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Global assessment of genomic regions required for growth in Mycobacterium tuberculosis.

ABSTRACT: Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.

SUBMITTER: Zhang YJ

PROVIDER: S-EPMC3460630 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Global assessment of genomic regions required for growth in Mycobacterium tuberculosis.

Zhang Yanjia J YJ Ioerger Thomas R TR Huttenhower Curtis C Long Jarukit E JE Sassetti Christopher M CM Sacchettini James C JC Rubin Eric J EJ

PLoS pathogens 20120927 9

Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains ...[more]

PMID: 23028335

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Project description:Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth and organized ?3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.IMPORTANCE Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12?months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH's global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases.

Project description:UNLABELLED:To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis ?lsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the ?lsr2 mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The ?lsr2 strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. IMPORTANCE:M. tuberculosis causes nearly two million deaths per year and infects nearly one-third of the world population. The success of this aerobic pathogen is due in part to its ability to successfully adapt to constantly changing oxygen availability throughout the infectious cycle, from the high oxygen tension during aerosol transmission to anaerobiosis within necrotic lesions. An understanding of how M. tuberculosis copes with these changes in oxygen tension is critical for its eventual eradication. Using a mutation in lsr2, we demonstrate that the Lsr2 protein present in all mycobacteria is a global transcriptional regulator in control of genes required for adaptation to changes in oxygen levels. M. tuberculosis lacking lsr2 was unable to adapt to both high and very low levels of oxygen and was defective in long-term anaerobic survival. Lsr2 was also required for disease pathology and for chronic infection in a mouse model of TB.

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Global assessment of genomic regions required for growth in Mycobacterium tuberculosis.

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Global assessment of genomic regions required for growth in Mycobacterium tuberculosis.

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