Clinically significant antibiotic resistance has evolved against virtually every antibiotic deployed. Yet the development of new classes of antibiotics has lagged far behind our growing need for such drugs. Rather than focusing on therapeutics that target in vitro viability, much like conventional antibiotics, an alternative approach is to target functions essential for infection, such as virulence factors required to cause host damage and disease. This approach has several potential advantages including expanding the repertoire of bacterial targets, preserving the host endogenous microbiome, and exerting less selective pressure, which may result in decreased resistance. We review new approaches to targeting virulence, discuss their advantages and disadvantages, and propose that in addition to targeting virulence, new antimicrobial development strategies should be expanded to include targeting bacterial gene functions that are essential for in vivo viability. We highlight both new advances in identifying these functions and prospects for antimicrobial discovery targeting this unexploited area.
Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.
These data suggest that APOE epsilon2 and epsilon4 might promote CAA-related hemorrhage through separate mechanisms: epsilon4 by enhancing amyloid deposition and epsilon2 by causing amyloid-laden vessels to undergo the vasculopathic changes that lead to rupture.
Zebrafish (Danio rerio) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study Pseudomonas aeruginosa pathogenesis and found that infection of zebrafish embryos with live P. aeruginosa (PA14 or PAO1) by microinjection results in embryonic death, unlike infection with Escherichia coli or heat-killed P. aeruginosa, which has no effect. Similar to studies with mice, P. aeruginosa mutants deficient in type three secretion (pscD) or quorum sensing (lasR and mvfR) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer P. aeruginosa cells than those required for embryos infected at 50 hpf, are susceptible to infection with lasR and pscD deletion mutants, and are moderately resistant to infection with an mvfR mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of P. aeruginosa, the role of individual components of host immune defense, and small-molecule modulators of infection.Pseudomonas aeruginosa, one of the most common causes of nosocomial infections in the United States, typically infects injured, burned, and immunocompromised patients and is the primary cause of mortality among cystic fibrosis patients. It is a ubiquitous, gram-negative bacterium adapted to a variety of niches, including water and soil in associations with other eukaryotic organisms. A number of evolutionarily divergent model hosts have been used to examine P. aeruginosa pathogenesis, including amoebae, plants, nematodes, insects, and rodents (25, 36, 37). While much has been learned about P. aeruginosa pathogenesis from these models, each model has different strengths and weaknesses. Invertebrate model hosts such as Caenorhabditis elegans offer greater genetic tractability than rodent models. Moreover, the size and life cycle of organisms like C. elegans enable experiments such as comprehensive genetic screens that require large numbers of animals, in contrast to rodent models, where such studies are often simply unfeasible due to cost and space requirements. The drawback to modeling human infections in invertebrate hosts is the dissimilarity between vertebrate and invertebrate immune responses. Invertebrate model hosts such as Drosophila melanogaster and C. elegans do not possess adaptive immunity, a true ...
New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.
Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets.
alpha2-Macroglobulin (A2M) is a proteinase inhibitor found in association with senile plaques (SP) in Alzheimer's disease (AD). A2M has been implicated biochemically in binding and degradation of the amyloid beta (Abeta) protein which accumulates in SP. We studied the relationship between Alzheimer's disease and a common A2M polymorphism, Val1000 (GTC)/Ile1000 (ATC), which occurs near the thiolester active site of the molecule. In an initial exploratory data set (90 controls and 171 Alzheimer's disease) we noted an increased frequency of the G/G genotype from 0.07 to 0.12. We therefore tested the hypothesis that the G/G genotype is over-represented in Alzheimer's disease in an additional independent data set: a group of 359 controls and 566 Alzheimer's disease patients. In the hypothesis testing cohort, the G/G genotype increased from 0.07 in controls to 0.12 in Alzheimer's disease (P < 0.05, Fisher's exact test). The odds ratio for Alzheimer's disease associated with the G/G genotype was 1.77 (1.16-2.70, P < 0.01) and in combination with APOE4 was 9.68 (95% CI 3.91-24.0, P < 0.001). The presence of the G allele was associated with an increase in Abeta burden in a small series. The A2M receptor, A2M-r/LRP, is a multifunctional receptor whose ligands include apolipoprotein E and the amyloid precursor protein. These four proteins have each been genetically linked to Alzheimer's disease, suggesting that they may participate in a common disease pathway.
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