Biology subjects

Gray, J.

Publications and source records attributed to Gray, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Bam complex associated proteins in Escherichia coli are functionally linked to peptidoglycan biosynthesis, membrane fluidity and DNA replication

Biogenesis of the bacterial outer membrane is key to bacterial survival and antibiotic resistance. Central to this is the {beta}-barrel assembly machine (Bam) complex and its associated chaperones, which are responsible for transport, folding and insertion of outer membrane proteins (OMPs). The Escherichia coli Bam complex is composed of two essential subunits, BamA and BamD, and three non-essential accessory lipoproteins, BamB, BamC and BamE. Optimal Bam function is further dependent on the non-essential periplasmic chaperones DegP, Skp and SurA. Despite intensive study, the specific function of these non-essential Bam-associated proteins is not fully understood. Here, we analysed {Delta}bamB, {Delta}bamC, {Delta}bamE, {Delta}surA, {Delta}skp and {Delta}degP knockout strains by phenotypic screening, conservation analysis and high-throughput genetics. We identify hundreds of synthetic-lethal interactions and reveal that Bam complex activity is impacted by changes in outer membrane lipid composition and that enterobacterial common antigen is essential in the absence of the chaperone SurA. We also show genes responsible for synthesis of peptidoglycan are synthetically-lethal with Bam accessory lipoprotein encoding genes. Together, our data indicates potential mechanisms for coordination of OMP biogenesis with other cellular growth processes such as LPS and peptidoglycan biogenesis.

microbiology↗

bioRxiv2023

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multi-drug resistant clones both alarmingly associated with high mortality. The molecular microbial genetics underpinning these recalcitrant K. pneumoniae infections is unclear, coupled with the emergence of lineages resistant to nearly all present day clinically important antimicrobials. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani medium), human urine and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

microbiology↗

bioRxiv2023

Oncogenic reactivation of young L1s is a hallmark of colon cancer

Transposable elements become increasingly active in both cancerous and aging cells, driven by loss of DNA methylation as cells divide. Here we leverage the epigenomes of colon cancers with matched adjacent tissue, in addition to non-cancerous normals and cell line models, to assess the role of transposable elements as drivers or passengers in cancer development. Using the baseline of activity from normal and adjacent tissue, we show that the youngest subfamilies of the LINE1 (L1) family exhibit a degree of activity and recurrence across patients that goes beyond what is expected from hypomethylation and cell division, suggesting an additional mechanism of oncogenic reactivation. We characterize this mechanism and find that the loss of the tumor suppressor PLZF drives young L1 reactivation in a cell-division-independent manner. PLZF de-repression exposes abundant motifs for tumor core factors in the L1 5UTR. Active young L1s act as oncogenic enhancers, interacting with oncogenes via gained chromatin loops. We uncover oncogenic L1 reactivation as a hallmark of colon cancer, where young L1s activate universally in our cohort at high levels of recurrence, act as enhancers to oncogenes, and become wired into the core regulatory circuitry of colon cancer.

genomics↗

bioRxiv2023

Masking, maintenance and mimicry: the interplay of cell-intrinsic and cell-extrinsic effects in evolutionary games

The temporal evolution of mutating pathogens in disease contexts arises from both the intrinsic properties of each subpopulation and the interactions among them, yet experimental inference often neglects the latter. Drug development studies commonly estimate selective advantages by comparing growth rates in monoculture, and in vitro monoculture dose-response curves are frequently used to justify or halt further investigation of a drug. Although many ecological models distinguish intrinsic from interaction-dependent growth rates, we show that simpler evolutionary game theory (EGT) frameworks can also be used to disentangle these contributions. We present a game-theoretic reparameterization of the replicator equation payoff matrix that separates intrinsic effects from interaction-driven contributions to frequency-dependent fitness. We also introduce an interaction-selection plot that facilitates the interpretation of the relative importance of between-population interactions compared with intrinsic evolutionary trade-offs. Using this framework, we map how interactions can mask, mirror, maintain, or mimic frequency-independent selection. We derive analytical conditions for these behaviors in both deterministic (replicator equation) and stochastic (Fokker-Planck-Kolmogorov) models, showing that simple conditions persist when mutation and noise are introduced. We validate these predictions using Wright-Fisher simulations. Applying our framework to published microbial and cancer co-culture data, we find that real systems span regimes dominated by either autonomous selection or interaction-driven effects, with interactions sometimes reversing or neutralizing frequency-independent fitness differences. Together, our results show that frequency-dependent effects can shape evolutionary dynamics in subtle and non-obvious ways, highlighting the importance of accounting for interactions when inferring fitness and predicting evolutionary outcomes.

evolutionary biology↗

bioRxiv2023

Centriole Remodeling Evolved into Centriole Degradation in Mouse Sperm

Centrioles are subcellular organelles found at the cilia base with an evolutionarily conserved structure and a shock absorber-like function. In sperm, centrioles are found at the flagellum base and are essential for embryo development in basal animals. Yet, sperm centrioles have evolved diverse forms, sometimes acting like a transmission system, as in cattle, and sometimes becoming dispensable, as in house mice. How the essential sperm centriole evolved to become dispensable in some organisms is unclear. Here, we test the hypothesis that this transition occurred through a cascade of evolutionary changes to the proteins, structure, and function of sperm centrioles and was possibly driven by sperm competition. We found that the final steps in this cascade are associated with a change in the primary structure of the centriolar luminal protein FAM161A in rodents. This information provides the first insight into the molecular mechanisms and adaptive evolution underlying a major evolutionary transition within the internal structure of the mammalian sperm neck. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/532086v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@bdbe1eorg.highwire.dtl.DTLVardef@10cc2b9org.highwire.dtl.DTLVardef@c42734org.highwire.dtl.DTLVardef@1e9c750_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Barrel-shaped centrioles are present in all rodents except members of the Muridae taxonomical family. - The FAM161A sequence evolved selectively in rodents and specifically in a murid subgroup. - The evolution of centriole degradation is correlated with the expression of a novel FAM161A isoform. - The ancestral centriole remodeling program involves elongation followed by redistribution. - The mouse centriole degradation program involves elongation followed by elimination.

cell biology↗

bioRxiv2023

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of [~]7 million cells profiled, and a spatially resolved transcriptomic dataset of [~]4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

neuroscience↗

bioRxiv2022

Temporal chromatin accessibility changes define transcriptional states essential for osteosarcoma metastasis

The metastasis-invasion cascade describes the series of steps required for a cancer cell to successfully spread from its primary tumor and ultimately grow within a secondary organ. Despite metastasis being a dynamic, multistep process, most omics studies to date have focused on comparing primary tumors to the metastatic deposits that define end-stage disease. This static approach means we lack information about the genomic and epigenomic changes that occur during the majority of tumor progression. One particularly understudied phase of tumor progression is metastatic colonization, during which cells must adapt to the new microenvironment of the secondary organ. Through temporal profiling of chromatin accessibility and gene expression in vivo, we identify dynamic changes in the epigenome that occur as osteosarcoma tumors form and grow within the lung microenvironment. Furthermore, we show through paired in vivo and in vitro CRISPR drop-out screens and pharmacological validation that the upstream transcription factors represent a class of metastasis-specific dependency genes. While current models depict lung colonization as a discrete step within the metastatic cascade, our study shows it is a defined trajectory through multiple epigenetic states, revealing new therapeutic opportunities undetectable with standard approaches.

cancer biology↗

bioRxiv2022

Integrative transcriptomic and proteomic meta-analysis of Zika viral infection reveals potential mechanisms for oncolytic therapy in neuroblastoma

BACKGROUNDPaediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) is neurotropic and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. METHODSHere, we perform an extensive meta-analysis of ZIKV infection studies to identify molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We summarise the neuroblastoma cell lines and ZIKV strains utilised and re-evaluate the infection data to deduce the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating transcriptomics, interaction proteomics, dependency factor and compound datasets we show the involvement of multiple host systems during ZIKV infection. RESULTSWe identify that most paediatric neuroblastoma cell lines are highly susceptible to ZIKV infection and that the PRVABC59 ZIKV strain is the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV is dependent on SREBP-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV nonstructural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. CONCLUSIONSOur work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials. KEYPOINTSO_LIThe Zika virus may provide the basis for an oncolytic virotherapy against Neuroblastoma C_LIO_LIMost paediatric neuroblastoma cell lines are susceptible to Zika viral infection C_LIO_LIWe identified molecular mechanisms that may induce the oncolytic response in Neuroblastoma C_LI Contribution to the fieldThe ability to both induce direct oncolysis and provoke an anti-tumoral immune response makes oncolytic virotherapy an attractive candidate to combat aggressive and heterogenous cancers, such as high-risk neuroblastoma. To progress oncolytic virotherapy to clinical trial it is essential to understand the host mechanisms the virus manipulates to kill cancer cells, alongside any pathology as a consequence of infection of normal cells. Here, we show that ZIKV efficiently infects and induces oncolysis of paediatric neuroblastoma cells and propose a potential TNF pathway-driven immune response. ZIKVs specificity for infection of nervous system cancer cells, while rarely causing nervous system-related pathology in young children, addresses many of its safety concerns. The inclusion of more effective and less toxic novel therapies, such as a potential ZIKV-based therapeutic, in multimodal treatment regimens will pave the way for improving patient long-term health and overall survival.

cancer biology↗

bioRxiv2022

Distinct tumor-immune ecologies in NSCLC patientspredict progression and define a clinical biomarker of therapy response

We investigated multiplexed histological images from nine patients (both pre- and on-treatment) with immunotherapy-refractory Non-Small Cell Lung Cancer (NSCLC) treated with an oral HDAC inhibitor (vorinostat) combined with a PD-1 inhibitor (pembrolizumab). Patient responses comprised of either stable disease (SD) or progressive disease (PD). We built an extensive multiplexed-image analysis pipeline involving both cell segmentation and quadrats, coupled with spatial statistics, machine learning, and deep learning to analyze the spatial and temporal features that predict disease progression and identify potential clinical biomarkers. We found that distinct spatial immune ecologies exist between SD and PD patients. We also demonstrate that tumors from PD patients are already characterized by an immune-suppressive environment prior to treatment. Finally, we show that the learned spatial ecologies can predict disease progression better than PD-L1 status alone, suggesting these ecologies can be used as potential companion biomarkers with PD-L1 in NSCLC. These findings will be investigated in a larger-cohort study generated from an ongoing clinical trial (NCT02638090) that includes a wider range of responses including complete and partial responders. Additionally, the computational infrastructure developed in this study can be generalized to any cancer type.

cancer biology↗

bioRxiv2022

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats unique traits.

developmental biology↗

bioRxiv2021

Peptidoglycan recycling contributes to outer membrane integrity and carbapenem tolerance in Acinetobacter baumannii

The Gram-negative cell envelope is an essential structure that not only protects the cell against lysis from the internal turgor, but also forms a barrier to limit entry of antibiotics. Some of our most potent bactericidal antibiotics, the {beta}-lactams, exploit the essentiality of the cell envelope by inhibiting its biosynthesis, typically inducing lysis and rapid death. However, many Gram-negative bacteria exhibit "antibiotic tolerance", the ability to sustain viability in the presence of {beta}-lactams for extended time periods. Despite several studies showing that antibiotic tolerance contributes directly to treatment failure, and is a steppingstone in acquisition of true resistance, the molecular factors that promote intrinsic tolerance are not well-understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. While typically reserved to combat multidrug resistant infections, carbapenem {beta}-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate high intrinsic tolerance to meropenem, form spheroplasts with the antibiotic and revert to normal growth after antibiotic removal. Using transcriptomics and genetics screens, we characterized novel tolerance factors and found that outer membrane integrity maintenance, drug efflux and peptidoglycan homeostasis collectively contribute to meropenem tolerance in A. baumannii. Furthermore, outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to meropenem tolerance in A. baumannii. ImportanceCarbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting an urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well-understood. Here, we characterized a role for peptidoglycan recycling in outer membrane integrity maintenance and carbapenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlights physiological processes that could be targeted to improve antimicrobial treatment.

microbiology↗

bioRxiv2021

Balance between protective and pathogenic immune responses to pneumonia in the neonatal lung enforced by gut microbiota.

While modern clinical practices like cesarean sections and perinatal antibiotics have improved infant survival, treatment with broad-spectrum antibiotics alters intestinal microbiota and causes dysbiosis. Infants exposed to perinatal antibiotics have an increased likelihood of life-threatening infections, including pneumonia. Here, we investigated how gut microbiota sculpt pulmonary immune responses, promoting recovery and resolution of infection in newborn rhesus macaques. Early-life antibiotic exposure, mirroring current clinical practices, interrupted the maturation of intestinal commensal bacteria and disrupted the developmental trajectory of the pulmonary immune system as assessed by single-cell proteomic and transcriptomic analyses of the pulmonary immune response. Early-life antibiotic exposure rendered newborn macaques susceptible to bacterial pneumonia, mediated by profound changes in neutrophil senescence, inflammatory signaling, and macrophage dysfunction. Pathogenic reprogramming of pulmonary immunity was reflected by a hyperinflammatory signature in all pulmonary immune cell subsets. Distinct patterns of immunoparalysis, including dysregulated antigen presentation in alveolar macrophages, impaired costimulatory function in T helper cells, and dysfunctional cytotoxic responses in natural killer (NK) cells, were coupled with a global loss of tissue-protective, homeostatic pathways in lungs of dysbiotic newborns. Fecal microbiota transfer corrected the broad immune maladaptations and protected against severe pneumonia. These data demonstrate the importance of intestinal microbiota in programming pulmonary immunity. Gut microbiota promote balance between pathways driving tissue repair and inflammatory responses, thereby leading to clinical recovery from infection in infants. One sentence summaryGut microbiota promote clinical recovery by reinforcing the balance between regenerative pathways driving tissue homeostasis and inflammatory responses limiting pathogens in infected neonatal lungs.

immunology↗

bioRxiv2021

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates

Malformations of or injuries to the developing lung are associated with perinatal morbidity and mortality with lifelong consequences for subsequent pulmonary health. One fetal exposure linked with poor health outcomes is chorioamnionitis, which impacts up to 25-40% of preterm births. Severe chorioamnionitis with prematurity is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may significantly alter developmental ontogeny of the lung. To test this hypothesis, we used intra-amniotic lipopolysaccharide (LPS, endotoxin) to generate experimental chorioamnionitis in prenatal Rhesus macaque (Macaca mulatta), a model which shares critical structural and temporal aspects of human lung development. Inflammatory injury directly disrupts the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis which was extensively disrupted by perinatal inflammation, leading to loss of gas exchange surface and alveolar simplification similar to that found in chronic lung disease of newborns. Blockade of IL1{beta} and TNF ameliorated endotoxin-induced inflammatory lung injury by blunting stromal response to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

developmental biology↗

bioRxiv2021

A potent myeloid response is rapidly activated in the lungs of premature Rhesus macaques exposed to intra-uterine inflammation

Intrauterine inflammation/infection (IUI), which is present in up to 40% of premature births, leads to elevated levels of pro-inflammatory mediators and microbial products within the amniotic fluid, which come in close contact to fetal mucosae. Yet, knowledge on the fetal mucosal responses to IUI exposure remains limited. To address these questions, we used a non-human primate model of IUI, in which pregnant Rhesus macaques received intra-amniotic (IA) LPS, compared with IA saline. We found that IA LPS exposure induced a robust and rapid inflammation of the fetal lung, but not the intestine. This inflammatory response was characterized by high levels of pro-inflammatory cytokines in the lung and the alveolar wash, and a potent myeloid cell response, dominated by neutrophils and monocytes/macrophages. scRNAseq analyses of fetal lungs showed that the infiltrating (neutrophils and inflammatory monocytes) and the resident (alveolar and interstitial macrophages) myeloid cells exhibited transcriptional profiles consistent with exposure to TLR ligands, as well as to cytokines, notably IL-1 and TNF. However, blocking IL-1 signaling or TNF, alone or simultaneously by administering inhibitors intra-amniotically and subcutaneously to the dam only partially blunted fetal lung inflammation. Together, our novel data indicate that the fetal innate immune system can mount a rapid multi-factorial mucosal innate response to IUI, responding both to direct signaling by bacterial products and to indirect cytokine-mediated pathways of activation. These data thus provide more mechanistic insights into the association between IUI exposure and the post-natal lung morbidities of the premature infant.

immunology↗

bioRxiv2021

DpaA detaches Braun's lipoprotein from peptidoglycan

Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Brauns lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Brauns lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

microbiology↗

bioRxiv2020

Proficiency Testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories

The adoption of whole genome sequencing (WGS) data over the past decade for pathogen surveillance, and decision-making for infectious diseases has rapidly transformed the landscape of clinical microbiology and public health. However, for successful transition to routine use of these techniques, it is crucial to ensure the WGS data generated meet defined quality standards for pathogen identification, typing, antimicrobial resistance detection and surveillance. Further, the ongoing development of these standards will ensure that the bioinformatic processes are capable of accurately identifying and characterising organisms of interest, and thereby facilitate the integration of WGS into routine clinical and public health laboratory setting. A pilot proficiency testing (PT) program for WGS of infectious agents was developed to facilitate widely applicable standardisation and benchmarking standards for WGS across a range of laboratories. The PT participating laboratories were required to generate WGS data from two bacterial isolates, and submit the raw data for independent bioinformatics analysis, as well as analyse the data with their own processes and answer relevant questions about the data. Overall, laboratories used a diverse range of bioinformatics tools and could generate and analyse high-quality data, either meeting or exceeding the minimum requirements. This pilot has provided valuable insight into the current state of genomics in clinical microbiology and public health laboratories across Australia. It will provide a baseline guide for the standardisation of WGS and enable the development of a PT program that allows an ongoing performance benchmark for accreditation of WGS-based test processes.

bioinformatics↗

bioRxiv2020

Four-dimensional characterization of the Babesia divergens asexual life cycle: from the trophozoite to the multiparasite stage

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double and quadruple trophozoites, paired and double paired-pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining the emerging technique cryo soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. As a result, the architecture and kinetic of the parasite population has been elucidated. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. Based on these observations, a four-dimensional (4D) asexual life cycle model has been designed highlighting the origin of the tetrad, double paired-pyriform and multiparasite stages and the transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next along the cycle. IMPORTANCEBabesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease, is increasing in frequency and geographical range, and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three and four dimensional (3D and 4D) imaging techniques to elucidate the origin, architecture, and kinetic of IE parasites. Unveil the nature of these parasites have provided a vision of the B. divergens asexual cycle in unprecedented detail and a key step to develop control strategies against babesiosis

microbiology↗

bioRxiv2020

Large-scale Characterization of Drug Responses of Clinically Relevant Proteins in Cancer Cell Lines

Perturbation biology is a powerful approach to developing quantitative models of cellular behaviors and gaining mechanistic insights into disease development. In recent years, large-scale resources for phenotypic and mRNA responses of cancer cell lines to perturbations have been generated. However, similar large-scale protein response resources are not available, resulting in a critical knowledge gap for elucidating oncogenic mechanisms and developing effective cancer therapies. Here we generated and compiled perturbed expression profiles of ~210 clinically relevant proteins in >12,000 cancer cell-line samples in response to >150 drug compounds using reverse-phase protein arrays. We show that integrating protein response signals substantially increases the predictive power for drug sensitivity and aids in gaining insights into mechanisms of drug resistance. We build a systematic map of protein-drug connectivity and develop an open-access, user-friendly data portal for community use. Our study provides a valuable information resource for a broad range of quantitative modeling and biomedical applications. HighlightsO_LIA large collection of cancer cell line protein responses to drug perturbations C_LIO_LIPerturbed protein responses greatly increase predictive power for drug sensitivity C_LIO_LIBuild a systematic map of protein-drug connectivity based on response profiles C_LIO_LIDevelop a user-friendly, interactive data portal for community use C_LI

systems biology↗