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bioRxiv2018

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes depending on the cell or tissue type. The virus Spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abl kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titers and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which can make experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titers by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying host cell pathways required for coronavirus infection. This will provide insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

microbiology↗

bioRxiv2018

A genomic island of Streptomyces coelicolor harbors the self-contained regulon of an ECF sigma factor

Streptomycetes constitute the largest genus of actinobacteria, living predominantly in soil and decaying vegetation. The bacteria are widely known for their filamentous morphologies and their capacity to synthesize antibiotics and other biologically active molecules. More than a decade ago, we and others identified 22 genomic islands that Streptomyces coelicolor M145 possesses and other Streptomyces strains lack. One of these genomic islands, Genomic Island (GI) 6, encodes an extracytoplasmic function (ECF) sigma factor that we were characterizing in separate work. Here we report that artificial induction of the ECF sigma factor, which is encoded by SCO3450, causes the transcription of approximately one-fourth of GI 6, or ~26 mostly contiguous genes, to increase. More than half of the regulon encodes putative enzymes involved in small molecule metabolism. A putative haloacid dehalogenase is present. Genes encoding two putative anti-sigma factors flank SCO3450, the three genes residing within the regulon. Our data suggest that the ECF sigma factor and its regulon are a self-contained transcriptional unit that can be transferred by horizontal gene transfer. To our knowledge, only one other example has been identified of an ECF sigma factor and its contiguous regulon appearing to be transferrable by horizontal gene transfer [18,19]. Because the regulon appears not to be induced by the 44 growth conditions recently examined by Byung-Kwan Cho and colleagues [20], if it confers fitness to S. coelicolor, the regulon likely does so in as-yet unknown situations. Those situations might range from scavenging to detoxification to even communication within microbial communities.\n\nIMPORTANCEStreptomyces bacteria grow as hyphae that colonize soil and differentiate into spores when nutrients become scarce. In their terrestrial habitats, the bacteria encounter diverse conditions. Presumably so that the bacteria can cope with those conditions, the chromosomes of streptomycetes are highly dynamic, varying greatly in structure not only between species but also between closely related strains of a single species. The bacteria also have large numbers of extracytoplasmic function (ECF) sigma factors, which undoubtedly help the microorganisms respond to the plethora of challenges coming from the environment. This work illustrates these two threads of Streptomyces biology dovetailing: Genetic adaptability through horizontal gene transfer seems to have enabled Streptomyces coelicolor to acquire a self-contained transcriptional unit that consists of an ECF sigma factor and its regulon. The suggested facile movement of the regulon between microbial hosts indicates the value of the metabolism of small molecules possibly mediated by the regulon.

microbiology↗

bioRxiv2018

Single Cell Observations Show Persister Cells Wake Based on Ribosome Content

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising naturally at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through seven sets of experiments), we used microscopy of single cells to determine that persisters have low levels of antibiotic-corrupting proteins and that their resuscitation is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found for persister cell resuscitation: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division, and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

microbiology↗

bioRxiv2018

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fibre. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60-68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.

microbiology↗

bioRxiv2018

Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach

The library preparation step is a major source of bias in NGS-based studies. Several PCR-related factors might negatively influence the application of NGS tools in environmental studies and diagnostics. Among the most understudied factors are DNA polymerases. In our study, we evaluated the effect of DNA polymerase type on the characterisation of bacterial communities, more precisely Legionella, using a genus-specific NGS approach. The assay with proof-reading high fidelity KAPA HiFi showed better amplification yield than the one with widely used non-proofreading HotStarTaq. Legionella community richness metrics were significantly overestimated with HotStarTaq. However, the choice of DNA polymerase did not significantly change the community profiling and composition. These results substantiate the use of proof-reading high fidelity DNA polymerases in NGS assays and highlight the need of considering the impact of different DNA polymerases in comparative studies and future guidelines for NGS-based diagnostic tools.

microbiology↗

bioRxiv2018

Microbial biogeography of 1,000 geothermal springs in New Zealand

Geothermal springs are model ecosystems to systematically investigate microbial biogeography as they i) represent discrete, homogenous habitats; ii) are abundantly distributed across multiple geographical scales; iii) span broad geochemical gradients; and iv) have simple community structures with reduced metazoan interactions. Taking advantage of these traits, we undertook the largest known consolidated study of geothermal ecosystems (http://1000springs.org.nz) to determine factors that influence biogeographical patterns. Rigorously standardised methodologies were used to measure microbial communities, 46 physicochemical parameters, and metadata from 1,019 hotspring samples across New Zealand. pH was found to be the primary influence on diversity in springs < 70 {degrees}C with community similarity decreasing with geographic distance. Surprisingly, community composition was dominated by two genera (Venenivibrio and Acidithiobacillus) in both average relative abundance (11.2 and 11.1 %) and prevalence (74.2 and 62.9 % respectively) across physicochemical spectrums of 13.9 - 100.6 {degrees}C and pH < 1 - 9.7. This study provides an unprecedented insight into the ecological conditions that drive community assembly in geothermal springs, and can be used as a foundation to improve the characterisation of global microbial biogeographical processes.

microbiology↗

bioRxiv2018

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role the sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute chronic cachexia development, pathology and potential reversal.

microbiology↗

bioRxiv2018

Comparative genomics and phylogeny unveil lineage diversification of Citrobacter rodentium polyvalent bacteriophage

Enteropathogenic Escherichia coli (EPEC) is a major etiology for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse has led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy. Due to the drawbacks of EPEC in vivo models, a surrogate is the mouse-restricted gut pathgoen Citrobacter rodentium. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus and CrRp10 is a new strain within the genus Tequatrovirus. Neither phage carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 and CrRp10 appear to have independently evolved from E. coli phages. CrRp3 appears to be the more potent being 24x more likely to find a host cell and has a shorter lytic cycle, while CrRp10 at MOI 0.001 was able to maintain bacterial density below the limit of detection after 18 h. We found that hypoxia (5% O2 and 5% CO2) inhibited CrRp3 ability to reverse exponential bacterial growth. It is unclear whether the subtle characteristic differences between CrRp3 and CrRp10 will influence treatment efficacy in future phage therapy in vivo investigations.

microbiology↗

bioRxiv2018

Viromic analysis of wastewater input to a river catchment reveals a diverse assemblage of RNA viruses

Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes, nor emerging and invasive viral species. In this study, we investigated the use of viromics, i.e. high-throughput sequencing of the biosphere viral fraction, to detect human/animal pathogenic RNA viruses in the Conwy river catchment area in Wales, UK. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater, estuarine river water and sediment, followed by RNASeq analysis on the Illumina HiSeq platform for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and assembled a complete norovirus GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional qRT-PCR. To our knowledge, this is the first environmentally-derived norovirus genome sequence to be available from a public database. The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested run-off from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included.\n\nImportanceEnteric viruses cause gastro-intestinal illness and are commonly transmitted through the faecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.

microbiology↗

bioRxiv2018

Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogensis

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection, and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was thought to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.\n\nIMPORTANCEThe baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also employed as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPVs ability to mobilize the host actin cytoskeleton within the cells nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cells nucleus, yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

microbiology↗

bioRxiv2018

Metatranscriptome analysis of the vaginal microbiota reveals potential mechanisms for recurrence and protection against metronidazole in bacterial vaginosis

Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the Lactobacillus spp. dominated microbial community towards a taxonomically diverse anaerobic community. For unknown reasons, some women do not respond to therapy. In our recent clinical study, out of 37 women diagnosed with BV, 31 were successfully treated with metronidazole, while 6 still had BV after treatment. To discover possible reasons for the lack of response in those patients, we performed a metatranscriptome analysis of their vaginal microbiota, comparing them to patients who responded. Seven out of 8 Cas genes of Gardnerella vaginalis were highly up-regulated in non-responding patients. Cas genes, in addition to protecting against phages, might be involved in DNA repair thus mitigating the bactericidal effect of DNA damaging agents like metronidazole. In the second part of our study, we analyzed the vaginal metatranscriptomes of four patients over three months and showed high in vivo expression of genes for pore-forming toxins in L. iners and of genes encoding enzymes for the production of hydrogen peroxide and D-lactate in L. crispatus.

microbiology↗

bioRxiv2018

A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors

Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.

microbiology↗

bioRxiv2018

Comparative transcriptomics reveals a conserved Bacterial Adaptive Phage Response (BAPR) to viral predation

Intrinsic and acquired defenses against bacteriophages, including Restriction/Modification, CRISPR/Cas, and Toxin/Anti-toxin systems have been intensely studied, with profound scientific impacts. However, adaptive defenses against phage infection analogous to adaptive resistance to antimicrobials have yet to be described. To identify such mechanisms, we applied an RNAseq-based, comparative transcriptomics approach in different Pseudomonas aeruginosa strains after independent infection by a set of divergent virulent bacteriophages. A common host-mediated adaptive stress response to phages was identified that includes the Pseudomonas Quinolone Signal, through which infected cells inform their neighbors of infection, and what may be a resistance mechanism that functions by reducing infection vigor. With host transcriptional machinery left intact, we also observe phage-mediated differential expression caused by phage-specific stresses and molecular mechanisms. These responses suggest the presence of a conserved Bacterial Adaptive Phage Response mechanism as a novel type of host defense mechanism, and which may explain transient forms of phage persistence.

microbiology↗

bioRxiv2018

Conserved collateral susceptibility networks in diverse clinical strains of Escherichia coli.

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, is a uniquely promising strategy that enables selection against resistance during treatment. However, using CS-informed therapy depends on conserved CS networks across genetically diverse bacterial strains. We examined CS conservation in 10 clinical strains of E. coli resistant to four clinically relevant antibiotics. Collateral susceptibilities of these 40 resistant mutants were then determined against a panel of 16 antibiotics. Multivariate statistical analyses demonstrate that resistance mechanisms, in particular efflux-related mutations, as well as relative fitness were principal contributors to collateral changes. Moreover, collateral responses shifted the mutant selection window suggesting that CS-informed therapies could affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that early detection of resistance mechanisms is important to accurately predict collateral antimicrobial responses.

microbiology↗

bioRxiv2018

A host-pathogen interaction screen identifies ada2 as a mediator of Candida glabrata defences against reactive oxygen species

Candida glabrata (C. glabrata) forms part of the normal human gut microbiota but can cause life-threatening invasive infections in immune-compromised individuals. C. glabrata displays high resistance to common azole antifungals, which necessitates new treatments. In this investigation, we identified five C. glabrata deletion mutants ({Delta}ada2, {Delta}bas1, {Delta}hir3,{Delta} ino2 and{Delta} met31) from a library of 196 transcription factor mutants that were unable to grow and activate an immune response in Drosophila larvae. This highlighted the importance of these transcription factors in C. glabrata infectivity. Further ex vivo investigation into these mutants revealed the requirement of C. glabrata ADA2 for oxidative stress tolerance. We confirmed this observation in vivo whereby growth of the C. glabrata {Delta}ada2 strain was permitted only in flies with suppressed production of reactive oxygen species (ROS). Conversely, overexpression of ADA2 promoted C. glabrata replication in infected wild type larvae resulting in larval killing. We propose that ADA2 orchestrates the response of C. glabrata against ROS-mediated immune defences during infection. With the need to find alternative antifungal treatment for C. glabrata infections, genes required for survival in the host environment, such as ADA2, provide promising potential targets.

microbiology↗

bioRxiv2018

Diverse spatial organization of photosynthetic membranes among purple nonsulfur bacteria

In diverse bacteria, proper cellular physiology requires the utilization of protein- or membrane-bound compartments that afford specific metabolic capabilities. One such compartment is the light-harvesting intracytoplasmic membrane (ICM) of purple nonsulfur bacteria (PNSB). Here we reveal that ICMs are subject to differential spatial organization among PNSB. We visualized ICMs in live cells of fourteen PNSB species by exploiting the natural autofluorescence of the photosynthetic machinery. We then quantitatively characterized ICM localization using automated computational analysis of autofluorescence patterns within single cells across the population. Our studies revealed that ICMs are localized in distinct subcellular patterns that differ between species; some PNSB elaborate ICMs throughout the cell, while others spatially restrict ICM to varying degrees. The most highly-restricted ICMs were localized in a specific pattern corresponding to progression of cell growth and division. An identical pattern of ICM restriction was conserved across at least two genera. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. This discovery of new diversity in bacterial cell organization has implications for understanding both the mechanisms underpinning spatial arrangement of bacterial compartments and the potential benefits of adopting different spatiotemporal patterns.

microbiology↗

bioRxiv2018

Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs in M. tuberculosis

Although drug resistance in M. tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in M. tuberculosis by constructing point mutations V219A, S292L in Rv1258c in the chromosome of M. tuberculosis and assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in M. tuberculosis and address their role in affecting treatment outcome in vivo.

microbiology↗

bioRxiv2018

Structure of the Core of the Type Three Secretion System ExportApparatus

Export of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 [A] by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.\n\nOne Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

microbiology↗