Potentiation of antibacterial activity of the MB-1 siderophore-monobactam conjugate using an efflux pump inhibitor.

Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability.

Adaptation-based resistance to siderophore-conjugated antibacterial agents by Pseudomonas aeruginosa.

Multidrug resistance in Gram-negative bacteria has become so threatening to human health that new antibacterial platforms are desperately needed to combat these deadly infections. The concept of siderophore conjugation, which facilitates compound uptake across the outer membrane by hijacking bacterial iron acquisition systems, has received significant attention in recent years. While standard in vitro MIC and resistance frequency methods demonstrate that these compounds are potent, broad-spectrum antibacterial agents whose activity should not be threatened by unacceptably high spontaneous resistance rates, recapitulation of these results in animal models can prove unreliable, partially because of the differences in iron availability in these different methods. Here, we describe the characterization of MB-1, a novel siderophore-conjugated monobactam that demonstrates excellent in vitro activity against Pseudomonas aeruginosa when tested using standard assay conditions. Unfortunately, the in vitro findings did not correlate with the in vivo results we obtained, as multiple strains were not effectively treated by MB-1 despite having low MICs. To address this, we also describe the development of new in vitro assays that were predictive of efficacy in mouse models, and we provide evidence that competition with native siderophores could contribute to the recalcitrance of some P. aeruginosa isolates in vivo.

Clinically relevant Gram-negative resistance mechanisms have no effect on the efficacy of MC-1, a novel siderophore-conjugated monocarbam.

The incidence of hospital-acquired infections with multidrug-resistant (MDR) Gram-negative pathogens is increasing at an alarming rate. Equally alarming is the overall lack of efficacious therapeutic options for clinicians, which is due primarily to the acquisition and development of various antibiotic resistance mechanisms that render these drugs ineffective. Among these mechanisms is the reduced permeability of the outer membrane, which prevents many marketed antibiotics from traversing this barrier. To circumvent this, recent drug discovery efforts have focused on conjugating a siderophore moiety to a pharmacologically active compound that has been designed to hijack the bacterial siderophore transport system and trick cells into importing the active drug by recognizing it as a nutritionally beneficial compound. MC-1, a novel siderophore-conjugated ß-lactam that promotes its own uptake into bacteria, has exquisite activity against many Gram-negative pathogens. While the inclusion of the siderophore was originally designed to facilitate outer membrane penetration into Gram-negative cells, here we show that this structural moiety also renders other clinically relevant antibiotic resistance mechanisms unable to affect MC-1 efficacy. Resistance frequency determinations and subsequent characterization of first-step resistant mutants identified PiuA, a TonB-dependent outer membrane siderophore receptor, as the primary means of MC-1 entry into Pseudomonas aeruginosa. While the MICs of these mutants were increased 32-fold relative to the parental strain in vitro, we show that this resistance phenotype is not relevant in vivo, as alternative siderophore-mediated uptake mechanisms compensated for the loss of PiuA under iron-limiting conditions.

COVID-19 Seroprevalence and Active Infection in an Asymptomatic Population.

In response to the COVID-19 pandemic, immediate and scalable testing solutions are needed to direct return to full capacity planning in the general public and across the Department of Defense (DoD). To fully understand the extent to which a population has been affected by COVID-19, active monitoring approaches require an estimation of overall seroprevalence in addition to accurate, affordable, and rapid tests to detect current SARS-CoV-2 infection. In this study, researchers in the Air Force Research Laboratory's 711th Human Performance Wing, Airman Systems Directorate evaluated the performance of various testing methods for the detection of SARS-CoV-2 antibodies and viral RNA in asymptomatic adults working at Wright-Patterson Air Force Base and the surrounding area during the period of 23 July 2020-23 Oct 2020. Altogether, there was a seroprevalance of 3.09% and an active infection rate of 0.5% (determined via the testing of saliva samples) amongst individuals tested, both of which were comparable to local and national averages at the time. This work also presents technical and non-technical assessments of various testing strategies as compared to the gold standard approaches (e.g., lateral flow assays vs. ELISA and RT-LAMP vs. RT-PCR) in order to explore orthogonal supply chains and fieldability. Exploration and validation of multiple testing strategies will allow the DoD and other workforces to make informed responses to COVID-19 and future pandemics.

The EF-hand calcium-binding protein calmyrin inhibits the transcriptional and DNA-binding activity of Pax3.

Pax3 is a member of the paired class homeodomain family of transcription factors and has been demonstrated to be an early marker in myogenic differentiation. To gain a better understanding of how protein-protein interactions regulate Pax3 transcriptional activity, we performed a yeast two-hybrid analysis to identify proteins that interact with Pax3. Screening of two cDNA libraries isolated nine independent clones that contained the complete encoding sequence of the EF-hand calcium-binding protein calmyrin. In this report, we demonstrate that calmyrin specifically interacts with Pax3 in vitro. In addition, we demonstrate that the interaction between Pax3 and calmyrin is mediated by the region of the Pax3 paired domain that is involved in making DNA contacts and the Pax3 octapeptide domain and its surrounding amino acid sequences. We also demonstrate that endogenous Pax3 and calmyrin are co-expressed in undifferentiated primary myoblasts and that calmyrin expression levels increase in the nucleus upon myogenic differentiation. Finally, we demonstrate that co-expression of calmyrin with Pax3 inhibits the transcriptional activity of Pax3 by inhibiting Pax3 from binding to its recognition DNA sequences. These results therefore suggest potential ways in which calcium, through its regulation of the EF-hand calcium-binding protein calmyrin, can alter the DNA-binding activity and subsequent transcriptional activity of Pax3.

Discovery and characterization of a novel class of pyrazolopyrimidinedione tRNA synthesis inhibitors.

A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure-activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC50s of 21.7 and 35 µM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 µM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens.

UNLABELLED: The problem of multidrug resistance in serious Gram-negative bacterial pathogens has escalated so severely that new cellular targets and pathways need to be exploited to avoid many of the preexisting antibiotic resistance mechanisms that are rapidly disseminating to new strains. The discovery of small-molecule inhibitors of LpxC, the enzyme responsible for the first committed step in the biosynthesis of lipid A, represents a clinically unprecedented strategy to specifically act against Gram-negative organisms such as Pseudomonas aeruginosa and members of the Enterobacteriaceae. In this report, we describe the microbiological characterization of LpxC-4, a recently disclosed inhibitor of this bacterial target, and demonstrate that its spectrum of activity extends to several of the pathogenic species that are most threatening to human health today. We also show that spontaneous generation of LpxC-4 resistance occurs at frequencies comparable to those seen with marketed antibiotics, and we provide an in-depth analysis of the mechanisms of resistance utilized by target pathogens. Interestingly, these isolates also served as tools to further our understanding of the regulation of lipid A biosynthesis and enabled the discovery that this process occurs very distinctly between P. aeruginosa and members of the Enterobacteriaceae. Finally, we demonstrate that LpxC-4 is efficacious in vivo against multiple strains in different models of bacterial infection and that the major first-step resistance mechanisms employed by the intended target organisms can still be effectively treated with this new inhibitor. IMPORTANCE: New antibiotics are needed for the effective treatment of serious infections caused by Gram-negative pathogens, and the responsibility of identifying new drug candidates rests squarely on the shoulders of the infectious disease community. The limited number of validated cellular targets and approaches, along with the increasing amount of antibiotic resistance that is spreading throughout the clinical environment, has prompted us to explore the utility of inhibitors of novel targets and pathways in these resistant organisms, since preexisting target-based resistance should be negligible. Lipid A biosynthesis is an essential process for the formation of lipopolysaccharide, which is a critical component of the Gram-negative outer membrane. In this report, we describe the in vitro and in vivo characterization of novel inhibitors of LpxC, an enzyme whose activity is required for proper lipid A biosynthesis, and demonstrate that our lead compound has the requisite attributes to warrant further consideration as a novel antibiotic.

Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek.

Human Daxx is a protein that functions, in part, as a transcriptional co-repressor through its interaction with a growing number of nuclear, DNA-associated proteins. To determine the mechanism by which hDaxx represses transcription, we used conventional chromatography to isolate endogenous hDaxx. We determined that hDaxx has an apparent molecular weight of 360 kDa, which is consistent with the fact that multiple domains of hDaxx are required for transcriptional repression and suggests that hDaxx associates with multiple proteins. Using co-fractionation and co-immunoprecipitation we demonstrate that hDaxx associates with proteins that are critical for transcriptional repression, such as histone deacetylase II, constituents of chromatin such as core histones H2A, H2B, H3 and H4, and Dek, a chromatin-associated protein reported to change the topology of DNA in chromatin in vitro. We also demonstrate a requirement for the SPT domain and the first paired amphipathic helix of hDaxx for its association with histone deacetylase II and acetylated histone H4, respectively. Finally, we provide evidence suggesting that the association of hDaxx with chromatin-related proteins is dependent on the post-translational phosphorylation status of hDaxx. A working model for the repressive action of hDaxx through its association with chromatin related proteins is presented.