Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

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

Elucidating the Pathological Mechanism of Histone H3.3 Mutations in Neurodevelopment

In eukaryotic cells, octamers of histone proteins intricately organize DNA, forming a macromolecule known as nucleosomes. A nucleosome is comprised of two copies of histone H2A, H2B, H4, and H3. Histone H3.3 (H3.3), a histone variant, is often found at actively transcribed loci. H3.3 plays a role in cellular inheritance as ablation of H3.3 expression leads to loss of active gene states and dysfunction of heterochromatin telomeric structures. H3F3A and H3F3B, the two genes are known to encode H3.3, are expressed in all human cells with higher expression in the gonads and brain. A recent publication detailed H3.3 as the causative gene in a neurodevelopmental disorder with craniofacial abnormalities. The underlying cause of the disease is unknown; however, H3.3 is involved in a wide range of central nervous system functions. This dissertation highlights work that aimed to profile the effects of these germline mutations. We performed a comprehensive screen on pathogenic H3.3 variants utilizing a combination of techniques and methodology, including tissue culture, bottom-up proteomics, and neurodevelopmental assays in Xenopus laevis. We found global alterations to histone post-translational modifications with significant alterations to histone acetylation on histone H2a, H3, and H4 peptides. In addition, performing quantitative proteomics in the 293T cells led us to determine that these mutations affect several cellular processes, such as RNA splicing, cell motility, neurofilament maintenance, folic acid metabolism, and post-synaptic density, all processes known to be dysregulated in other neurological syndromes. Utilizing the model organism Xenopus, we introduced two of the pathogenic variants into embryos. We observed reduced craniofacial cartilage, abnormal head shape, and impaired motility due to kinked tails in the mutant tadpoles. We performed quantitative proteomics on these tadpoles and found pathways related to carbon metabolism and amino acid degradation upregulated. Lastly, our transcriptomic analyses corroborated some of these finds and an upregulation of the TGF-beta signaling pathway in one of the mutants. This work provides the first mechanistic study of these germline H3.3 mutations, introduces pathways dysregulated that can be further studied. Understanding the basic biology of these mutations will shed light on the molecular mechanisms of H3.3 in neurodevelopment

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.</p

Evolutionary trade-offs underlie the multi-faceted virulence of <em>Staphylococcus aureus</em>

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.</p

The identification of six novel toxicity-affecting loci.

<p>Transposon mutagenesis was used to functionally verify the role of loci associated with toxicity by genome-wide association study (GWAS; PLINK). Mean of six replicates are presented, error bars represent the 95% confidence intervals. The mutation in <i>agrB</i> is provided as a negative control. To access this data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002229#pbio.1002229.s002" target="_blank">S2 Data</a>.</p

Toxicity levels and disease origin distributed across the USA300 collection.

<p>The clinical origin (carriage = C, SSTI = S, or blood = B) and the toxicity (low = green, medium = orange or high = red) of 134 USA300 isolates are mapped onto a maximum likelihood tree based on their whole genome sequence.</p

Low levels of toxicity are associated with invasive <i>S</i>. <i>aureus</i> infections across two collections of clinical isolates.

<p><b>a:</b> Individual isolates from seven time points were assayed for toxicity and their genome sequenced. Early nose cultures (ENCs) from months 1, 2, 4, 6, and 8 were all highly toxic. Late nose culture (LNC) isolates at month 12 and bloodstream isolates following bacteraemia (late blood culture, LBC) were all significantly less toxic than the ENC isolates. <b>b</b> and <b>c:</b> The toxicity of 134 USA300 isolates were assayed, and those from bacteraemia were on average significantly less toxic (measured as percent T2 and THP1 cell death (b and c, respectively)) than from carriage or SSTI <i>(p <</i> 0.01 ANOVA). The medians are presented as horizontal bars, with the boxes and whiskers showing the 1st and 3rd quartile and interquartile ranges. To access this data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002229#pbio.1002229.s001" target="_blank">S1 Data</a>.</p

The highly toxic isolates are less fit than the low-toxicity isolates in the presence of human serum.

<p>We quantified the relative fitness of six high- and six low-toxicity isolates from the single patient collection (A), 10 high- and 10 low-toxicity isolates from the USA300 collection (B) and an Agr wild type and mutant isogenic pair (C) in BHI with and without 5% (vol/vol) human serum. The medians are presented as horizontal bars, with the boxes and whiskers showing the 1st and 3rd quartile and interquartile ranges. <i>p</i>-values are indicated in the text. To access this data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002229#pbio.1002229.s004" target="_blank">S4 Data</a>.</p