Characterization of the flanking region of the Shiga toxin operon in Stx2a bacteriophages reveals a diversity of the NanS-p sialate O-acetylesterase gene.

Shiga toxin-producing E. coli (STEC) are diarrheagenic strains that can cause bloody diarrhea and hemolytic-uremic syndrome. Their main virulence factor, the Shiga toxin (Stx), is encoded by phages integrated into the bacterial chromosome. Stx phages are widely diverse and carry many genes with limited or unknown function. As the toxin subtype Stx2a is associated with highly pathogenic strains, this study was mainly focused on the characterization of the stx flanking region of Stx2a phages. Of particular interest was a sialate O-acetylesterase (NanS-p), which has been described previously to be encoded downstream stx in some phage genomes and may confer a growth advantage for STEC. Complete DNA sequences of Stx2a phages and prophages were retrieved from the GenBank database, and the genomic regions from anti-terminator Q to holin S genes were bioinformatically analyzed. Predicted NanSp sequences from phages encoding other Stx subtypes were also studied. Additionally, expression of nanS-p was quantified by qPCR in strains selected from our laboratory collection. The analysis of Stx2a phage genomes showed that all carried the Q, stx2a, nanS-p and S genes, but with allele diversity and other sequence differences. In particular, sequence differences were detected in each of the three domains of NanS-p esterases encoded by Stx2a phages and other Stx phages; however, nanS-p was not identified in the Stx2e, Stx2f and Stx2g phages analyzed. The expression of nanS-p increased in most stx2a-positive strains under phage inducing conditions, as was previously shown for stx2a. As the present work showed diversity at the Q-S region among Stx phages, and particularly in the encoded NanS-p enzyme, future studies will be necessary to evaluate if NanS-p variants differ in their activity and to assess the impact of the absence of nanS-p in certain Stx phages.

Population replacement of benzimidazole-resistant Haemonchus contortus with susceptible strains: evidence of changes in the resistance status.

The spread of anthelmintic resistance (AR) in nematode populations threatens the viability of sheep production systems worldwide, and warrants the adoption of sensitive, practical, and standardized tests to detect AR. The aim of this study was to characterize the replacement of an Haemonchus contortus population resistant to benzimidazoles (BZDs) by a susceptible one, by means of both phenotypic and genotypic techniques. Phenotypic methods to assess BZD resistance included in vivo tests, such as the fecal egg count reduction test (FECRT), and in vitro tests, such as the egg hatch assay (EHA). Additionally, genotypification of polymorphisms associated with BZD resistance by sequencing a fragment of the isotype 1 ß-tubulin gene was carried out. The initial, BZD-resistant population (initial Balcarce population) exhibited an egg count reduction (ECR) of 59.3%. Following refugium replacement, the final population (final Balcarce population) exhibited an ECR of 95.2%. For the initial Balcarce population, the median effective dose (ED50) for the EHA was 0.607 µg thiabendazole (TBZ)/mL, with a rate of eclosion at a discriminating dose (EDD) of 0.1 µg TBZ/mL of 76.73%. For the final Balcarce population, ED50 was 0.02 µg TBZ/mL, and EDD was 1.97%. In the initial population, 93% of the analyzed individuals exhibited genotypic combinations associated with BZD resistance (53% Phe/Phe167-Tyr/Tyr200, 37% Phe/Tyr167-Phe/Tyr200, and 3% Phe/Tyr167-Glu/Leu198). Conversely, no combination associated with resistance was found in individuals from the final population. All of the tests were useful for detecting AR to BZDs. The results from the genetic and phenotypical studies were consistent, and the resulting information greatly aided in interpreting the outcomes of the population replacement and the potential impact of this strategy on management of AR.

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif.

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

AQPX-cluster aquaporins and aquaglyceroporins are asymmetrically distributed in trypanosomes.

Major Intrinsic Proteins (MIPs) are membrane channels that permeate water and other small solutes. Some trypanosomatid MIPs mediate the uptake of antiparasitic compounds, placing them as potential drug targets. However, a thorough study of the diversity of these channels is still missing. Here we place trypanosomatid channels in the sequence-function space of the large MIP superfamily through a sequence similarity network. This analysis exposes that trypanosomatid aquaporins integrate a distant cluster from the currently defined MIP families, here named aquaporin X (AQPX). Our phylogenetic analyses reveal that trypanosomatid MIPs distribute exclusively between aquaglyceroporin (GLP) and AQPX, being the AQPX family expanded in the Metakinetoplastina common ancestor before the origin of the parasitic order Trypanosomatida. Synteny analysis shows how African trypanosomes specifically lost AQPXs, whereas American trypanosomes specifically lost GLPs. AQPXs diverge from already described MIPs on crucial residues. Together, our results expose the diversity of trypanosomatid MIPs and will aid further functional, structural, and physiological research needed to face the potentiality of the AQPXs as gateways for trypanocidal drugs.

Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for Caenorhabditis elegans neuron classes.

Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data and on a molecular atlas describing the expression pattern of ∼800 genes at the single cell resolution, we designed an iterative clustering analysis aiming to match each cell-cluster to the ∼100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 97 of the 118 neuron classes to a cluster. Sixty two clusters were assigned to a single neuron class and 15 clusters grouped neuron classes sharing close molecular signatures. Pseudotime analysis revealed a maturation process occurring in some neurons (e.g. PDA) during the L2 stage. Based on the molecular profiles of all identified neurons, we predicted cell fate regulators and experimentally validated unc-86 for the normal differentiation of RMG neurons. Furthermore, we observed that different classes of genes functionally diversify sensory neurons, interneurons and motorneurons. Finally, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations.

Genetic dissection of neuropeptide cell biology at high and low activity in a defined sensory neuron.

Neuropeptides are ubiquitous modulators of behavior and physiology. They are packaged in specialized secretory organelles called dense core vesicles (DCVs) that are released upon neural stimulation. Unlike synaptic vesicles, which can be recycled and refilled close to release sites, DCVs must be replenished by de novo synthesis in the cell body. Here, we dissect DCV cell biology in vivo in a Caenorhabditis elegans sensory neuron whose tonic activity we can control using a natural stimulus. We express fluorescently tagged neuropeptides in the neuron and define parameters that describe their subcellular distribution. We measure these parameters at high and low neural activity in 187 mutants defective in proteins implicated in membrane traffic, neuroendocrine secretion, and neuronal or synaptic activity. Using unsupervised hierarchical clustering methods, we analyze these data and identify 62 groups of genes with similar mutant phenotypes. We explore the function of a subset of these groups. We recapitulate many previous findings, validating our paradigm. We uncover a large battery of proteins involved in recycling DCV membrane proteins, something hitherto poorly explored. We show that the unfolded protein response promotes DCV production, which may contribute to intertissue communication of stress. We also find evidence that different mechanisms of priming and exocytosis may operate at high and low neural activity. Our work provides a defined framework to study DCV biology at different neural activity levels.