Francesca Forti

Mycobacterium tuberculosis FurA Autoregulates Its Own Expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.

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-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, 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.

Genetic analysis of the immunity region of phage‐plasmid P4

In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418‐8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity‐defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

Background: Mycobacterium tuberculosis PknB plays a critical role in modulating cell division and cell wall synthesis. Results: We present a comprehensive evaluation of the importance of various domains of PknB in modulating cell survival. Conclusion: The intracellular kinase domain and extracytoplasmic PASTA domains of PknB are essential for cell survival. Significance: PknB is essential for both in vitro growth and survival of the pathogen in vivo. The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.

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 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.

Mycobacterium smegmatis RNase J is a 5-3 exo-/endoribonuclease and both RNase J and RNase E are involved in ribosomal RNA maturation

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.

Mycobacterium tuberculosis RNA polymerase-binding protein A (RbpA) and its interactions with sigma factors.

Background: RNA polymerase-binding protein A (RbpA) plays an unknown essential role in Mycobacterium tuberculosis. Results: The structure of RbpA was solved using NMR. Conclusion: RbpA binds sigma factors A and B via its conserved N and C termini. Significance: The identified interactions shed light on the function of RbpA in regulating transcription. RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the β subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σA and the stress-induced σB as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σB via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell.

Isolation of conditional expression mutants in Mycobacterium tuberculosis by transposon mutagenesis

In Mycobacterium tuberculosis identification of essential genes has been hampered by the scarcity of suitable genetic tools for genome wide screenings. We constructed two Himar1 transposon derivatives in which the Streptomyces pristinamycin I-inducible ptr promoter was inserted at one transposon end in outward orientation. These transposons, Tn-pip/pptr (which harbours the promoter and its repressor pip gene) and Tn-pptr (which depends on a host expressing the pip gene), were inserted in the thermosensitive mycobacteriophage phAE87. After transduction into M. tuberculosis H37Rv, hygromycin resistant clones were selected in the presence of pristinamycin, screened for inducer dependent growth, and the transposon insertion point mapped by sequencing. Out of 3530 Hyg(R) mutants tested, we obtained 14 (0.4%) single insertion conditional mutants. In three (leuA, mazE6, rne) pptr was located upstream of genes whose function had been assessed by experimental evidence, whereas in seven the transposon targeted genes (ftsK, glf, infB, metC, pyrD, secY, and tuf) whose function had been assigned by similarity with homologous genes and four ORFs of unknown function (Rv0883c, Rv1478, Rv2050 and Rv2204c). These results validate our mutagenesis system and provide previously unavailable conditional expression mutants in genes of known, putative and unknown functions for genetic and physiological studies.

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

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 of Mycobacterium 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.

Design of a broad-range bacteriophage cocktail that reduces Pseudomonas aeruginosa biofilms and treats acute infections in two animal models.

ABSTRACT 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 of Pseudomonas 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 lyse P. aeruginosa both 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 the Galleria larva 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.