A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance

A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue.

Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target

Climate, demography, immunology, and virology combine to drive two decades of dengue virus dynamics in Cambodia

The incidence of dengue virus disease has increased globally across the past half-century, with highest number of cases ever reported in 2019 and again in 2023. We analyzed climatological, epidemiological, and phylogenomic data to investigate drivers of two decades of dengue in Cambodia, an understudied endemic setting. Using epidemiological models fit to a 19-y dataset, we first demonstrate that climate-driven transmission alone is insufficient to explain three epidemics across the time series. We then use wavelet decomposition to highlight enhanced annual and multiannual synchronicity in dengue cycles between provinces in epidemic years, suggesting a role for climate in homogenizing dynamics across space and time. Assuming reported cases correspond to symptomatic secondary infections, we next use an age-structured catalytic model to estimate a declining force of infection for dengue through time, which elevates the mean age of reported cases in Cambodia. Reported cases in >70-y-old individuals in the 2019 epidemic are best explained when also allowing for waning multitypic immunity and repeat symptomatic infections in older patients. We support this work with phylogenetic analysis of 192 dengue virus (DENV) genomes that we sequenced between 2019 and 2022, which document emergence of DENV-2 Cosmopolitan Genotype-II into Cambodia. This lineage demonstrates phylogenetic homogeneity across wide geographic areas, consistent with invasion behavior and in contrast to high phylogenetic diversity exhibited by endemic DENV-1. Finally, we simulate an age-structured, mechanistic model of dengue dynamics to demonstrate how expansion of an antigenically distinct lineage that evades preexisting multitypic immunity effectively reproduces the older-age infections witnessed in our data

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Genomic approaches to investigate the control of gene expression in the human malaria parasite, Plasmodium falciparum

The recent advances in genomic sequencing technologies in the past decade have enabled the unprecedented ability to investigate infectious diseases and organisms that were traditionally difficult to study in the laboratory. In particular, the human malaria parasite, Plasmodium falciparum, is a unique challenge because of the difficulty to grow these obligate intracellular parasites during the complex blood stages. Yet despite these challenges, genomic approaches have allowed us to investigate the parasite in an unbiased and comprehensive manner. The work described here uses DNA sequencing, RNA sequencing, high-throughput screening, and bioinformatics tools to understand the fundamental biological processes of the malaria parasite such as mechanisms of drug resistance, regulation of translation, and determinants of translational efficiency. A mechanistic understanding of how the parasite can escape antimalarial drug pressure is valuable for future drug development and the design of new drugs that avoid known parasite resistance mechanisms. To this end, we have developed a bioinformatics pipeline that will identify mutations such as copy number variations or single nucleotide polymorphisms that confer resistance to various drugs in vitro. The process of translation in malaria is poorly understood as the technologies for measuring translation or protein products are limited to measuring only the most abundant proteins. To address this problem, we took a genome-wide approach of ribosome profiling which quantitatively measures transcription and translation simultaneously for the entire genome. This study led to many interesting questions revolving around how the P. falciparum ribosome functions differently from other eukaryotic ribosomes and what cis-acting sequences determine the efficiency of translation. To answer these questions, we developed an in vitro translation system derived from cell free lysates of P. falciparum to both screen for drug compounds that specifically inhibit malaria translation and not other eukaryotic translation and we have employed this assay to find specific sequences found primarily on the 5’ untranslated region of an mRNA that can modulate translation. In total, this work addresses the regulation of gene expression in the asexual blood stages of this medically relevant parasite