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

The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation

<p>Abstract</p> <p>Background</p> <p>In <it>Mycobacterium tuberculosis </it>and in <it>Mycobacterium smegmatis </it>the <it>furA</it>-<it>katG </it>loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In <it>M. tuberculosis furA-katG </it>constitute a single operon, whereas in <it>M. smegmatis </it>a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the <it>furA </it>gene, corresponds to transcription initiation from the <it>furA </it>promoter; the second one is the <it>katG </it>mRNA 5' end, located in the terminal part of <it>furA</it>.</p> <p>Results</p> <p>In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the <it>M. smegmatis </it>region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of <it>M. tuberculosis </it>and <it>M. smegmatis </it>were inserted in a plasmid between the <it>sigA </it>promoter and the <it>lacZ </it>reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the <it>katG </it>translation start codon, increased beta-galactosidase activity and stabilized the <it>lacZ </it>transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of <it>M. smegmatis </it>was followed by an increasing number of <it>katG </it>codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the <it>katG </it>transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing.</p> <p>Conclusion</p> <p>This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The <it>furA-katG </it>mRNA is transcribed from the <it>furA </it>promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA.</p

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

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)