Peter Sander

Mycobacterium tuberculosis prevents inflammasome activation.

Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

Fitness Cost of Chromosomal Drug Resistance-Conferring Mutations

ABSTRACT To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

A Single 16S Ribosomal RNA Substitution Is Responsible for Resistance to Amikacin and Other 2-Deoxystreptamine Aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae

Twenty-six clinical isolates of Mycobacterium abscessus resistant to amikacin were identified. Most isolates were from patients with posttympanostomy tube placement otitis media or patients with cystic fibrosis who had received aminoglycoside therapy. Isolates were highly resistant (MICs > 1024 microg/mL) to amikacin, kanamycin, gentamicin, tobramycin, and neomycin (all 2-deoxystreptamine aminoglycosides) but not to streptomycin. Sequencing of their 16S ribosomal (r) RNA revealed that 16 (94%) of 17 had an A-->G mutation at position 1408. In vitro-selected amikacin-resistant mutants of M. abscessus and Mycobacterium chelonae had the same resistance phenotype, and 15 mutants all had the same A-->G substitution at position 1408. Introducing an rRNA operon from Mycobacterium smegmatis with a mutated A-->G at this position into a single functional allelic rRNA mutant of M. smegmatis produced the same aminoglycoside resistance phenotype. These studies demonstrate this 16S rRNA mutation is responsible for amikacin resistance in M. abscessus, which has only one copy of the rRNA operon.

RPSL+ : A DOMINANT SELECTABLE MARKER FOR GENE REPLACEMENT IN MYCOBACTERIA

Molecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double‐crossover events by homologous recombination. Here we describe a vector‐host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wild‐type rpsL gene coding for ribosomal protein S12 in a streptomycin‐resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin‐resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin‐sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one‐step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single‐crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.

A synthetic mammalian gene circuit reveals antituberculosis compounds

Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In Mycobacterium tuberculosis EthR binds to a specific operator (OethR) thereby repressing ethA and preventing EthA-catalyzed conversion of the prodrug ethionamide, which increases the resistance of the pathogen to this last-line-of-defense treatment. We have designed a synthetic mammalian gene circuit that senses the EthR–OethR interaction in human cells and produces a quantitative reporter gene expression readout. Challenging of the synthetic network with compounds of a rationally designed chemical library revealed 2-phenylethyl-butyrate as a nontoxic substance that abolished EthRs repressor function inside human cells, in mice, and within M. tuberculosis where it triggered derepression of ethA and increased the sensitivity of this pathogen to ethionamide. The discovery of antituberculosis compounds by using synthetic mammalian gene circuits may establish a new line of defense against multidrug-resistant M. tuberculosis.

Fitness of antibiotic-resistant microorganisms and compensatory mutations

come of different selections indicates that it is important to investigate clinical isolates with drug resistance or to use relevant animal models to investigate which of the possible resistance mutations are selected under in vivo conditions. A high level of selection for drug-resistance mutations without involving the cost of decreased fitness would seem to exist in vivo. These results argue against the belief that compensatory mutations are of clinical relevance, at least for drug resistance due to mutational target alterations. Two main strategies have been proposed to alleviate the growing burden of diseases caused by drug resistant bacteria: to reduce the use of antibiotics and to develop new drugs. The success of both strategies depends on a more refined understanding of the biology of antibiotic drug resistance. In vivo selection for resistance mutations with unaltered fitness would limit substantially the value of strategies based solely on the restrictive use of antibiotics, as these are unlikely to result in the elimination of resistant strains from reservoirs or transmission cycles. Increased knowledge and investigations of the biology of drug resistance is likely to have a considerable effect on the etic and germ cell tumors, developing within cell populations with a high degree of biological diversity, exhibit great complexity. However, the common features of tumors—to expand a clone of cells with a growth advantage and to adapt through genetic changes—provide cancers with a survival advantage. A systems analysis of all these biological processes reveals features similar to the interplay between order and chaos. There are many examples of complex adaptive systems in biology, such as those discussed by Andrei Cucuianu and those that I describe in my article. The complex adaptive system is a central feature of the pathology of cancer, although the strategies used to reach this complexity are different.

Structural basis for selectivity and toxicity of ribosomal antibiotics

Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.

The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild‐type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.

Lipoprotein processing is required for virulence of Mycobacterium tuberculosis

Lipoproteins are a subgroup of secreted bacterial proteins characterized by a lipidated N‐terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (LspA). The study of LspA function has been limited mainly to non‐pathogenic microorganisms. To study a potential role for LspA in the pathogenesis of bacterial infections, we have disrupted lspA by allelic replacement in Mycobacterium tuberculosis, one of the worlds most devastating pathogens. Despite the presence of an impermeable lipid outer layer, it was found that LspA was dispensable for growth under in vitro culture conditions. In contrast, the mutant was markedly attenuated in virulence models of tuberculosis. Our findings establish lipoprotein metabolism as a major virulence determinant of tuberculosis and define a role for lipoprotein processing in bacterial pathogenesis. In addition, these results hint at a promising new target for therapeutic intervention, as a highly specific inhibitor of bacterial lipoprotein signal peptidases is available.

Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.