Total Synthesis of the Reported Structure of Cahuitamycin A: Insights into an Elusive Natural Product Scaffold.

In a 2016 screen of natural product extracts, a new family of natural products, the cahuitamycins, was discovered and found to inhibit biofilm formation in the human pathogen Acinetobacter baumannii. The proposed molecular structures contained an unusual piperazic acid residue, which piqued interest related to their structure/function and biosynthesis. Herein we disclose the first total synthesis of the proposed structure of cahuitamycin A in a 12-step longest linear sequence and 18% overall yield. Comparison of spectral and biological data of the authentic natural product and synthetic compound revealed inconsistentancies with the isolated metabolite. We therefore executed the diverted total synthesis of three isomeric compounds, which were also found to be disparate from the isolated natural product. This work sets the stage for future synthetic and biochemical investigations of an important class of natural products.

Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen with a high mortality rate due to multi-drug-resistant strains. The synthesis and uptake of the iron-chelating siderophores acinetobactin (Acb) and preacinetobactin (pre-Acb) have been shown to be essential for virulence. Here, we report the kinetic and structural characterization of BauF, a flavin-dependent siderophore-interacting protein (SIP) required for the reduction of Fe(III) bound to Acb/pre-Acb and release of Fe(II). Stopped-flow spectrophotometric studies of the reductive half-reaction show that BauF forms a stable neutral flavin semiquinone intermediate. Reduction with NAD(P)H is very slow (k obs, 0.001 s-1) and commensurate with the rate of reduction by photobleaching, suggesting that NAD(P)H are not the physiological partners of BauF. The reduced BauF was oxidized by Acb-Fe (k obs, 0.02 s-1) and oxazole pre-Acb-Fe (ox-pre-Acb-Fe) (k obs, 0.08 s-1), a rigid analogue of pre-Acb, at a rate 3-11 times faster than that with molecular oxygen alone. The structure of FAD-bound BauF was solved at 2.85 Å and was found to share a similarity to Shewanella SIPs. The biochemical and structural data presented here validate the role of BauF in A. baumannii iron assimilation and provide information important for drug design.

Broad Spectrum Antibiotic Xanthocillin X Effectively Kills Acinetobacter baumannii via Dysregulation of Heme Biosynthesis.

Isonitrile natural products exhibit promising antibacterial activities. However, their mechanism of action (MoA) remains largely unknown. Based on the nanomolar potency of xanthocillin X (Xan) against diverse difficult-to-treat Gram-negative bacteria, including the critical priority pathogen Acinetobacter baumannii, we performed in-depth studies to decipher its MoA. While neither metal binding nor cellular protein targets were detected as relevant for Xan's antibiotic effects, sequencing of resistant strains revealed a conserved mutation in the heme biosynthesis enzyme porphobilinogen synthase (PbgS). This mutation caused impaired enzymatic efficiency indicative of reduced heme production. This discovery led to the validation of an untapped mechanism, by which direct heme sequestration of Xan prevents its binding into cognate enzyme pockets resulting in uncontrolled cofactor biosynthesis, accumulation of porphyrins, and corresponding stress with deleterious effects for bacterial viability. Thus, Xan represents a promising antibiotic displaying activity even against multidrug resistant strains, while exhibiting low toxicity to human cells.

Connecting iron acquisition and biofilm formation in the ESKAPE pathogens as a strategy for combatting antibiotic resistance.

The rise of antibiotic resistant bacteria has become a problem of global concern. Of particular interest are the ESKAPE pathogens, species with high rates of multi-drug resistant infections. Novel antibiotic mechanisms of action are necessary to compliment traditional therapeutics. Recent research has focused on targeting virulence factors as a method of combatting infection without creating selective pressure for resistance or damaging the host commensal microbiome. Some investigations into one such virulence behavior, iron acquisition, have displayed additional effects on another virulence behavior, biofilm formation. The use of exogenous iron-chelators, gallium as an iron mimic, and inhibition of siderophore-mediated iron acquisition are all strategies for disturbing iron-homeostasis that have implicated effects on biofilms. However, the exact nature of this connection remains ambiguous. Herein we summarize these findings and identify opportunities for further investigation.

From General to Specific: Can Pseudomonas Primary Metabolism Be Exploited for Narrow-Spectrum Antibiotics?

The spread of antimicrobial resistance is a major threat to human health, and patients requiring prolonged antibiotic exposure are in desperate need of new therapeutic strategies. It has been hypothesized that tailoring our antibiotics to inhibit molecular targets specific to pathogens might stem the spread of resistance. A prime candidate for such a strategy is Pseudomonas aeruginosa, which can be found in the lungs of nearly all adult cystic fibrosis patients and, due to chronic exposure to antibiotics, has a high rate of multidrug-resistant strains. Although much research has been done on P. aeruginosa virulence factors as narrow-spectrum targets, less attention has been paid to primary carbon metabolism being leveraged for pathogen-specific mechanisms. However, early studies show that primary metabolic pathways, although shared amongst all organisms, contain intricacies specific to Pseudomonas species that have potential for antibiotic exploitation. Here we lay out some of this work in the hopes that it inspires researchers to continue developing a knowledge base for future antibiotic discovery to build upon and include a case study of a Pseudomonas primary metabolic pathway that has been targeted by small molecules in a species-specific manner.

Identification of Specific and Nonspecific Inhibitors of Bacillus anthracis Type†III Pantothenate Kinase (PanK).

Antibiotics with novel mechanisms of action are desperately needed to combat the increasing rates of multidrug-resistant infections. Bacterial pantothenate kinase (PanK) has emerged as a target of interest to cut off the biosynthesis of coenzyme A. Herein we report the results of an in vitro high-throughput screen of over 10 000 small molecules against Bacillus anthracis PanK, as well as a follow-up screen of hits against PanK isolated from Pseudomonas aeruginosa and Burkholderia cenocepacia. Nine hits are structurally categorized and analyzed to set the stage for future drug development.

Crystal Structure of the Siderophore Binding Protein BauB Bound to an Unusual 2:1 Complex Between Acinetobactin and Ferric Iron.

The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multidrug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin. Acinetobactin is unusual in that it is first biosynthesized in an oxazoline form called preacinetobactin that spontaneously isomerizes to the final isoxazolidinone acinetobactin. Interestingly, both isomers can bind iron and both support growth of A. baumannii. To address how the two isomers chelate their ferric cargo and how the complexes are used by A. baumannii, structural studies were carried out with the ferric acinetobactin complex and its periplasmic siderophore binding protein BauB. Herein, we present the crystal structure of BauB bound to a bis-tridentate (Fe3+L2) siderophore complex. Additionally, we present binding studies that show multiple variants of acinetobactin bind BauB with no apparent change in affinity. These results are consistent with the structural model that depicts few direct polar interactions between BauB and the acinetobactin backbone. This structural and functional characterization of acinetobactin and its requisite binding protein BauB provides insight that could be exploited to target this critical iron acquisition system and provide a novel approach to treat infections caused by this important multidrug resistant pathogen.

Biologically Inspired Total Synthesis of Ulbactin F, an Iron-Binding Natural Product.

Natural products from environmental microbiomes provide exquisite templates for elucidating biological activity in the search for new drugs. A recently discovered marine Brevibacillus sp. metabolite, ulbactin F, was found to inhibit tumor cell migration and invasion at IC50 < 3 µM. Herein, we disclose the first total synthesis of ulbactin F and epi-ulbactin F, which was modeled after the biosynthetic pathway. The scaffold bears structural similarity to siderophores of human pathogens but contains a novel tricyclic ring system derived from cysteine. We have found that ulbactin F forms low-affinity metal complexes, with a preference for Fe3+ and Cu2+, which may hint both at its environmental role and its antimetastatic mechanism of action.

Rigid Oxazole Acinetobactin Analog Blocks Siderophore Cycling in Acinetobacter baumannii.

The emergence of multidrug resistant (MDR) Gram-negative bacterial pathogens has raised global concern. Nontraditional therapeutic strategies, including antivirulence approaches, are gaining traction as a means of applying less selective pressure for resistance in vivo. Here, we show that rigidifying the structure of the siderophore preacinetobactin from MDR Acinetobacter baumannii via oxidation of the phenolate-oxazoline moiety to a phenolate-oxazole results in a potent inhibitor of siderophore transport and imparts a bacteriostatic effect at low micromolar concentrations under infection-like conditions.

Structure-function studies of acinetobactin analogs.

Pathogenic Acinetobacter baumannii excrete the siderophore pre-acinetobactin as an iron-scavenging virulence factor. Pre-acinetobactin is a 2,3-dihydroxy-phenyl oxazoline that undergoes pH-dependent isomerization to the isooxazolidinone form acinetobactin in order to expand the pH range for iron acquisition by A. baumannii. In this study we establish important structure-function relationships for the kinetics of isomerization, iron(iii) binding, and siderophore utilization by A. baumannii. We showed that electronic properties of the phenyl oxazoline influence isomerization kinetics and iron(iii) binding. We found that iron(iii) chelation was directly correlated with A. baumannii utilization. Our studies provide important structural and mechanistic insight for understanding how pathogenic A. baumannii uses pre-acinetobactin as a 2-for-1 iron-scavenging siderophore virulence factor.

Acinetobactin Isomerization Enables Adaptive Iron Acquisition in Acinetobacter baumannii through pH-Triggered Siderophore Swapping.

Pathogenic strains of Acinetobacter baumannii excrete multiple siderophores that enhance iron scavenging from host sources. The oxazoline siderophore pre-acinetobactin undergoes an unusual non-enzymatic isomerization, producing the isoxazolidinone acinetobactin. In this study, we explored the kinetics, mechanism, and biological consequence of this siderophore swapping. Pre-acinetobactin is excreted to the extracellular space where the isomerization to acinetobactin occurs with a pH-rate profile consistent with 5-exo-tet cyclization at C5' with clean stereochemical inversion. Pre-acinetobactin persists at pH <6, and acinetobactin is rapidly formed at pH >7, matching each siderophore's pH preference for iron(III) chelation and A. baumannii growth promotion. Acinetobactin isomerization provides two siderophores for the price of one, enabling A. baumannii to sequester iron over a broad pH range likely to be encountered during the course of an infection.

Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism's putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.