Anne E. Clatworthy

Targeting virulence: a new paradigm for antimicrobial therapy

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.

Pseudomonas aeruginosa Infection of Zebrafish Involves both Host and Pathogen Determinants

ABSTRACT 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.

Expression and alternate splicing of apolipoprotein E receptor 2 in brain.

Apolipoprotein E isoforms affect the risk of developing Alzheimers disease. Apolipoprotein E-associated risk may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. We examined the brain expression of the most recently identified member of this receptor family, apolipoprotein E receptor 2, in human brain and placenta. We analysed apolipoprotein E receptor 2 messenger RNA by reverse transcription-polymerase chain reaction and apolipoprotein E receptor 2 protein by immunohistochemistry. Four exons of the apolipoprotein E receptor 2 message were alternately spliced in both fetal and adult brain tissue. Exon 5, encoding three of the seven ligand binding repeats, was absent in the apolipoprotein E receptor 2 messenger RNA examined. Apolipoprotein E receptor 2 messages lacking exon 8, encoding an epidermal growth factor precursor repeat, exon 15, encoding the O-glycosylation region, or exon 18, encoding a cytoplasmic domain, were also present as minor splice variants in the brain and placenta. No differences were observed in the pattern of apolipoprotein E receptor 2 splicing between control and Alzheimer brains. Immunohistochemistry of mouse brain showed that apolipoprotein E receptor 2 was expressed in neurons throughout the brain, with strong expression in pyramidal neurons of the hippocampus, granule cells of the dentate gyrus, cortical neurons and Purkinje cells of the cerebellum. Thus, apolipoprotein E receptor 2 is the fourth apolipoprotein E receptor identified on neuronal cells.

Molecular Recognition of Human CD1b Antigen Complexes: Evidence for a Common Pattern of Interaction with αβ TCRs

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the α1 and α2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the α helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both α helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.

V(D)J recombination and RAG-mediated transposition in yeast

Antigen receptor genes are assembled during lymphoid development by a specialized recombination reaction normally observed only in cells of the vertebrate immune system. Here, we show that expression in Saccharomyces cerevisiae of murine RAG1 and RAG2, the lymphoid-specific components of the V(D)J recombinase, is sufficient to induce V(D)J cleavage and rejoining in this lower eukaryote. The RAG proteins cleave recombination substrates introduced into yeast cells, generating signal ends that can be joined to form signal joints. These signal joints are precise, as in mammalian cells, and their formation is dependent on a yeast nonhomologous end-joining protein, the XRCC4 homolog LIF1. Moreover, joining of SmaI-generated blunt ends is generally imprecise in the yeast strain used here, suggesting that the RAG proteins influence signal-end joining. Cleaved signal ends are also transposed into new sites in DNA, allowing RAG-induced transposition to be studied in vivo.

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32

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.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase.

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 enzymes 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.

The Sensor Kinase KinB Regulates Virulence in Acute Pseudomonas aeruginosa Infection

Two-component sensors are widely used by bacteria to sense and respond to the environment. Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its unique ability to adapt to multiple environments, including the human host. Several of these two-component sensors, such as GacS and RetS, have been shown to play roles in virulence in rodent infection models. However, the role and function of the majority of these two-component sensors remain unknown. Danio rerio is a recently characterized model host for pathogenesis-related studies that is amenable to higher-throughput analysis than mammalian models. Using zebrafish embryos as a model host, we have systematically tested the role of 60 two-component sensors and identified 6 sensors that are required for P. aeruginosa virulence. We found that KinB is required for acute infection in zebrafish embryos and regulates a number of virulence-associated phenotypes, including quorum sensing, biofilm formation, and motility. Its regulation of these phenotypes is independent of its kinase activity and its known response regulator AlgB, suggesting that it does not fit the canonical two-component sensor-response regulator model.

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to β-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore.

Lack of association of presenilin-1 intron-8 polymorphism with neuropathological features of Alzheimer's disease

Over 45 mutations within the coding region of presenilin-1 (PS-1) are associated with an autosomal dominant form of Alzheimers disease. Recently allele 1 of a polymorphism within intron-8 was reported to be in disequilibrium with Alzheimers disease in a group of patients with sporadic Alzheimers disease. This association has been replicated in some, but not all, studies. To determine whether the PS-1 intronic polymorphism is overrepresented in Alzheimers disease in an autopsy-proven series, and to examine whether allele 1 is associated with a specific neuropathological phenotype, polymerase chain reaction based technique was used to assess the genotype in 85 cases of Alzheimers disease. The resulting genotypes were compared with age of onset, duration of illness, and quantitative neuropathological measures of Abeta(total), Abeta(1-40), Abeta(1-42), neurofibrillary tangle number and neuron number. The 1/1 genotype did not associate with any differences in the clinical or neuropathological phenotype. These data suggest that the PS-1 intron-8 polymorphism does not strongly impact the clinical or neuropathologic features of Alzheimers disease.