Eric J. Rubin

Genes required for mycobacterial growth defined by high density mutagenesis

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non‐essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

Genetic requirements for mycobacterial survival during infection

Despite the importance of tuberculosis as a public health problem, we know relatively little about the molecular mechanisms used by the causative organism, Mycobacterium tuberculosis, to persist in the host. To define these mechanisms, we have mutated virtually every nonessential gene of M. tuberculosis and determined the effect disrupting each gene on the growth rate of this pathogen during infection. A total of 194 genes that are specifically required for mycobacterial growth in vivo were identified. The behavior of these mutants provides a detailed view of the changing environment that the bacterium encounters as infection proceeds. A surprisingly large fraction of these genes are unique to mycobacteria and closely related species, indicating that many of the strategies used by this unusual group of organisms are fundamentally different from other pathogens.

Comprehensive identification of conditionally essential genes in mycobacteria

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette–Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in vero cells

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP‐ribosylates a eukaryotic GTP‐binding protein of the ras superfamily. We show now that the bacterially‐expressed product of the human rhoC gene is ADP‐ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP‐ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae

A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.

PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project

Introduction: The Blueprint Programmed Death Ligand 1 (PD‐L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial‐academic collaborative partnership to provide information on the analytical and clinical comparability of four PD‐L1 IHC assays used in clinical trials. Methods: A total of 39 NSCLC tumors were stained with four PD‐L1 IHC assays (22C3, 28‐8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. Results: Analytical comparison demonstrated that the percentage of PD‐L1–stained tumor cells was comparable when the 22C3, 28‐8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD‐L1 classification would be made depending on which assay/scoring system was used. Conclusions: The Blueprint PD‐L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD‐L1 expression for three assays, interchanging assays and cutoffs would lead to “misclassification” of PD‐L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy‐related PD‐L1 cutoffs.

Bacterial Growth and Cell Division: a Mycobacterial Perspective

SUMMARY The genus Mycobacterium is best known for its two major pathogenic species, M. tuberculosis and M. leprae, the causative agents of two of the worlds oldest diseases, tuberculosis and leprosy, respectively. M. tuberculosis kills approximately two million people each year and is thought to latently infect one-third of the worlds population. One of the most remarkable features of the nonsporulating M. tuberculosis is its ability to remain dormant within an individual for decades before reactivating into active tuberculosis. Thus, control of cell division is a critical part of the disease. The mycobacterial cell wall has unique characteristics and is impermeable to a number of compounds, a feature in part responsible for inherent resistance to numerous drugs. The complexity of the cell wall represents a challenge to the organism, requiring specialized mechanisms to allow cell division to occur. Besides these mycobacterial specializations, all bacteria face some common challenges when they divide. First, they must maintain their normal architecture during and after cell division. In the case of mycobacteria, that means synthesizing the many layers of complex cell wall and maintaining their rod shape. Second, they need to coordinate synthesis and breakdown of cell wall components to maintain integrity throughout division. Finally, they need to regulate cell division in response to environmental stimuli. Here we discuss these challenges and the mechanisms that mycobacteria employ to meet them. Because these organisms are difficult to study, in many cases we extrapolate from information known for gram-negative bacteria or more closely related GC-rich gram-positive organisms.

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

The regulation of liver apolipoprotein (apo) A-I gene expression by fibrates was studied in human apo A-I transgenic mice containing a human genomic DNA fragment driving apo A-I expression in liver. Treatment with fenofibrate (0.5% wt/wt) for 7 d increased plasma human apo A-I levels up to 750% and HDL-cholesterol levels up to 200% with a shift to larger particles. The increase in human apo A-I plasma levels was time and dose dependent and was already evident after 3 d at the highest dose (0.5% wt/wt) of fenofibrate. In contrast, plasma mouse apo A-I concentration was decreased after fenofibrate in nontransgenic mice. The increase in plasma human apo A-I levels after fenofibrate treatment was associated with a 97% increase in hepatic human apo A-I mRNA, whereas mouse apo A-I mRNA levels decreased to 51%. In nontransgenic mice, a similar down-regulation of hepatic apo A-I mRNA levels was observed. Nuclear run-on experiments demonstrated that the increase in human apo A-I and the decrease in mouse apo A-I gene expression after fenofibrate occurred at the transcriptional level. Since part of the effects of fibrates are mediated through the nuclear receptor PPAR (peroxisome proliferator-activated receptor), the expression of the acyl CoA oxidase (ACO) gene was measured as a control of PPAR activation. Both in transgenic and nontransgenic mice, fenofibrate induced ACO mRNA levels up to sixfold. When transgenic mice were treated with gemfibrozil (0.5% wt/wt) plasma human apo A-I and HDL-cholesterol levels increased 32 and 73%, respectively, above control levels. The weaker effect of this compound on human apo A-I and HDL-cholesterol levels correlated with a less pronounced impact on ACO mRNA levels (a threefold increase) suggesting that the level of induction of human apo A-I gene is related to the PPAR activating potency of the fibrate used. Treatment of human primary hepatocytes with fenofibric acid (500 microM) provoked an 83 and 50% increase in apo A-I secretion and mRNA levels, respectively, supporting that a direct action of fibrates on liver human apo A-I production leads to the observed increase in plasma apo A4 and HDL-cholesterol.

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.