Inhibition of bacterial growth by peptide nucleic acids complementary to bacterial rRNA

Nowadays antibiotics are overused in human treatment, agriculture and animal breading. Bacteria can effectively acquire resistance when exposed to antibiotics so it is crucial to find new antimicrobial drugs to combat antibiotic-resistant pathogens. The ribosome is one of the bacterial targets for clinically used antibiotics. However, the ribosome can be also sterically blocked by complementary oligomers for example peptide nucleic acids (PNA). PNAs are synthetic analogues of DNA with a neutral peptide-mimicking backbone. Importantly, they are not digested by enzymes such nucleases and proteases. Antisense modified oligonucleotides such PNA, which inhibit bacteria translation, could become useful tools for controlling in the future the spread of bacteria The main goal of this research was to design PNA sequences that would bind to bacterial rRNA and inhibit bacterial growth. I designed four PNA sequences complementary to rRNA and control sequences that included different versions of PNA with peptide, without and with fluorescein what gives together nineteen conjugates tested during my studies. I verified the sequence-specific hybridization of PNAs with isolated rRNA from Escherichia coli. I determined that PNA inhibits translation in a bacterial cell-free translation/transcription system. Then, I showed that PNA targeted to functional site in the ribosome inhibits the growth of E. coli cultures. Furthermore, I proved by agarose gel electrophoresis that PNA binds rRNA present in isolated total RNA, and also binds 70S ribosomes. The results of my study indicate that targeting rRNA with PNA could be a promising way to inhibit bacterial growth and to search for new target sites in bacterial RNA.Zastosowanie antybiotyków na szeroką skalę, w leczeniu ludzi i hodowli zwierząt, przyczyniło się do rozwinięcia oporności bakterii na konwencjonalne antybiotyki. Ważne jest, aby znaleźć środki za pomocą których można będzie zwalczać oporne szczepy bakteryjne. Docelowym miejscem działania dla wielu klas antybiotyków jest rybosom bakteryjny. Oligonukleotydy, stosowane do zahamowania procesu syntezy białek niezbędnych do życia bakterii, mogą okazać się pomocne w zwalczaniu chorób bakteryjnych. Peptydowe kwasy nukleinowe (PNA) są syntetycznymi analogami kwasów nukleinowych, które zawierają neutralny szkielet, są niewrażliwe na działanie enzymów degradujących kwasy nukleinowe i białka oraz tworzą stabilne kompleksy zarówno z DNA jak i RNA. Głównym celem mojej rozprawy doktorskiej było poszukiwanie oligonukleotydów PNA wiążących się do rybosomowego RNA (rRNA), które wydajnie hamowałyby wzrost bakterii Escherichia coli. Zaprojektowałam cztery sekwencje PNA komplementarne do rRNA i ich warianty z peptydem umożliwiającym dostarczanie do komórki bakteryjnej, a także z fluoresceiną oraz sekwencje kontrolne. Łącznie pracowałam z dziewiętnastoma oligonukleotydami PNA. Wykazałam, że zaprojektowane oligomery PNA oddziałują komplementarnie z określonymi fragmentami rRNA E. coli. Udowodniłam również, że PNA komplementarne do rRNA hamuje proces bakteryjnej translacji oraz wzrost komórek bakteryjnych. Ponadto pokazałam, że PNA wiąże się z izolowanymi rybosomami bakteryjnymi. Otrzymane wyniki mogą być pomocne w trakcie poszukiwania nowych miejsc docelowych w rRNA bakterii dla cząsteczek przeciwbakteryjnych, a także wskazują, że PNA komplementarne do wybranych miejsc w rRNA mogą skutecznie hamować wzrost komórek bakteryjnych

Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets

We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer

Adhesion proteins--an impact on skeletal myoblast differentiation.

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro

Expression of mRNAs encoding integrin alpha3 and other adhesion proteins at 24 and 48 hours after transfection of MPCs-derived myoblasts with siRNA-alpha3. sqRT-PCR analysis.

<p>NT – control, not transfected myoblasts, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%.</p

Changes in expression and localization of adhesion proteins during <i>Soleus</i> muscle regeneration.

<p>A - level of mRNAs encoding adhesion proteins analyzed by sqRT-PCR. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%. B – localization of integrin alpha3 (red) in intact and in regenerating muscle at day 3, 5, 7, 14. Nuclei – blue. White arrows show degenerating myofibers. Scale bars 50 µm.C –colocalization of Pax7, MyoD, myogenin (MG) (red) with integrin alpha3 (green) in intact muscle and at day 1, 3 and 5 of regeneration (intact, d1, d3, d5 respectively). White arrows - myoblasts, pink – muscle fiber nuclei, yellow - MyoD, myogenin and integrin alpha3 negative cells. D – control staing with secondary antibodies only. Nuclei – blue. Scale bars 50 µm. E - localization of integrin beta1, ADAM12, CD9, or M-cadherin (green) at day 3 of regeneration. Nuclei – blue. Scale bars 50 µm. F – immunoblotting analysis of M-cadherin (M-cad), integrin beta1, ADAM12 (AD12), CD9 and integrin alpha3 level during muscle regeneration (days 1, 3, 7, 14).</p

Downregulation of alpha3 integrin expression reduces fusion of MPCs-derived myoblasts.

<p>A - Pappenheim's staining of fusing myoblasts. B – total number of nuclei calculated at day 12 of myoblast culture. C –index of fusion analyzed at day 12 of culture in control and experimental myoblasts shown as a percentage of myotube nuclei per number of all nuclei. D - proportion of 2–4, 5–7, 8–10 and >11 nuclear myotubes per number of all myotubes, respectively. NT – control, not transfected myoblasts, TR – control myoblasts cultured in medium supplemented with transfection reagent, siRNA-cont - control myoblasts transfected with scramble siRNA, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Error bars indicate SEM, results were analyzed by Kruskal-Wallis test. Kruskal-Wallis One Way Analysis of Variance showed differences between experimental groups which were considered statistically significant when p<0.05 (marked with asterisks). * p≤0.05.</p

Downregulation of integrin alpha3 reduces the C2C12 ability to fuse with control myoblasts.

<p>A – control - myoblasts transfected with scramble siRNA-AlexaRed (red) cultured with myoblasts stained with QTracker (green), experiment+siRNA-alpha3– myoblasts cotransfected with scramble siRNA-AlexaRed (red) and siRNA-alpha3 cultured with control myoblasts stained with QTracker (green). Hybrid myotubes are yellow. Scale bars 20 µm. B –number of hybrid myotubes formed by control myoblasts transfected with scramble siRNA-AlexaRed and control myoblasts stained with QTracker (control) compared with the number of hybrid myotubes formed by myoblasts co-transfected with scramble siRNA-AlexaRed and siRNA-alpha3 and control myoblasts stained with QTracker (siRNA-alpha3) (day 11 of culture). Error bars indicate SEM, results were analyzed by Student's test and differences were considered statistically significant when p<0.05 (marked with asterisks). ** p≤0.01.</p