriptional regulation mechanism of Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5' -end by RNase III, rather than the primary pnp transcript (RNase III-dependent models) and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δpnp rnc(+) and Δpnp Δrnc strains with a chromosomal pnp-lacZ translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream of the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1, but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1.
In Escherichia coli, polynucleotide phosphorylase (PNPase, encoded by pnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of pnp mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability
Temperature-dependent regulation of the lpxT gene in Escherichia coli and Pseudomonas aeruginosa
No full textThe LpxT protein modifies the outer membrane of Gram negative bacteria by transferring a phosphate group from undecaprenyl-pyrophosphate to the lipid A moiety of the lipopolysaccharide. Recently, we found that the expression of the lpxT gene of Pseudomonas aeruginosa (Pa) is post-transcriptionally regulated by an RNA thermometer (RNAT). An RNAT is a thermo-labile secondary structure that entraps the mRNA Translation Initiation Region (TIR) at low temperature, thus inhibiting ribosome binding. As temperature increases, the RNAT unfolds allowing mRNA translation. The bioinformatic analysis of the Escherichia coli (Ec) lpxT 5’-untranslated region (5’-UTR) shows that the TIR may be sequestered in a stem-loop structure with features typically found in some RNATs, thus suggesting a conserved regulatory strategy for Ec and Pa lpxT. On the other hand, Ec lpxT mRNA was claimed to be a direct target of the sRNA MicA, which was proposed to pair ca. 90 nt upstream of the lpxT AUG and negatively regulate lpxT expression. To assess whether Ec lpxT is regulated by temperature and/or MicA, we have assayed the expression of different lpxT-GFP translational fusions in the BW25113 strain and in its isogenic ∆micA derivative at 28° and 42°C. Moreover, we analysed in the same strains and conditions the transcription profile of the chromosomal lpxT locus. We found that i) the lpxT transcript starts 29 nt upstream of the ORF start codon; ii) the presence of the physiological lpxT 5’-UTR confers thermo-dependent expression to the reporter construct; ii) lpxT-GFP transcripts with a longer 5’-UTR, encompassing the putative MicA interaction site, are poorly expressed at any temperature; iii) MicA does not affect the expression of the reporter constructs. On the whole our results suggest that Ec lpxT may be regulated by a new RNAT. Experiments are in progress in our lab to characterize the Ec lpxT putative RNAT structure and function
Exploring pyrazinamide derivatives as novel Pseudomonas aeruginosa inhibitors: unexploited antibacterial molecules for a new antibiotics target
No full textBackground and Rationale
P. aeruginosa is the most common pathogen in cystic fibrosis lung infection with a high negative impact on lung functionality and patients’ mortality. The increasing diffusion of multi-resistant strains demands for the development of new anti-Pa agents. The ribosomal protein S1 (encoded by rpsA) is a promising target for new antibacterial drugs. In Escherichia coli, S1 has an essential role in translation. S1 is highly conserved among Gram negative bacteria and absent in mammalian cells. Recently, it has been found that pyrazinamide (PZA), a first-line tuberculosis drug, targets S1 protein. PZA derivatives were developed by Bracco SpA in the early ‘60s and roughly characterized for antibacterial activity; interestingly, in preliminary tests, the derivative B2320 seemed to be active against Pseudomonas aeruginosa (Pa). B2320 target in Pa is currently unknown.
Hypothesis and objectives
1. Characterizing the anti-Pa activity of B2320.
2. Exploring Pa S1 as a potential target for new antibacterials.
3. Setting-up an E. coli biosensor strain for the screening of S1 inhibitors.
Results and their significance
1. B2320 has been tested for anti-bacterial activity both against Pseudomonas lab strains and a collection of clinical isolates from CF patients in different growth conditions. We found that although only high concentrations of B2320 impaired aerobic growth of most P. aeruginosa strains, the sensitivity to B2320 was increased by stress conditions and in vivo, in the infection model Galleria mellonella. The genome of a clinical isolate particularly sensitive to B2320 and of a B2320-resistant derivative were sequenced.
2. rpsA essentiality in P. aeruginosa has been addressed by site-directed mutagenesis. Our data strongly support rpsA essentiality in P. aeruginosa.
3. We have set up a fluorescence-based assay to find inhibitors of S1-dependent translation initiation in E. coli. The screen allows to identify compounds interfering with an essential and bacteria specific step of gene expression, discriminating from molecules with nonspecific cell toxicity. Moreover, as we have adapted the assay to P. aeruginosa, cell penetration properties of the active compounds identified in the primary screening could be easily assessed. Since our screen is technically very simple and appears to be robust, it is potentially adaptable to High-Throughput-Screening campaigns
S1 ribosomal protein over-expression inhibits RNase E-dependent decay in Escherichia coli
No full textIt is commonly accepted that in bacteria transcription, translation and degradation of RNA are tightly coordinated processes; however, the molecular bases of this interconnection are poorly understood. S1 is the largest E. coli ribosomal protein; it is very abundant and weakly associated with the 30S ribosomal subunit. Both its over-expression and depletion impair bacterial growth. In a previous work we showed that S1 over-expression leads to a general increase in mRNA stability. To further characterize this phenomenon, we tested the role of 5'-UTR in S1-dependent stabilization. As a model transcript, we chose the cspE mRNA, which is sensitive to S1 stabilization. We showed by EMSA that S1 still binds a cspE RNA lacking the 5'-UTR (cspE-Del), albeit with lower affinity relative to the complete mRNA (cspE-wt), suggesting that for this mRNA different S1 binding sites may exist. In vivo, cspE-Del was less stable than cspE-wt RNA. Upon S1 induction, both the transcripts were stabilized; moreover, different RNA decay intermediates, deriving from cspE-Del degradation at the 5'-end, were detected, whereas no cspE-wt degradation products were present. In an rne thermosensitive mutant expressing cspE-Del, these decay intermediates were produced and stabilized, suggesting that RNase E may be involved in their degradation but not in their production. On the whole, our data suggest that S1 may interact both with the 5'-UTR and/or the 3'-UTR and prevent RNase E-dependent mRNA degradation but not the RNase E-independent endonucleolytic pathway operating on the leaderless cspE mRNA
A whole-cell assay for specific inhibitors of translation initiation in bacteria
No full textThe bacterial translational apparatus is an ideal target for the search of new antibiotics. In fact, it performs an essential process carried out by a large number of potential subtargets for antibiotic action. Moreover, it is sufficiently different in several molecular details from the apparatus of Eukarya and Archaea to generally ensure specificity for the bacterial domain. This applies in particular to translation initiation, which is the most different step in the process. In bacteria, the 30S ribosomal subunit directly binds to the translation initiation region, a site within the messenger RNA (mRNA) 5'-untranslated region (5'-UTR). 30S binding is mediated by the interaction of both the 16S ribosomal RNA and the ribosomal protein S1 with specific regions of the mRNA 5'-UTR. An alternative, S1-independent pathway is enjoyed by leaderless mRNAs (i.e., transcripts devoid of a 5'-UTR). We have developed a simple fluorescence-based whole-cell assay in Escherichia coli to find inhibitors of the canonical S1-dependent translation initiation pathway. The assay has been set up both in a common E. coli laboratory strain and in a strain with an outer membrane permeability defect. Compared with other whole-cell assays for antibacterials, the major advantages of the screen described here are high sensitivity and specificity
Phage therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients
No full textPseudomonas aeruginosa is the most common pathogen found in the lung of cystic fibrosis patients. The use of phage therapy could help in fighting the alarming diffusion of antibiotic multi-resistant strains.
A number of new phages were isolated from sewage samples in Milan, and tested for growth on a panel of P. aeruginosa strains collected in Italian hospitals. Comprehensively, we analyzed 23 new phages on 57 clinical or environmental P. aeruginosa strains. Six phages belonging to different classes, i.e. Myoviridae, Podoviridae and Siphoviridae, as assessed by TEM analysis, were mixed in a cocktail. The host range of the phage cocktail was larger than that of individual phages. Infection in liquid culture of strain PAO1 indicated that the phage cocktail efficiently killed the bacterial cells, although resistant mutants appeared at the end. The ability to infect P. aeruginosa growing in biofilm demonstrated that the phage cocktail was able to reduce the biomass of a preformed biofilm. DNA was extracted from the selected phages and send for whole genome shotgun sequencing using the Illumina MiSeq platform at the CNR IBBE institute in Bari.
This project is financed by Italian Foundation of Cystic Fibrosis (# 17/2015)
S1 ribosomal protein and the interplay between translation and mRNA decay
No full textIn bacteria, transcription, translation and mRNA decay are tightly interconnected processes; however, little is known about specific factors and molecular mechanisms involved in their co-ordination. The ribosomal protein S1, an “atypical” ribosomal protein weakly associated with the 30S subunit of Escherichia coli ribosome, has been implicated in translation, transcription and control of RNA stability. It is thus a good candidate for playing a role in the interplay among these processes. We have addressed S1 function by assaying translation and decay of model full-length and leaderless mRNAs upon modulation of S1 intracellular concentration (from depletion to overexpression). We have shown that S1 over-expression leads to polysome disappearance and translation inhibition. Moreover, in the same condition, RNase E-dependent decay of both the cspE+ and leaderless ΔL-cspE mRNAs is prevented. Conversely, cleavage of ΔL-cspE mRNA by an unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and stabilize mRNA through the specific inhibition of RNase E-dependent decay
Exploring pyrazinamide derivatives as novel Pseudomonas aeruginosa inhibitors: unexploited antibacterial molecules for a new antibiotics target
No full textBackground
The ribosomal protein S1 (encoded by rpsA) is a promising target for new antibacterial drugs. In Escherichia coli, S1 has an essential role in translation. S1 is highly conserved among Gram negative bacteria and absent in mammalian cells. Recently, it has been found that pyrazinamide (PZA), a first-line tuberculosis drug, targets S1 protein. PZA derivatives were developed by Bracco SpA in the early ‘60s and roughly characterized for antibacterial activity; interestingly, in preliminary tests, the derivative B2320 seemed to be active against Pseudomonas aeruginosa (Pa). B2320 target in Pa is currently unknown.
Hypothesis and objectives
1. Characterizing the anti-Pa activity of B2320.
2. Exploring Pa S1 as a potential target for new antibacterials.
3. Setting-up an E. coli biosensor strain for the screening of S1 inhibitors.
Essential methods
1. B2320 will be tested for anti-bacterial activity both against Pseudomonas lab strains and a collection of clinical isolates from cystic fibrosis patients. The mechanism of action of the compound will be investigated.
2. A Pa strain with rpsA conditional expression will be constructed in order to assess its essentiality.
3. A fluorescence-based assay to find inhibitors of S1-dependent translation initiation will be set up in E. coli, transferred in Pa and used to screen a collection of heterogeneous chemical compounds for translation inhibitors.
Preliminary results
1. B2320 activity against PAO1 and PA14 lab strains has been tested in different growth conditions. Interestingly, the more virulent PA14 strain seems more sensitive to B2320 than PAO1 strain.
2. The mutant is under construction. We have mapped the 5’-end of rpsA transcript in Pa. This has been instrumental in designing the construct for rpsA mutation.
3. We have developed a simple whole-cell assay in E. coli that allows discriminating antibiotics inhibiting different translation steps.
Expected results and their significance
P. aeruginosa is the most common pathogen in CF lung infection with a high negative impact on lung functionality and patients’ mortality. The increasing diffusion of multi-resistant strains demands for the development of new anti-Pa agents. These could be identified both among PZA derivatives and by our whole-cell screening of libraries of chemical compounds. Moreover, if Pa S1 will result to be essential as expected, it would be a robust target for the design of new drugs
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