Next generation antibiotic combinations to combat pan-drug resistant Klebsiella pneumoniae.

Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-ß-lactamases. Unexpectedly, the ß-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug ß-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated ß-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel ß-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.

Machine Learning-Led Optimization of Combination Therapy: Confronting the Public Health Threat of Extensively Drug Resistant Gram-Negative Bacteria.

Developing optimized regimens for combination antibiotic therapy is challenging and often performed empirically over many clinical studies. Novel implementation of a hybrid machine-learning pharmacokinetic/pharmacodynamic/toxicodynamic (ML-PK/PD/TD) approach optimizes combination therapy using human PK/TD data along with in vitro PD data. This study utilized human population PK (PopPK) of aztreonam, ceftazidime/avibactam, and polymyxin B along with in vitro PDs from the Hollow Fiber Infection Model (HFIM) to derive optimal multi-drug regimens de novo through implementation of a genetic algorithm (GA). The mechanism-based PD model was constructed based on 7-day HFIM experiments across 4 clinical, extensively drug resistant Klebsiella pneumoniae isolates. GA-led optimization was performed using 13 different fitness functions to compare the effects of different efficacy (60%, 70%, 80%, or 90% of simulated subjects achieving bacterial counts of 102 CFU/mL) and toxicity (66% of simulated subjects having a target polymyxin B area under the concentration-time curve [AUC] of 100 mg·h/L and aztreonam AUC of 1,332 mg·h/L) on the optimized regimen. All regimens, except those most heavily weighted for toxicity prevention, were able to achieve the target efficacy threshold (102 CFU/mL). Overall, GA-based regimen optimization using preclinical data from animal-sparing in vitro studies and human PopPK produced clinically relevant dosage regimens similar to those developed empirically over many years for all three antibiotics. Taken together, these data provide significant insight into new therapeutic approaches incorporating ML to regimen design and treatment of resistant bacterial infections.

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

Morphotype-specific effector functions of Cryptococcus neoformans PUM1.

The basidiomycete fungal pathogen Cryptococcus neoformans requires the PUF protein, Pum1, for hyphal morphogenesis during sexual development. In this study we found that Pum1 was auto-repressive under growth as yeast, but that auto-repression was relieved during filamentous growth through utilization of an alternative transcription start site driven by the master filamentation regulator Znf2. In addition, Pum1 was required to stabilize the ZNF2 mRNA through an indirect mechanism suggesting that Znf2 and Pum1 each positively regulate the expression of the other to achieve the filamentous morphotype required for sexual development in Cryptococcus.

Puf4 regulates both splicing and decay of HXL1 mRNA encoding the unfolded protein response transcription factor in Cryptococcus neoformans.

The endoplasmic reticulum (ER) responds to errors in protein folding or processing by induction of the unfolded protein response (UPR). During conditions of ER stress, unconventional splicing of an mRNA encoding the UPR-responsive transcription factor occurs at the ER surface, resulting in activation of the UPR. UPR activation is necessary for adaptation to ER stress and for the pathogenic fungus Cryptococcus neoformans is an absolute requirement for temperature adaptation and virulence. In this study, we have determined that C. neoformans has co-opted a conserved PUF RNA binding protein to regulate the posttranscriptional processing of the HXL1 mRNA encoding the UPR transcription factor. PUF elements were identified in both the 5' and 3' untranslated regions of the HXL1 transcript, and both elements bound Puf4. Deletion of PUF4 resulted in delayed unconventional splicing of HXL1 mRNA and delayed induction of Hxl1 target genes. In addition, the HXL1 transcript was stabilized in the absence of Puf4. The puf4Δ mutant exhibited temperature sensitivity but was as virulent as the wild type, despite a reduction in fungal burden in the brains of infected mice. Our results reveal a novel regulatory role in which a PUF protein influences the unconventional splicing of the mRNA encoding the UPR-responsive transcription factor. These data suggest a unique role for a PUF protein in controlling UPR kinetics via the posttranscriptional regulation of the mRNA encoding the UPR transcription factor Hxl1.

Hydroxyurea enhances post-fusion hyphal extension during sexual development in C. neoformans var. grubii.

Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.