Single Amino Acid Polymorphisms Research Articles

Prediction of Deleterious Single Amino Acid Polymorphisms with a Consensus Holdout Sampler.

Single Amino Acid Polymorphisms (SAPs) or nonsynonymous Single Nucleotide Variants (nsSNVs) are the most common genetic variations. They result from missense mutations where a single base pair substitution changes the genetic code in such a way that the triplet of bases (codon) at a given position is coding a different amino acid. Since genetic mutations sometimes cause genetic diseases, it is important to comprehend and foresee which variations are harmful and which ones are neutral (not causing changes in the phenotype). This can be posed as a classification problem. Computational methods using machine intelligence are gradually replacing repetitive and exceedingly overpriced mutagenic tests. By and large, uneven quality, deficiencies, and irregularities of nsSNVs datasets debase the convenience of artificial intelligence-based methods. Subsequently, strong and more exact approaches are needed to address these problems. In the present work paper, we show a consensus classifier built on the holdout sampler, which appears strong and precise and outflanks all other popular methods. We produced 100 holdouts to test the structures and diverse classification variables of diverse classifiers during the training phase. The finest performing holdouts were chosen to develop a consensus classifier and tested using a k-fold (1 ≤ k ≤5) cross-validation method. We also examined which protein properties have the biggest impact on the precise prediction of the effects of nsSNVs. Our Consensus Holdout Sampler outflanks other popular algorithms, and gives excellent results, highly accurate with low standard deviation. The advantage of our method emerges from using a tree of holdouts, where diverse LM/AI-based programs are sampled in diverse ways.

A single amino acid polymorphism in natural Metchnikowin alleles of Drosophila results in systemic immunity and life history tradeoffs.

Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be rapidly evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that insect AMPs tend to evolve more slowly than the genome average. Metchikowin (Mtk) is a Drosophila AMP that has a single amino acid residue that segregates as either proline (P) or arginine (R) in populations of four different species, some of which diverged more than 10 million years ago. These results suggest that there is a distinct functional importance to each allele. The most likely hypotheses are driven by two main questions: does each allele have a different efficacy against different specific pathogens (specificity hypothesis)? Or, is one allele a more potent antimicrobial, but with a host fitness cost (autoimmune hypothesis)? To assess their functional differences, we created D. melanogaster lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing and performed a series of life history and infection assays to assess them. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. Females show some results that contrast with males, with Mtk alleles either not contributing to survival or with the P allele outperforming the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results are consistent with both the specificity hypothesis (either allele can perform better against certain pathogens depending on context), and the autoimmune hypothesis (the R allele is generally the more potent antimicrobial in males, and carries a fitness cost). These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP.

Single Amino Acid Polymorphisms in the Fasciola hepatica Carboxylesterase Type B Gene and Their Potential Role in Anthelmintic Resistance.

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand-protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme's interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme's catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

DNA polymerase δ: A single Pol31 polymorphism suppresses the strain background-specific lethality of Pol32 inactivation in Saccharomyces cerevisiae

The evolutionarily conserved DNA polymerase delta (Polδ) plays several essential roles in eukaryotic DNA replication and repair, responsible for the synthesis of the lagging-strand, lower replicative mutagenesis via its proof-reading exonuclease activity and synthetizes both strands during break-induced replication. In Saccharomyces cerevisiae, the Polδ protein complex consists of three subunits encoded by the POL3, POL31 and POL32 genes. Surprisingly, in contrast to POL3 and POL31, the POL32 gene deletion was found to be viable but lethal in all other eukaryotes, raising the question to which extent the viability of the POL32 deletion in S. cerevisiae was species specific. To address this issue, we inactivated the POL32 gene in 10 evolutionary close or distant S. cerevisiae strains and found that POL32 was either essential (3 strains including SK1), non-essential (5 strains including the reference S288C strain) or confers a slow-growth phenotype (2 strains). Whole-genome sequencing of S288C/SK1 pol32∆ meiotic segregants identified the lethal/suppressor effect of the single Pol31-C43Y polymorphism. Consistently, the introduction of the Pol31–43C allele in the SK1 and West African (WA) pol32∆ mutants was sufficient to restore cell viability and wild-type growth upon introduction of two copies of POL31–43C in the SK1 haploid strain. Reciprocally, introduction of the SK1 POL31–43Y allele in the S288C pol32∆ mutant was lethal. Sequence analyses of the POL31 polymorphisms in the 1,011 yeasts genome dataset correlates with the strict occurrence of the POL31–43Y allele in the yeast African palm wine clade. Differently, the single Pol31-E400G polymorphism confers pol32∆ lethality in the Malaysian strain. In the yeast two-hybrid assay, we observed a weakened interaction between Pol3 and Pol31–43Y versus Pol31–43C suggesting an insufficient level of the Polδ holoenzyme stability/activity. Thus, the enigmatic non-essentiality of Pol32 in S. cerevisiae results from single Pol31 amino acid polymorphism and is clade rather than species specific.

The Reovirus σ1 Attachment Protein Influences the Stability of Its Entry Intermediate.

Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.

That H9N2 avian influenza viruses circulating in different regions gather in the same live-poultry market poses a potential threat to public health.

H9N2 avian influenza viruses are endemic and persistent in China, but those that are prevalent in different provinces are also causes of wide epidemics, related to the spread of wild birds and the cross-regional trade in live poultry. For the past 4 years, beginning in 2018, we have sampled a live-poultry market in Foshan, Guangdong, in this ongoing study. In addition to the prevalence of H9N2 avian influenza viruses in China during this period, we identified isolates from the same market belonging to clade A and clade B, which diverged in 2012-2013, and clade C, which diverged in 2014-2016, respectively. An analysis of population dynamics revealed that, after a critical divergence period from 2014 to 2016, the genetic diversity of H9N2 viruses peaked in 2017. Our spatiotemporal dynamics analysis found that clade A, B, and C, which maintain high rates of evolution, have different prevalence ranges and transmission paths. Clades A and B were mainly prevalent in East China in the early stage, and then spread to Southern China, becoming epidemic with clade C. Strains from different regions converge at the same live-poultry market to communicate, which may be one reasons the H9N2 viruses are difficult to eradicate and increasingly dominant throughout China. Selection pressure and molecular analysis have demonstrated that single amino acid polymorphisms at key receptor binding sites 156, 160, and 190 under positive selection pressure, suggesting that H9N2 viruses are undergoing mutations to adapt to new hosts. Live-poultry markets are important because people who visit them have frequent contact with poultry, H9N2 viruses from different regions converge at these markets and spread through contact between live birds and humans, generating increased risks of human exposure to these viruses and threatening public health safety. Thus, it is important to reducing the cross-regional trade of live poultry and strengthening the monitoring of avian influenza viruses in live-poultry markets to reduce the spread of avian influenza viruses.

Modulation of Mechanical Properties of Short Bioinspired Peptide Materials by Single Amino-Acid Mutations.

The occurrence of modular peptide repeats in load-bearing (structural) proteins is common in nature, with distinctive peptide sequences that often remain conserved across different phylogenetic lineages. These highly conserved peptide sequences endow specific mechanical properties to the material, such as toughness or elasticity. Here, using bioinformatic tools and phylogenetic analysis, we have identified the GX8 peptide with the sequence GLYGGYGX (where X can be any residue) in a wide range of organisms. By simple mutation of the X residue, we demonstrate that GX8 can be self-assembled into various supramolecular structures, exhibiting vastly different physicochemical and viscoelastic properties, from liquid-like coacervate microdroplets to hydrogels to stiff solid materials. A combination of spectroscopic, electron microscopy, mechanical, and molecular dynamics studies is employed to obtain insights into molecular scale interactions driving self-assembly of GX8 peptides, underscoring that π-π stacking and hydrophobic interactions are the drivers of peptide self-assembly, whereas the X residue determines the extent of hydrogen bonding that regulates the macroscopic mechanical response. This study highlights the ability of single amino-acid polymorphism to tune the supramolecular assembly and bulk material properties of GX8 peptides, enabling us to cover a broad range of potential biomedical applications such as hydrogels for tissue engineering or coacervates for drug delivery.

Intact Transition Epitope Mapping-Force Differences between Original and Unusual Residues (ITEM-FOUR).

Antibody-based point-of-care diagnostics have become indispensable for modern medicine. In-depth analysis of antibody recognition mechanisms is the key to tailoring the accuracy and precision of test results, which themselves are crucial for targeted and personalized therapy. A rapid and robust method is desired by which binding strengths between antigens and antibodies of concern can be fine-mapped with amino acid residue resolution to examine the assumedly serious effects of single amino acid polymorphisms on insufficiencies of antibody-based detection capabilities of, e.g., life-threatening conditions such as myocardial infarction. The experimental ITEM-FOUR approach makes use of modern mass spectrometry instrumentation to investigate intact immune complexes in the gas phase. ITEM-FOUR together with molecular dynamics simulations, enables the determination of the influences of individually exchanged amino acid residues within a defined epitope on an immune complex's binding strength. Wild-type and mutated epitope peptides were ranked according to their experimentally determined dissociation enthalpies relative to each other, thereby revealing which single amino acid polymorphism caused weakened, impaired, and even abolished antibody binding. Investigating a diagnostically relevant human cardiac Troponin I epitope for which seven nonsynonymous single nucleotide polymorphisms are known to exist in the human population tackles a medically relevant but hitherto unsolved problem of current antibody-based point-of-care diagnostics.

Deep coverage proteome analysis of hair shaft for forensic individual identification

Hair shaft is one of the most common biological evidence found at crime scenes. However, due to the biogenic degradation of nuclear DNA in hair shaft, it is difficult to achieve individual identification through routine DNA analysis. In contrast, the proteins in hair shaft are stable and contain genetic polymorphisms in the form of single amino acid polymorphisms (SAPs), translated from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome. However, the number of SAPs detected still cannot meet the requirements of practical applications. This paper developed a deep coverage proteome analysis method by combining a three-step sequential ionic liquid-based protein extraction and 2D-RPLC-MS/MS with high and low pH to identify both variant and reference SAPs from 2-cm-long hair shafts. We identified 632 ± 243 protein groups from 10 individuals, with the average number of SAPs reaching 167 ± 21/person. These were further used to calculate random match probabilities (RMPs), a widely accepted forensic statistical term for human identification. The RMPs ranged from 6.53 × 10–4 to 3.10 × 10–14 (median = 2.62 × 10–8) when calculated with frequency of matching nsSNP genotype data from exomes, and ranged from 2.62 × 10–3 to 2.07 × 10–10 (median = 4.88 × 10–6) with SAP genotype frequency. All these results indicate that the deep coverage proteomics method is beneficial for improving SAP-based forensic individual identification in hair shaft, with great potential in crime investigation.

Using Forward and Reverse Genetics to Understand Calcium Dysregulation in Enteric Viral Virulence

Acute gastroenteritis (AGE) remains the second leading cause of death among children under the age of 5 worldwide. AGE becomes fatal when pathogen‐associated upregulation of secretory activity across the intestinal epithelium causes severe volume depletion, hypovolemic shock, and multi‐system failure. While enteric viruses remain the most common cause of fatal AGE in kids, the drivers of their virulence (i.e. how they increase secretory activity) remain poorly understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in kids, release adenosine diphosphate (ADP) to induce coordinated signals known as “intercellular calcium waves,” which spread through uninfected neighboring cells. This dysregulates calcium signaling pathways, which enhances fluid secretion from uninfected cells and thereby contributes to volume depletion. Pharmacological blockade of calcium waves in infected mouse pups decreases disease severity, suggesting they are integral to rotavirus virulence. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. Here, we report data showing that recombinant expression of a single rotavirus protein, non‐structural protein 4 (NSP4), is sufficient to activate intercellular calcium waves. Using the rotavirus reverse genetics system, we show that the diminished calcium wave phenotype associated with an attenuated strain of porcine rotavirus segregates with the NSP4 gene upon reassortment. Furthermore, we show that this attenuation is attributable to a single amino acid polymorphism at the c‐terminal end of NSP4. This implicates the c‐terminal end and a putative PDZ‐binding motif therein as key upstream mediators of intercellular calcium waves. These findings deepen our understanding of enteric virus pathogenesis, characterize a new role for rotavirus non‐structural protein 4, offer novel targets for anti‐secretory therapeutics, and expand foundational knowledge to support the development of improved live‐attenuated vaccines.

In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography, species differentiation, and sequence typing

Burkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species. We performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer β-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n = 101) and Burkholderia mallei (n = 26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n = 1,442) and B. mallei (n = 81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST. Conserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species.

Maintenance of the Shigella sonnei Virulence Plasmid Is Dependent on Its Repertoire and Amino Acid Sequence of Toxin-Antitoxin Systems.

ABSTRACTShigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an ∼210 kb plasmid that encodes a type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here, we investigated the molecular basis for the emergence of avirulence in S. sonnei and showed that avirulence mainly results from pINV loss, which is consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin-antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri. We showed that the introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while the single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contributed to pINV loss. Avirulence also resulted from deletions of T3SS-associated genes in pINV through recombination between insertion sequences (ISs) on the plasmid. These events differed from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrated that TA systems and ISs influenced plasmid dynamics and loss in S. sonnei and could be exploited for the design and evaluation of vaccines.IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrializing countries and is an emerging, multidrug-resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory through loss of its virulence plasmid (pINV). Here, we deciphered the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin-antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri. Our findings highlighted how subtle differences in plasmids in closely related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei. This should facilitate research on this bacterium and vaccine development.

Identification of Disease-specific Single Amino Acid Polymorphisms Using a Simple Random Forest at Protein-level

Background: The number of human genetic variants deposited into publicly available databases has been increasing exponentially. Among these variants, non-synonymous single nucleotide polymorphisms (nsSNPs), also known as single Amino Acid Polymorphisms (SAPs), have been demonstrated to be strongly correlated with phenotypic variations of traits/diseases. Objective: However, the detailed mechanisms governing the disease association of SAPs remain unclear. Thus, further investigation of new attributes and improvement of the prediction becomes more and more urgent since amount of unknown disease-related SAPs need to be investigated. Methods: Based on the principle of Random Forest (RF), we firstly constructed a new effective prediction model for SAPs associated with a particular disease from protein sequences. Four usual sequence signature extractions were separately performed to select the optimal features. Then SAP peptide lengths from 12 to 202 were also optimized. Results: The optimal models achieve higher than 90% accuracy and Area Under the Curve (AUC) of over 0.9 on all 11 external testing datasets. Finally, the good performance on an independent test set with an accuracy higher than 95% proves the superiority of our method. Conclusion: In this paper, based on Random Forest (RF), we constructed 11 disease-association prediction models for SAPs from the protein sequence level. All models yield prediction accuracy higher than 90% and Area Under the Curve (AUC) more than 0.9. Our method only using the information of protein sequences are more universal than those that depend on some additional information or predictions about the proteins.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair.

Cooperation between receptors from the nucleotide-binding, leucine-rich repeats (NLR) superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses, whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum.

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

Prophylactic Administration of Antibodies Specific for the HPA-1a Epitope Prevents Alloimmunization and Platelet Destruction in a Murine Model of Fetal Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited antigens present on the surface of fetal and neonatal platelets. The human platelet alloantigen HPA-1a (formerly known as the Pl A1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism near the amino terminus of the integrin b3 subunit (known as GPIIIa in the platelet literature) serves as the central target for alloantibody binding, leading to clearance of both fetal and neonatal platelets, thrombocytopenia, and in the most severe cases, spontaneous- or trauma-induced intracranial hemorrhage. Unlike hemolytic disease of the newborn, which occurs in pregnancies subsequent to parturition-induced alloimmunization, an estimated 25% to 50% of FNAIT cases occur without warning during gestation of the first pregnancy. Though long proposed, there are currently no approved therapies for the prevention of FNAIT. We recently described the development of transgenic mice expressing the human HPA-1a allogeneic epitope on a murine GPIIIa backbone. Transfusion of such platelets into wild-type female mice induced the generation of high-titer anti-HPA-1a alloantibodies that can cross the placenta and recapitulate many of the relevant clinical features of FNAIT. To test the hypothesis that rapid elimination of fetal HPA-1a positive platelets from the circulation of a mother who is HPA-1a negative might prevent maternal alloimmunization and the development of FNAIT, we administered either a hyperimmune plasma-derived polyclonal anti-HPA-1a antibody derived from females having previous cases of FNAIT (termed RLYB211), or a novel human monoclonal antibody directed against the HPA-1a epitope (termed RLYB212), to wild-type female mice prior to challenging them with HPA-1a-positive murine platelets. RLYB211 and RLYB212 were each able to effect the rapid removal of HPA-1a-positive platelets from murine circulation and prevent the development of anti-HPA-1a alloantibodies. Importantly, wild-type female mice pretreated with RLYB211 prior to exposure to HPA-1a-positive platelets, and then impregnated by HPA-1a-positive males, gave birth to HPA-1a-positive pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish the potential for prophylactic polyclonal and monoclonal anti-HPA-1a antibody therapy for the prevention of FNAIT in humans. DisclosuresSheridan: Rallybio: Current Employment, Current equity holder in publicly-traded company. Newman: Rallybio: Consultancy, Research Funding.

Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Articles of Significant Interest in This Issue

Filoviruses utilize a complex entry pathway to initiate infection, which requires extensive proteolytic processing of the viral glycoprotein (GP) in the endosome/lysosome prior to membrane fusion. Lennemann et al. (e01073-21) have identified a single amino acid polymorphism in early isolates of Sudan virus (SUDV) GP at position 95 which has a significant impact on GP-mediated entry due to changes in protein stability. This polymorphism, located in the base of GP, alters the efficacy of inhibitors of GP-mediated entry in a species-specific manner. This work highlights unique characteristics of GPs among filovirus species, which may pose a challenge for the development of pan-filovirus antivirals.

Elucidation of familial relationships using hair shaft proteomics

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual’s genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

Allele-based analysis revealed the critical functions of region 277-297 in the NorA efflux pump of Staphylococcus aureus.

The NorA efflux pump in Staphylococcus aureus mediates resistance to many fluoroquinolone (FQ) antibiotics. Three norA alleles with high sequence similarity are found in various S. aureus strains exhibiting different FQ resistance profiles. This study aimed to elucidate the underlying molecular basis for the varying efflux activity of these three allelic variations. The norA genotypes of 20 S. aureus isolates were analysed. Multiple alignments and conservative analyses were conducted to explore the evolutionary variations. After heterologous expression in Escherichia coli, seven mutants were constructed for MIC tests, efflux activity and conformational change measurements. Three NorA alleles were identified that displayed different FQ MICs and varying efflux activity for ethidium bromide, with the NorAII protein showing the strongest activity. A total of 29 single amino acid polymorphisms were identified by conservative analysis within three allelic peptides, with seven sites densely distributed in the 277-297 region. Mutations of these seven residues in NorAII all significantly impaired drug resistance and efflux activity, and three key mutants showed conformational changes in fluorescence resonance energy transfer (FRET) analysis. Evolutionary variations of the 277-297 region could be a major explanation for the functional difference of three norA alleles and serve as a potential target for the development of novel NorA inhibitors.