Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonymous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa, and L. lactis as their primary host. We use the concept of a “genome landscape,” which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such as GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.     

Optimizing clinical dosing of combination broadly neutralizing antibodies for HIV prevention

Broadly neutralizing antibodies (bNAbs) are promising agents to prevent HIV infection and achieve HIV remission without antiretroviral therapy (ART). As with ART, bNAb combinations are likely needed to cover HIV’s extensive diversity. Not all bNAbs are identical in terms of their breadth, potency, and in vivo longevity (half-life). Given these differences, it is important to optimally select the composition, or dose ratio, of combination bNAb therapies for future clinical studies. We developed a model that synthesizes 1) pharmacokinetics, 2) potency against a wide HIV diversity, 3) interaction models for how drugs work together, and 4) correlates that translate in vitro potency to clinical protection. We found optimization requires drug-specific balances between potency, longevity, and interaction type. As an example, tradeoffs between longevity and potency are shown by comparing a combination therapy to a bi-specific antibody (a single protein merging both bNAbs) that takes the better potency but the worse longevity of the two components. Then, we illustrate a realistic dose ratio optimization of a triple combination of VRC07, 3BNC117, and 10–1074 bNAbs. We apply protection estimates derived from both a non-human primate (NHP) challenge study meta-analysis and the human antibody mediated prevention (AMP) trials. In both cases, we find a 2:1:1 dose emphasizing VRC07 is nearly optimal. Our approach can be immediately applied to optimize the next generation of combination antibody prevention and cure studies.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

A comparison of two techniques for measuring and crossdating tree rings

Crossdating is the core principle of dendrochronology. Our study compared two techniques for measuring and crossdating tree rings using Juniperus virginiana L. (eastern redcedar) as a case study. We used a pseudo 2 × 2 study design comparing the traditional skeleton plot/sliding measuring stage technique to a semi-automatic image analysis program across two technicians. Crossdating was evaluated in COFECHA. Raw measurements of total, earlywood, and latewood widths from the two methods were analyzed using the Verify for Windows program, ANOVA, and correlation matrices. Total ring width and earlywood width were well correlated between techniques and technicians but questionable ring boundaries from image analysis program should be checked under a stereoscope. Juniperus virginiana latewood widths were significantly different between techniques and technicians; therefore, we do not recommend combining latewood measurements from species with limited latewood variability for dendrochronological analysis. A standard definition of the earlywood-latewood boundary that can be replicated across technicians is needed to combine latewood measurements from the sliding measuring stage and image analysis systems.     

Comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions.

Orthopoxviruses are among the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, two orthopoxviruses with different pathogenic potentials, human monkeypox virus and vaccinia virus, were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by high-resolution reversed-phase nano-LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST and X! Tandem resulted in the confident identification of hundreds of monkeypox, vaccinia, and copurified host-cell proteins. The unfractionated samples were additionally analyzed by LC-MS using an LTQ-Orbitrap, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially abundant Orthopoxvirus proteins are discussed. Data, processed results, and protocols are available at http://www.proteomicsresource.org/.     

Correction to: Experimental evidence of minimal effects on octocoral hosts caused by the introduced ophiuroid Ophiothela mirabilis

Coral Reefs - A correction to this paper has been published: https://doi.org/10.1007/s00338-021-02110-0     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.