Pablo Bifani

Plasmodium cynomolgi is the closest relative of P. vivax and the primary experimental model for relapsing malaria, hypnozoite biology, and blood-stage drug susceptibility. Yet existing reference genomes remain fragmented, leaving structurally complex, AT-rich regions largely unresolved. We generated a chromosome-scale genome assembly for the K4-A7 cloned line of P. cynomolgi Berok by combining Hi-C chromosome conformation capture, Oxford Nanopore long reads, PacBio, and Illumina sequencing. The assembly spans 14 chromosomes plus mitochondrial and apicoplast genomes, with only seven unplaced minor contigs, the fewest for any non- P. falciparum Plasmodium genome, and an N50 of 3.06 Mb. Critically, this hybrid strategy resolved approximately 8 Mb of extremely AT-rich (~ 20% GC) sequence onto chromosomes 4, 8, and 13, anchoring what were previously unplaced or absent contigs into a continuous chromosomal framework. These subtelomere-like expansions (SLEs) constitute ~ 26.5% of the chromosomal genome and are enriched for PIR/VIR, STP1, variable surface antigen, and methyltransferase pseudogene families. Despite low gene density, SLE-encoded genes are transcriptionally active and show stage-specific expression across the erythrocytic cycle. Integrated lifecycle transcriptomics across 7,006 genes revealed a ~ 54-hour erythrocytic cycle with a "just-in-time" transcriptional cascade closely resembling that of P. vivax . Phylogenomic analyses and pairwise amino acid comparisons across more than 2,600 single-copy orthologs show that Berok forms a deeply diverged P. cynomolgi lineage, suggesting a distinct subspecies. This assembly establishes a high-resolution genomic foundation for comparative malaria biology, drug discovery, and the study of subtelomeric architecture, host adaptation, and lineage boundaries in primate Plasmodium .

The zoonotic parasite Plasmodium knowlesi has emerged as a widespread cause of human malaria in Southeast Asia. We assembled a high-resolution transcriptome of zoonotic P. knowlesi across its intraerythrocytic developmental cycle and combined transcriptomics, quantitative proteomics and thermal stability measurements to compare parasites cultured in macaque and human red blood cells. We found that P. knowlesi accelerates through the ring stage to settle at the same developmental pace as P. falciparum during the trophozoite and schizont stages. Subsequent multi-omic analysis revealed broad transcriptional and proteomic adaptations of P. knowlesi to the human host cell environment. Parasites cultured in human cells expressed glutathione synthetase earlier and displayed changes in upstream processes that include NADPH production by the pentose phosphate pathway and glutamine synthesis. These changes were accompanied by further differences in the expression of ribosomal subunits and mitochondrial respiratory complexes. Together, our findings offer a systems-level view of non-genetic adaptations of P. knowlesi to the human red blood cell environment.

The rise of drug-resistant Klebsiella pneumoniae infections is prompting a re-evaluation of phage therapy as an alternative treatment. Phage therapy has achieved successful outcomes, yet important limitations persist, including the rapid emergence of phage resistance and the time required to source suitable phages. Combining multiple phages into a phage cocktail based on genetic diversity, morphology or distinct infection mechanisms is a common strategy to mitigate resistance. Phage pairing based on these criteria neither mitigates nor delays the emergence of resistance, using CG23 hypervirulent Klebsiella pneumoniae (hv Kp ) EC0675 as a model. In CG23 Kp strains, resistance arises through disruption of the capsular biosynthetic pathway, leading to capsule impairment and global cross-resistance to all capsule-active phages. Combining phages active against encapsulated wild-type bacteria with those active against capsule-deficient escape mutants can overcome this constraint, improving bacterial suppression in vitro and reducing bacterial burdens across multiple organs in a mouse systemic infection model. In parallel, phage-driven capsule loss increased susceptibility of hv Kp EC0675 to selected antibiotics targeting the bacterial envelope or cell wall in vitro . These findings highlight the contribution of capsule-associated resistance to phage cross-resistance in the CG23 Kp strains examined, and support further evaluation of phage combinations targeting both wild-type bacteria and resistant derivatives, including their use alongside selected antibiotics, informing future clinical translation.

Plasmodium cynomolgi is the closest relative of P. vivax and the primary experimental model for relapsing malaria, hypnozoite biology, and blood-stage drug susceptibility. Yet existing reference genomes remain fragmented, leaving structurally complex, AT-rich regions largely unresolved. We generated a chromosome-scale genome assembly for the K4-A7 cloned line of P. cynomolgi Berok by combining Hi-C chromosome conformation capture, Oxford Nanopore long reads, PacBio, and Illumina sequencing. The assembly spans 14 chromosomes plus mitochondrial and apicoplast genomes, with only seven unplaced minor contigs, the fewest for any non- P. falciparum Plasmodium genome, and an N50 of 3.06 Mb. Critically, this hybrid strategy resolved approximately 8 Mb of extremely AT-rich (∼20% GC) sequence onto chromosomes 4, 8, and 13, anchoring what were previously unplaced or absent contigs into a continuous chromosomal framework. These subtelomere-like expansions (SLEs) constitute ∼26.5% of the chromosomal genome and are enriched for PIR/VIR, STP1, variable surface antigen, and methyltransferase pseudogene families. Despite low gene density, SLE-encoded genes are transcriptionally active and show stage-specific expression across the erythrocytic cycle. Integrated lifecycle transcriptomics across 7,006 genes revealed a ∼54-hour erythrocytic cycle with a “just-in-time” transcriptional cascade closely resembling that of P. vivax . Phylogenomic analyses and pairwise amino acid comparisons across more than 2,600 single-copy orthologs show that Berok forms a deeply diverged P. cynomolgi lineage, suggesting a distinct subspecies. This assembly establishes a high-resolution genomic foundation for comparative malaria biology, drug discovery, and the study of subtelomeric architecture, host adaptation, and lineage boundaries in primate Plasmodium .

Exploring bacteriophage structural diversity is essential for understanding phage biology and for advancing phage-based therapies. Here, we determine the cryo-electron microscopy structure of Jabs, providing, to our knowledge, the first high-resolution view of a phage infecting the multidrug-resistant human pathogen Mycobacterium abscessus . Although Jabs displays the canonical organization of a siphophage, its virion combines several unusual architectural features. The T=9 icosahedral capsid is assembled from two distinct major capsid proteins, with one forming the hexons and the other the pentons, revealing an unprecedented capsid assembly strategy among icosahedral phages. An extensive network of ∼1,700 disulfide bonds stabilize individual structural components and covalently links the capsid, connector, tail, and adhesion device into a continuous assembly. At the distal end of the tail, an elaborate and conformationally dynamic adhesion device comprises multiple candidate receptor-binding proteins organized into complex multidomain architectures, including carbohydrate-binding modules and β-sandwich hetero- and homotrimers resembling the receptor-binding proteins of phages infecting lactic acid bacteria. Together, these findings expand our understanding of phage structural diversity and provide a framework for investigating phage-host interactions and guiding the engineering of therapeutic phages.

Tuberculosis (TB) is a major global health threat, with millions of new cases diagnosed annually. Rifampicin is a cornerstone drug in the current standard of care for TB treatment. Recently, rifapentine, an analog of rifampicin, has been considered as an alternative to rifampicin primarily due to its longer half-life and shorter 4-month TB treatment regimens. Despite some encouraging advantages in the clinical setting, the rifamycin resistance mutation frequency and type have yet to be systematically investigated. The mutation identity and frequency could directly impact clinical outcomes and the epidemiology of TB resistance. Our objective was to compare the in vitro antimicrobial properties of rifapentine and rifampicin across four Mycobacterium tuberculosis strains, namely H37Rv, CDC1551, HN878, and W4, a lineage 2 (Beijing) genotype, and to determine whether resistance differed between the two drugs. We compared the frequency of resistance between rifampicin and rifapentine at various concentrations, and evaluated the frequency of different mutation types in the four chosen TB strains via Sanger sequencing. Overall, no significant differences were observed in the frequency of resistance or mutation identity between rifampicin and rifapentine; however, strain-dependent differences were noted. H37Rv showed a higher frequency of resistance at all concentrations tested in both drugs compared to CDC1551 and the Beijing strains W4 and HN878. The profound overlap in in vitro properties between the two antimicrobials provides valuable insight for the ongoing evaluation of rifapentine as a viable alternative to rifampicin in the standard of care for TB treatment. IMPORTANCE: Recent clinical trials demonstrate that a 4-month regimen combining rifapentine, isoniazid, pyrazinamide, and moxifloxacin is non-inferior to the standard 6-month rifampicin-based regimen. This shorter regimen, with less frequent rifamycin administration, may enhance patient compliance and treatment outcomes. However, two pivotal questions warrant attention before proceeding with a switch to rifapentine-based treatments. First, we must ensure that rifapentine maintains comparable or lower rates of rifamycin resistance, as an increased rate of resistance mutations would undermine the new regimen’s viability. Second, we need to confirm whether current molecular diagnostic tools can accurately detect rifapentine resistance with equivalent sensitivity and specificity. Thus, any rifapentine-specific mutations and their frequency must be identified and incorporated into existing molecular diagnostic platforms if novel. In our investigation, we found that the mutation frequency and repertoire were not significantly different between rifampicin and rifapentine within strains. Current diagnostic methods should be equally sensitive to rifapentine and rifampicin resistance.

Background and Aims: Understanding regulatory interactions between hepatitis B virus (HBV) and host factors is essential for the development of next generation host-directed antiviral therapies and the achievement of a functional HBV cure. Here, we investigated HBV-induced alterations in host gene expression in primary human hepatocytes (PHH) to identify host factors exploited by the virus for replication and persistence. Whole-transcriptome sequencing (WTS) of HBV-infected PHH identified host pathways with potential roles in the HBV life cycle. RNA interference-based functional screening of dysregulated candidate genes identified cyclin L1 (CCNL1) as a key host factor. RNAi-mediated knockdown of CCNL1 reduced HBV gene expression, including hepatitis B surface antigen (HBsAg). Mechanistically, CCNL1 regulates phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) at serine 2 (S2), consistent with a role in transcriptional regulation. CCNL1 knockdown further reduced the binding of total and phospho- (Ser2/Ser5) RNAPII, pan-acetylated histone H3 (H3ac), and H3K27ac to HBV covalently closed circular DNA (cccDNA), indicating impaired cccDNA-dependent transcription. In addition, CCNL1 expression was elevated in chronic hepatitis B patients compared with those with resolved infection. Collectively, these data demonstrate that CCNL1 promotes HBV transcription and replication through modulation of RNAPII phosphorylation and chromatin-associated transcriptional activity, identifying CCNL1 as a potential host susceptibility factor for HBV. Importance: Hepatitis B virus infection remains a major threat to human health in areas with high prevalence. There is need to fully understand the complex interactions between the virus and human host factors/processes to support ongoing efforts to develop anti-HBV therapies that can be used with existing therapies to achieve a better cure. HBV relies on host cellular factors and biological processes to establish and maintain efficient infection, making host–virus interactions attractive targets for therapeutic intervention. Thus, identifying host factors that support and/or restrict HBV infection is essential for understanding the molecular basis of chronic HBV infection and for developing host-targeting anti-HBV drugs. This study identifies cyclin L1 (CCNL1) as a host susceptibility factor that promotes HBV transcription and replication through regulation of RNA polymerase II activity and or post-transcriptional mechanisms.

Mycobacterium abscessus infections represent a growing global health concern due to their severe pathology and difficulty of treatment, largely driven by their intrinsic antimicrobial resistance. While phage therapy has emerged as a promising alternative approach, studies have predominantly focused on glycopeptidolipids (GPL)-deficient M. abscessus rough variants instead of the GPL-producing smooth variants predominant in Asia. Here, we aim to develop phage cocktails targeting both smooth and rough morphotypes. In the process, we found that phage treatment of smooth variants can select for rough morphotype switching from smooth-to-rough variants in vitro and in vivo, resulting in phage resistance associated with mutations within the GPL biosynthetic locus. We validated our findings in vitro and in vivo, suggesting a two-layered phage combination that surpasses single-phage treatments and potentially improves clinical phage cocktail strategies. This work underlines the need to better understand mechanisms of phage resistance in phage therapy and associated potential adverse effects and solutions. Phage resistance in M. abscessus through morphotype switching is clinically significant, as it may complicate treatment outcomes but could be averted with proper phage combinations.

Machine learning has revolutionized protein structure and interaction prediction, yet its full potential for drug discovery is still emerging. In this study, we show that denoise diffusion-based co-folding methods—such as AlphaFold3 and Boltz-1/2—not only achieve highly accurate protein-ligand interaction predictions but can also separate active compounds from inactive ones. We introduce a simple and effective strategy, pairwise competitive docking, which ranks candidate molecules by directly comparing their relative binding to a protein’s target site. Applied to 17 protein benchmark systems, the method generated rankings consistent with experimental trends, although the degree of agreement varied considerably by system, with concordance indices ranging from 0.52 (indicating no meaningful correlation) to 0.89 (indicating strong correlation). Notably, our rankings showed strong agreement with Boltz-2 affinity predictions, positioning our method as a practical alternative for inhibitor prioritization. Finally, we show how pairwise competitive docking can accelerate the identification of promising hits within a large chemical library and guide the de novo design of inhibitors with improved predicted potency. Collectively, these findings highlight how modern machine-learning models can make structure-based drug design faster, more reliable, and more cost-effective than relying solely on experimental workflows.

Machine learning has transformed how we predict protein structures and interactions, but its full potential in drug discovery is only beginning to be realized. In this study, we demonstrate that advanced deep learning tools —such as AlphaFold3 and Boltz-1/2— not only predict protein-ligand interactions with high accuracy but can also separate active drug compounds from inactive ones. We present a straightforward strategy called pairwise competitive docking , which ranks drug candidates by directly comparing how well they bind to a protein’s target site. When applied to both bacterial and human enzymes, this method produced rankings that closely matched experimental results. We further show how this approach can guide the design of improved antibiotics and speed up the discovery of promising drug candidates from large chemical libraries. Overall, our findings highlight how machine learning can make structure-based drug design faster, more reliable, and more cost-effective.