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Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB–TcC subcomplex that makes a hollow shell. This TcB–TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB–TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

Abstract The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.

Abstract In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiāng virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

The Spt-Ada-Gcn Acetyltransferase (SAGA) complex is a highly conserved co-activator found across eukaryotes. It is composed of a number of modules which can vary between species, but all contain the core module. Hfi1 (known as TADA1 in Homo sapiens) is one of the proteins that forms the core module, and has been shown to play an important role in maintaining complex structural integrity in both brewer’s yeast and humans. In this study we successfully identified the gene encoding this protein in the important fungal pathogen, Cryptococcus neoformans, and named it HFI1. The hfi1Δ mutant is highly pleiotropic in vitro, influencing phenotypes, ranging from temperature sensitivity and melanin production to caffeine resistance and titan cell morphogenesis. In the absence of Hfi1, the transcription of several other SAGA genes is impacted, as is the acetylation and deubiquination of several histone residues. Importantly, loss of the gene significantly impacts virulence in a murine inhalation model of cryptococcosis. In summary, we have established that Hfi1 modulates multiple pathways that directly affect virulence and survival in C. neoformans, and provided deeper insight into the importance of the non-enzymatic components of the SAGA complex.

Abstract Background A subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks. Methods We performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry. Results Our sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus. Conclusions Genetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines. Graphical Abstract

IntroductionBreeding for tick resistance is a sustainable alternative to control cattle ticks due to widespread resistance to acaricidal drugs and the lack of a protective vaccine. The most accurate method used to characterise the phenotype for tick resistance in field studies is the standard tick count, but this is labour-intensive and can be hazardous to the operator. Efficient genetic selection requires reliable phenotyping or biomarker(s) for accurately identifying tick-resistant cattle. Although breed-specific genes associated with tick resistance have been identified, the mechanisms behind tick resistance have not yet been fully characterised.MethodsThis study applied quantitative proteomics to examine the differential abundance of serum and skin proteins using samples from naïve tick-resistant and -susceptible Brangus cattle at two-time points following tick exposure. The proteins were digested into peptides, followed by identification and quantification using sequential window acquisition of all theoretical fragment ion mass spectrometry.ResultsResistant naïve cattle had a suite of proteins associated with immune response, blood coagulation and wound healing that were significantly (adjusted P < 10- 5) more abundant compared with susceptible naïve cattle. These proteins included complement factors (C3, C4, C4a), alpha-1-acid glycoprotein (AGP), beta-2-glycoprotein-1, keratins (KRT1 & KRT3) and fibrinogens (alpha & beta). The mass spectrometry findings were validated by identifying differences in the relative abundance of selected serum proteins with ELISA. The proteins showing a significantly different abundance in resistant cattle following early and prolonged tick exposures (compared to resistant naïve) were associated with immune response, blood coagulation, homeostasis, and wound healing. In contrast, susceptible cattle developed some of these responses only after prolonged tick exposure.DiscussionResistant cattle were able to transmigrate immune-response related proteins towards the tick bite sites, which may prevent tick feeding. Significantly differentially abundant proteins identified in this research in resistant naïve cattle may provide a rapid and efficient protective response to tick infestation. Physical barrier (skin integrity and wound healing) mechanisms and systemic immune responses were key contributors to resistance. Immune response-related proteins such as C4, C4a, AGP and CGN1 (naïve samples), CD14, GC and AGP (post-infestation) should be further investigated as potential biomarkers for tick resistance.

Abstract Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

Perdomo et al. fed mosquitos (Aedes aegypti) a diet spiked with human blood miRNA, hsa-miR-21-5p and used proteomics analysis to show differential protein expression in female Ae. aegypti mosquitoes after feeding. They showed that hsa-miR-21-5p positively regulated vitellogenin, which plays a role in egg production, thus could have effects on Ae. aegypti progeny production.

Zacchi et al. attempt to bridge the gap between understanding the impact of process variables and determination of critical post-translational modifications on a highly complex glycoprotein, factor IX. They implement an LC-MS/MS approach based on data independent acquisition which allows measurement of all detectable peptides, even with heterogeneous modifications.

Abstract Brewing science is undergoing a renaissance with the use of modern analytical chemistry and microbiology techniques. However, these modern analytical tools and techniques are not necessarily aligned with the scale and scope of brewing science. In particular, brewing processes can be time consuming, ingredient intensive, and require specialised technical equipment. These drawbacks compound with the need for appropriate numbers of replicates for adequately powered experimental design. Here, we describe a micro-scale mash method that can be performed using a common laboratory benchtop shaker/incubator, allowing for high throughput mashing and easy sample replication for statistical analysis. Proteomic profiles at both the protein and peptide levels were consistent between the 1 mL micro-mash and a 23 L Braumeister mash, and both mash scales produced wort with equivalent fermentable sugar and free amino acid profiles. The experimental flexibility offered by our micro-mash method allowed us to investigate the effects of altered mash parameters on the beer brewing proteome.

The pathogenesis of Streptococcus pyogenes (GAS) causing scarlet fever has been associated with the presence of prophages, such as ΦHKU.vir, and their products. Here, the authors characterize the exotoxins SpeC and Spd1 of ΦHKU.vir and show these to act synergistically to facilitate nasopharyngeal colonization in mice.

Mass spectrometry glycoproteomics is rapidly maturing, allowing unprecedented insights into the diversity and functions of protein glycosylation. However, quantitative glycoproteomics remains challenging. We developed GlypNirO, an automated software pipeline which integrates the complementary outputs of Byonic and Proteome Discoverer to allow high-throughput automated quantitative glycoproteomic data analysis. The output of GlypNirO is clearly structured, allowing manual interrogation, and is also appropriate for input into diverse statistical workflows. We used GlypNirO to analyse a published plasma glycoproteome dataset and identified changes in site-specific N- and O-glycosylation occupancy and structure associated with hepatocellular carcinoma as putative biomarkers of disease.

Abstract Aims The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno‐protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno‐protection. Materials and methods To examine the effects of increased podocyte oligosaccharyltransferase‐48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase‐48kDa subunit abundance in podocytes driven by the podocin promoter. Results Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra‐resolution microscopy of podocytes revealed denudation of foot processes where there was co‐localization of oligosaccharyltransferase‐48kDa subunit and advanced glycation end‐products. Conclusions These studies indicate that increased podocyte expression of oligosaccharyltransferase‐48 kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function.

Fermented beverages have been consumed for millennia and today support a global industry producing diverse products. Saccharomyces yeasts currently dominate the fermented beverage industry, but consumer demands for alternative products with a variety of sensory profiles and actual or perceived health benefits are driving the diversification and use of non-Saccharomyces yeasts. The diversity of flavours, aromas, and other sensory characteristics that can be obtained by using non-Saccharomyces yeasts in fermentation is, in large part, due to the diverse secondary metabolites they produce compared to conventional Saccharomyces yeast. Here, we review the use of metabolomic analyses of non-Saccharomyces yeasts to explore their impact on the sensory characteristics of fermented beverages. We highlight several key species currently used in the industry, including Brettanomyces, Torulaspora, Lachancea, and Saccharomycodes, and emphasize the future potential for the use of non-Saccharomyces yeasts in the production of diverse fermented beverages.

Dystonia is the third most common movement disorder, but its diagnosis and treatment remain challenging. One of the most severe types of dystonia is early-onset torsion dystonia (EOTD). The best studied and validated EOTD-associated mutation, torsinAΔE, is a deletion of a C-terminal glutamate residue in the AAA+ ATPase torsinA. TorsinA appears to be an endoplasmic reticulum (ER)/nuclear envelope chaperone with multiple roles in the secretory pathway and in determining subcellular architecture. Many functions are disabled in the torsinAΔE variant, and torsinAΔE is also less stable than wild-type torsinA and is a substrate for ER-associated degradation. Nevertheless, the molecular factors involved in the biogenesis and degradation of torsinA and torsinAΔE have not been fully explored. To identify conserved cellular factors that can alter torsinAΔE protein levels, we designed a new high-throughput, automated, genome-wide screen utilizing our validated Saccharomyces cerevisiae torsinA expression system. By analyzing the yeast non-essential gene deletion collection, we identified 365 deletion strains with altered torsinAΔE steady-state levels. One notable hit was EUG1, which encodes a member of the protein disulfide isomerase family (PDIs). PDIs reside in the ER and catalyze the formation of disulfide bonds, mediate protein quality control and aid in nascent protein folding. We validated the role of select human PDIs in torsinA biogenesis in mammalian cells and found that overexpression of PDIs reduced the levels of torsinA and torsinAΔE. Together, our data report the first genome-wide screen to identify cellular factors that alter expression levels of the EOTD-associated protein torsinAΔE. More generally, the identified hits help in dissecting the cellular machinery involved in folding and degrading a torsinA variant, and constitute potential therapeutic factors for EOTD. This screen can also be readily adapted to identify factors impacting the levels of any protein of interest, considerably expanding the applicability of yeast in both basic and applied research.

Abstract Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation’sequon’. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.

Abstract Subtilase cytotoxin (SubAB) of Escherichia coli is an AB5 class bacterial toxin. The pentameric B subunit (SubB) binds the cellular carbohydrate receptor, α2–3-linked N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is not expressed on normal human cells, but is expressed by cancer cells. Elevated Neu5Gc has been observed in breast, ovarian, prostate, colon and lung cancer. The presence of Neu5Gc is prognostically important, and correlates with invasiveness, metastasis and tumour grade. Neu5Gc binding by SubB suggests that it may have utility as a diagnostic tool for the detection Neu5Gc tumor antigens. Native SubB has 20-fold less binding to N-acetlylneuraminic acid (Neu5Ac); over 30-fold less if the Neu5Gc linkage was changed from α2–3 to α2–6. Using molecular modeling approaches, site directed mutations were made to reduce the α2–3 $${\boldsymbol{\gg }}$$ ≫ α2–6-linkage preference, while maintaining or enhancing the selectivity of SubB for Neu5Gc over Neu5Ac. Surface plasmon resonance and glycan array analysis showed that the SubBΔS106/ΔT107 mutant displayed improved specificity towards Neu5Gc and bound to α2–6-linked Neu5Gc. SubBΔS106/ΔT107 could discriminate NeuGc- over Neu5Ac-glycoconjugates in ELISA. These data suggest that improved SubB mutants offer a new tool for the testing of biological samples, particularly serum and other fluids from individuals with cancer or suspected of having cancer.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

Foam-related parameters are associated with beer quality and dependent, among others, on the protein content. This study aimed to develop a machine learning (ML) model to predict the pattern and presence of 54 proteins. Triplicates of 24 beer samples were analyzed through proteomics. Furthermore, samples were analyzed using the RoboBEER to evaluate 15 physical parameters (color, foam, and bubbles), and a portable near-infrared (NIR) device. Proteins were grouped according to their molecular weight (MW), and a matrix was developed to assess only the significant correlations (p < 0.05) with the physical parameters. Two ML models were developed using the NIR (Model 1), and RoboBEER (Model 2) data as inputs to predict the relative quantification of 54 proteins. Proteins in the 0–20 kDa group were negatively correlated with the maximum volume of foam (MaxVol; r = −0.57) and total lifetime of foam (TLTF; r = −0.58), while those within 20–40 kDa had a positive correlation with MaxVol (r = 0.47) and TLTF (r = 0.47). Model 1 was not as accurate (testing r = 0.68; overall r = 0.89) as Model 2 (testing r = 0.90; overall r = 0.93), which may serve as a reliable and affordable method to incorporate the relative quantification of important proteins to explain beer quality.

ABSTRACT Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases. IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phase variable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

ABSTRACT Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

Vegemite is an iconic Australian food spread made from spent brewers’ yeast extract, which has been reported to be used as an ingredient in illegal home brewing. In this study, we tested the utility of Vegemite and the similar spread Marmite in promoting fermentation. We could not culture microorganisms from either Vegemite or Marmite, consistent with these food-grade spreads being essentially sterile. To test if the addition of Vegemite or Marmite could assist in fermentation when additional viable yeast was also present, solutions containing glucose and a range of concentrations of either Vegemite or Marmite were inoculated with brewers’ yeast. No fermentation occurred in any condition without addition of extra brewer’s yeast. Fermentation did not occur when yeast was inoculated into solutions containing only glucose, but progressed efficiently with when Vegemite or Marmite was also added. Gas Chromatography confirmed that ethanol was present at ∼3% v/v post-fermentation in all samples which contained glucose, Vegemite or Marmite, and brewers’ yeast. Trace amounts of methanol were also detected. Mass spectrometry proteomics identified abundant intracellular yeast proteins and barley proteins in Vegemite and Marmite, and abundant secreted yeast proteins from actively growing yeast in those samples to which extra brewers’ yeast had been added. We estimate that the real-world cost of home brewed “Vegemite Beer” would be very low. Our results show that Vegemite or other yeast extract spreads could provide cheap and readily available sources of nutrient supplementation to increase the efficiency of fermentation in home brewing or other settings.

Carbohydrate analyses are often challenging due to the structural complexity of these molecules, as well as the lack of suitable analytical tools for distinguishing the vast number of possible isomers. The coupled technique, ion mobility-mass spectrometry (IM-MS), has been in use for two decades for the analysis of complex biomolecules, and in recent years it has emerged as a powerful technique for the analysis of carbohydrates. For carbohydrates, most studies have focused on the separation and characterization of isomers in biological samples. IM-MS is capable of separating isomeric ions by drift time, and further characterizing them by mass analysis. Applications of IM-MS in carbohydrate analysis are extremely useful and important for understanding many biological mechanisms and for the determination of disease states, although efforts are still needed for higher sensitivity and resolution.

Flocculation is one of the most important characteristics of brewing yeast as it allows for the easy and cheap removal of cells after fermentation. The genes responsible for both the Flo1 and NewFlo flocculation phenotypes are well characterized. However, the relationship between Flo protein abundance and flocculation efficiency is poorly understood. In this present study, we used mass spectrometry proteomics to compare the cell wall and whole cell proteomes of commercial yeast strains with diverse flocculation behaviors. We found that the relative abundance of Flo1/5 or Flo10 in the cell wall was correlated with the ability of these yeast strains to flocculate. Analysis of whole cell proteomes identified differences in the proteomes of yeast strains and identified the potential for high metabolic diversity. Characterization of the cell wall and whole cell proteomes during fermentation showed high levels of Flo10 in cells that settled early during fermentation. Our data reveal the diversity of the cell wall and global proteomes of brewing yeast, highlighting the potential biochemical diversity present in yeast that can be utilized in the production of fermented beverages.