Daniela Ghisotti

The alarming diffusion of multidrug-resistant (MDR) bacterial strains requires investigations on nonantibiotic therapies. Among such therapies, the use of bacteriophages (phages) as antimicrobial agents, namely, phage therapy, is a promising treatment strategy supported by the findings of recent successful compassionate treatments in Europe and the United States. In this work, we combined host range and genomic information to design a 6-phage cocktail killing several clinical strains ofPseudomonas aeruginosa, including those collected from Italian cystic fibrosis (CF) patients, and analyzed the cocktail performance. We demonstrated that the cocktail composed of four novel phages (PYO2, DEV, E215 and E217) and two previously characterized phages (PAK_P1 and PAK_P4) was able to lyseP. aeruginosaboth in planktonic liquid cultures and in biofilms. In addition, we showed that the phage cocktail could cure acute respiratory infection in mice and treat bacteremia in wax moth (Galleria mellonella) larvae. Furthermore, administration of the cocktail to larvae prior to bacterial infection provided prophylaxis. In this regard, the efficiency of the phage cocktail was found to be unaffected by the MDR or mucoid phenotype of the pseudomonal strain. The cocktail was found to be superior to the individual phages in destroying biofilms and providing a faster treatment in mice. We also found theGallerialarva model to be cost-effective for testing the susceptibility of clinical strains to phages, suggesting that it could be implemented in the frame of developing personalized phage therapies.

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth ofMycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

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 5′/3′ 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 5′ 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 proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S -nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM , encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

Summary: The presence of very different sets of enzymes, and in particular the presence of RNase E and RNase J, has been used to explain significant differences in RNA metabolism between the two model organisms Escherichia coli and Bacillus subtilis. However, these studies might have somewhat polarized our view of RNA metabolism. Here, we identified a RNase J in Mycobacterium smegmatis that has both 5′‐3′ exo‐ and endonucleolytic activity. This enzyme coexists with RNase E in this organism, a configuration that enabled us to study how these two key nucleases collaborate. We demonstrate that RNase E is responsible for the processing of the furA‐katG transcript in M. smegmatis and that both RNase E and RNase J are involved in the 5′ end processing of all ribosomal RNAs. In contrast to B. subtilis, the activity of RNase J, although required in vivo for 23S rRNA maturation, is not essential in M. smegmatis. We show that the pathways for ribosomal RNA maturation in M. smegmatis are quite different from those observed in E. coli and in B. subtilis. Studying organisms containing different combinations of key ribonucleases can thus significantly broaden our view of the possible strategies that exist to direct RNA metabolism.

In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis 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 furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA.

In the Escherichia coli satellite phage P4, transcription starting from P_LE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t₁₅₁ site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at P_LE and P_LL promoters, respectively, and terminating at the intracistronic t₁₅₁ site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and α genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t₁₅₁ transcription termination site within the 7633–7361 region between orf151 and gene cnr. A potential stem–loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414–7385.