Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria

Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100?400 nm, genome size in the 50?70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30uC, eight of them failed to propagate at 37uC. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.Fil: Stella, Emma Julieta. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia

Complete genome sequences of nine mycobacteriophages

Genome analyses of a large number of mycobacteriophages, bacterial viruses that infect members of the genus Mycobacterium, yielded novel enzymes and tools for the genetic manipulation of mycobacteria. We report here the complete genome sequences of nine mycobacteriophages, including a new singleton, isolated using Mycobacterium smegmatis mc(2)155 as a host strain.Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.médicas. Escuela de Cs.médicas. Cátedra de Microbiología,parasitología y Virologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Suárez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Cs.médicas. Escuela de Cs.médicas. Cátedra de Microbiología,parasitología y Virologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Teran, Lucrecia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Raya, Raul Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.médicas. Escuela de Cs.médicas. Cátedra de Microbiología,parasitología y Virologia; Argentin

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. Smegmatis mutants

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor (s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.Fil: Suárez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentin

ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC50 of 8 ìM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugsFil: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de la Iglesia, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bazet Lyonnet, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lin, Ting Wang. University of California; Estados UnidosFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Tsai, Shiou Chuan. University of California; Estados UnidosFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

Background: Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts. Results: We report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30–40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl2, mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content. Conclusion: This is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His)6-tag location and lysis buffer additives (e.g.N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.Fil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Boncompain, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Peressutti Bacci, Natalia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentin

Mutations in the anti-sigma H factor RshA confer resistance to econazole and clotrimazole in Mycobacterium smegmatis

Azole drugs such as econazole, are active on Mycobacterium tuberculosis and Mycobacterium smegmatis; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system mmpL5-mmpS5 conferred resistance to econazole in M. tuberculosis. We herein report that an azole-resistant mutant screen in M. smegmatis rendered mutations in rshA, encoding a non-essential anti-sigma H protein.Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: de la Iglesia, Agustina Inés. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Figueroa, Virginia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Di Capua, Cecilia Beatriz. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Ioerger, Thomas R.. Texas A&M University; Estados UnidosFil: Parish, Tanya. Infectious Disease Research Institute. TB Discovery Research; Estados Unido

A katG S315T or an ahpC promoter mutation mediate Mycobacterium tuberculosis resistance to 2-thiophen carboxylic acid hydrazide, an inhibitor resembling the anti-tubercular drugs Isoniazid and Ethionamide

Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Belardinelli, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. State University of Colorado - Fort Collins; Estados UnidosFil: Tong, Ping. University College Dublin; Irlanda. University of Edinburgh; Reino UnidoFil: Loftus, Brendan. University College Dublin; IrlandaFil: Recio Balsells, Alejandro Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Labadie, Guillermo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Gordon, Stephen V.. University College Dublin; IrlandaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario; Argentin

Virulence factors of the mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. Introduction.Fil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Gioffré, Andrea Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Sabio y García, Julia Verónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Escuela de Ciencias Médicas. Cátedra de Microbiología, Parasitología y Virología; ArgentinaFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Impact of the deletion of the six mce operons in Mycobacterium smegmatis

The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4 operon is involved in cholesterol transport in M. smegmatis.Fil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Osella, Ana Virginia. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Stella, Emma Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Bianco, María Verónica. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Santangelo, María de la Paz. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Sassetti, Cristopher. Massachusetts Institute of Technology; Estados UnidosFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados UnidosFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin