Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration

Genome-wide discovery of small RNAs in Mycobacterium tuberculosis

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 59/39 UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 59 end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane

Phage therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients

Pseudomonas 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)

Assess the utility of the PIP-ON system in M. tb and overexpress or silence several mycobacterial genes

In this system the gene of interest is under transcriptional control of the Streptomyces Pip protein, the pristinamycin sensitive repressor. The pip gene of S. coelicolor has been cloned downstream of a constitutive mycobacterial promoter (the mutant pfurA102 of M. tb), and introduced in Mycobacterium smegmatis (see pMYS696 in Fig. 1). Furthermore, a Pip-ON system has been constructed, by cloning the S. pristinaespiralis ptr promoter upstream of a reporter gene (lacZ), in the above plasmid (see pMY718 in Fig. 1). These constructs have been introduced in M. smegmatis and beta-galactosidase activity measured: the activity expressed from pMY718 is very low (less than 10 Miller units) and inducible, in a dose dependent manner, by addition of pristinamycin

Essentiality testing with inducible promoters

Objectives: Assess the utility of the PIP-ON system in M.tb. Progress: The PIP-ON system was transferred in M. tb where we could show a very efficient repression of Pip on the ptr promoter. In response to pristinamicin we could see a dose dependent induction of the promoter obtaining a maximum induction of about 100 fold with 150 ng/ml of pristinamicin. The construction of plasmids where the expression of pknB, glnA1 and fad32 genes and of their antisense RNA is under the ptr promoter control and thus responding to pristinamicin is under way. An alternative approach using a TET/PIP repressible system was developed. The reporter gene is represented by the same one used for the PIP ON system (lacZ under ptr promoter transcriptional control). In this case the PIP repressor is provided in trans under the control of a TET dependant promoter. In the absence of inducer PIP should not be produced and the ptr promoter should be expressed constitutively. Addition of tetracycline should induce the expression of the PIP repressor structural gene causing the repression of transcription of the ptr promoter. Removal of tetracycline and/or addition of pristinamycin should restore its transcription. The system, introduced in M. smegmatis did not give the expected results probably due to the high strength of the TET-dependant promoter used in these constructs. We decided to place PIP under the control of a weaker promoter. We chose a mutated furA promoter previously shown to express PIP at functional level. The promoter was mutagenized to introduce two tet-operators, one immediately upstream the -35 region and the other between the -35 and the -10, placed upstream the PIP conding region and finally cloned into an integrative vector containing a lacZ gene under ptr- promoter transcriptional control. This plasmid has been sequenced and introduced in a M smegmatis strain expressing the TET repressor from a replicative plasmid and is actually under analysi

Pristinamycin-inducible gene regulation in mycobacteria

In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr \u2013Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be overexpressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria

Uno scritto poco noto di Danilo & Sandri

Volume: 22Start Page: 113End Page: 13