Biology subjects

Hu, B.

Publications and source records attributed to Hu, B..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Development and application of an uncapped mRNA platform

A novel uncapped mRNA platform was developed. Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), S{delta}T-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1[~]400). Quantifiable target protein expression was achieved in vitro and in vivo with eGFP-and Fluc-mRNA-LNP. S{delta}T-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, S{delta}T-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1m{psi}TP) in the induction of antibodies via S{delta}T-mRNA-LNP. Our uncapped, process-simplified, and economical mRNA platform may have broad utility in vaccines and protein replacement drugs.

bioengineering↗

bioRxiv2022

Genomic screening reveals UBA1 as a potent and druggable target in c-MYC-high TNBC models

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer-related deaths, despite accounting for only 10%-15% of total breast cancer cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Targeting UBA1 with the first in-class UBA1 inhibitor TAK-243 induced unresolvable ER-stress and activating transcription factor 4 (ATF4)-mediated upregulation of pro-apoptotic NOXA, leading to cell death. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. In an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. Lastly, c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. We posit UBA1 is an important new target in TNBC expressing high levels of c-MYC. SignificanceGenomic screening of TNBC cell lines revealed broad sensitivity to depletion of the E1 ubiquitin enzyme, UBA1. Disrupting UBA1 with the first in-class inhibitor TAK-243 in TNBC models induces ER-stress through an ATF4-NOXA axis that is dependent on c-MYC, leading to apoptosis, in vitro and in vivo, primary tumor growth inhibition and metastatic inhibition.

cancer biology↗

bioRxiv2022

HIV integration in the human brain is linked to microglial activation and 3D genome remodeling

Exploration of genome organization and function in the HIV infected brain is critical to aid in the development of treatments for HIV-associated neurocognitive disorder (HAND) and HIV cure strategies. Here, we generated a resource comprised of single nuclei transcriptomics, complemented by cell-type-specific Hi-C chromosomal conformation ( 3D genome) and viral integration site sequencing (IS-seq) in frontal brain tissues from individuals with HIV encephalitis (HIVE), HIV-infected people without encephalitis (HIV+), and HIV uninfected (HIV-) controls. We observed profound 3D genomic reorganization of open/repressive (A/B) compartment structures encompassing 6.4% of the HIVE microglial genome that was associated with transcriptomic reprogramming, including down-regulation of homeostasis and synapse-related functions and robust activation of interferon signaling and cell migratory pathways. HIV RNA was detected in 0.003% of all nuclei in HIVE brain, predominantly in the most activated microglia where it ranked as the second most highly expressed transcript. Microglia from HIV+ brains showed, to a lesser extent, similar transcriptional alterations. IS-seq recovered 1,221 insertion events in glial nuclei that were enriched for chromosomal domains newly mobilized into a permissive chromatin environment in HIVE microglia. Brain and peripheral myeloid cell integration revealed a preference overall for transcription-permissive chromatin, but robust differences in the frequency of recurrent insertions, intergenic integration, and enrichment for pre-integration complex-associated factors at integration sites. Our resource highlights critical differences in the genomic patterns of HIV infection in brain versus blood and points to a dynamic interrelationship between inflammation-associated 3D genome remodeling and successful integration in brain.

neuroscience↗

bioRxiv2022

Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants

Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from human to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, in order to evaluate the impact of S mutations, we constructed 20 Hela cell lines stably expressing ACE2 orthologs from different animals, and prepared 27 pseudotyped SARS-CoV-2 carrying different spike mutants, among which 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.135), Lambda (B.1.429) and Mu (B.1.525). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammals ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.

microbiology↗

bioRxiv2022

Every individual makes a difference: A trinity derived from linking individual brain morphometry, connectivity and mentalising ability

Mentalising ability, indexed as the ability to understand others beliefs, feelings, intentions, thoughts and traits, is a pivotal and fundamental component of human social cognition. However, considering the multifaceted nature of mentalising ability, little research has focused on characterising individual differences in different mentalising components. And even less research has been devoted to investigating how the variance in the structural and functional patterns of the amygdala and hippocampus, two vital subcortical regions of the social brain, are related to inter-individual variability in mentalising ability. Here, as a first step toward filling these gaps, we exploited inter-subject representational similarity analysis (IS-RSA) to assess relationships between amygdala and hippocampal morphometry (surface-based multivariate morphometry statistics, MMS), connectivity (resting-state functional connectivity, rs-FC) and mentalising ability (interactive mentalisation questionnaire (IMQ) scores) across the participants (N = 24). In IS-RSA, we proposed a novel pipeline, i.e., computing patching and pooling operations-based surface distance (CPP-SD), to obtain a decent representation for high-dimensional MMS data. On this basis, we found significant correlations (i.e., secondorder isomorphisms) between these three distinct modalities, indicating that a trinity existed in idiosyncratic patterns of brain morphometry, connectivity and mentalising ability. Notably, a region-related mentalising specificity emerged from these associations: self-self and self-other mentalisation are more related to the hippocampus, while other-self mentalisation shows a closer link with the amygdala. Furthermore, by utilising the dyadic regression analysis, we observed significant interactions such that subject pairs with similar morphometry had even greater mentalising similarity if they were also similar in rs-FC. Altogether, we demonstrated the feasibility and illustrated the promise of using IS-RSA to study individual differences, deepening our understanding of how individual brains give rise to their mentalising abilities.

neuroscience↗

bioRxiv2022

Bipartite recruitment of PCH2 and COMET to the synaptonemal complex drives chromosome axis reconstruction leading to crossover restriction

Chromosome axis-associated HORMA domain proteins (HORMADs), e.g., ASY1 in Arabidopsis, are crucial for meiotic recombination. ASY1, as other HORMADs, is assembled on the axis at early meiosis and depleted when homologous chromosomes synapse. Puzzlingly, both processes are catalyzed by AAA+ ATPase PCH2 together with its cofactor COMET. Here, we show that the ASY1 remodeling complex is temporally and spatially differently assembled. While PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis, PCH2 is recruited by the transverse filament protein ZYP1 and brought to the ASY1-bound COMET assuring the timely removal of ASY1 during chromosome synapsis. Since we found that the PCH2 homolog TRIP13 also binds to the ZYP1 homolog SYCP1 in mouse, we postulate that this mechanism is conserved among eukaryotes. Deleting the PCH2 binding site of ZYP1 led to a failure of ASY1 removal. Interestingly, the placement of one obligatory crossover per homologous chromosome pair, compromised by ZYP1 depletion, is largely restored in this engineered zyp1 allele suggesting that crossover assurance is promoted by synapsis. In contrast, the engineered zyp1 allele, similar to the zyp1 null mutant, showed elevated type I crossover numbers indicating that PCH2-mediated eviction of ASY1 from the axis restricts crossover formation.

cell biology↗

bioRxiv2022

The Arabidopsis Hop1 homolog ASY1 mediates cross-over assurance and interference

The chromosome axis plays a crucial role in meiotic recombination. Here, we study the function of ASY1, the Arabidopsis homolog of the yeast chromosome axis associated component Hop1. Specifically, we characterized cross-over (CO) distribution in female and male meiosis by deep sequencing of the progeny of an allelic series of asy1 mutants. Combining data from nearly 1000 individual plants, we find that reduced ASY1 activity leads to genomic instability and sometimes drastic genomic rearrangements. We further observed that COs are less frequent and appear in more distal chromosomal regions in plants with no or reduced ASY1 activity, consistent with previous analyses. However, our sequencing approach revealed that the reduction in CO number is not as dramatic as suggested by cytological analyses. Analysis of double mutants of asy1 with mutants with three other CO factors, MUS81, MSH4 and MSH5 as well as the determination of foci number of the CO regulator MLH1 demonstrates that the majority of the COs in asy1, similar to the situation in the wildtype, largely belong to the class I, which are subject to interference. However, these COs are redistributed in asy1 mutants and typically appear much closer than in the wildtype. Hence, ASY1 plays a key role in CO interference that spaces COs along a chromosome. Conversely, since a large proportion of chromosomes do not receive any CO, we conclude that CO assurance, the process that ensures the obligatory assignment of one CO per chromosome, is also affected in asy1 mutants. Significant statementThe regulation of the number and placement of cross-overs (COs) during meiosis is critical to ensure meiotic fidelity and promote new genetic combinations. Here, we investigated the function of one of the proteins of the chromosome axis, which plays a key role in CO formation: ASY1. Our results show that COs in asy1 mutants are positioned closer to each other than in the wildtype and that, despite a roughly similar number of COs, not every chromosome receives a CO. With this, our results shed light on the mechanisms regulating two important but still poorly understood aspects of meiosis: CO assurance, which safeguards at least one CO per chromosome pair, and CO interference, which prevents two COs from occurring close to each other.

cell biology↗

bioRxiv2021

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and nucleosome assembly

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during early embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and early embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in early embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in expression genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine early development.

developmental biology↗

bioRxiv2021

Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

cell biology↗

bioRxiv2021

Identification, analysis, and confirmation of seed storability-related loci in Dongxiang wild rice (Oryza rufipogon Griff.)

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) has strong seed storability and identifying its elite gene resources may facilitate genetic improvements in rice seed storability. In this study, we developed two backcross inbred lines (BILs) populations, with DXWR as a common donor parent and two rice varieties (F6 and R974) as recipient parents. Bulked segregant analysis via whole genome sequencing (BSA-seq) was used to identify seed storability-related loci in the DXWR and F6 population. Two main genomic regions containing 18,550,000-20,870,000 bp on chromosome 4 and 7,860,000-9,780,000 bp on chromosome 9 were identified as candidate loci of DXWR seed storability; these overlapped partially with seed storability-related quantitative trait loci (QTLs) discovered in previous studies, suggesting that these loci may provide important regions for isolating the responsible genes. In total, 448 annotated genes were predicted within the identified regions, of which 274 and 82 had nonsynonymous and frameshift mutations, respectively. We detected extensive metabolic activities and cellular processes during seed storability and confirmed the effects of the seed storability-related candidate loci using four BILs from DXWR and R974. These results may facilitate the cloning of DXWR seed storability-related genes, thereby elucidating rice seed storability and its improvement potential.

genomics↗

bioRxiv2021

Drug specific transcriptomic signatures in healthy human subject iPSC derived cardiomyocytes

Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focused on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced pluripotent stem cell-derived cardiomyocytes. Using bulk transcriptomics profiles, we applied singular value decomposition to identify drug-selective patterns in cell lines obtained from multiple healthy human subjects. Cellular pathways affected by highly cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Whole genome sequences of the cell lines, using outlier responses enabled us to correctly reidentify a genomic variant associated with anthracycline cardiotoxicity and predict genomic variants potentially associated with TKI cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale predictive understanding of cardiotoxicity for drug development and patient stratification. One sentence summaryGenes, pathways, and cell types of the human heart associated with antineoplastic drug cardiotoxicity.

systems biology↗

bioRxiv2021

The exosomes derived from urine of premature infants target MAPK8 via miR-30a-5p to protect cisplatin-induced acute kidney injury in mice

AimAcute kidney injury (AKI), a global public health issue, not only causes millions of deaths every year, but is also a susceptible factor for chronic kidney disease (CKD). Nephrotoxic drugs are an important cause of AKI. There is still a lack of effective and satisfactory prevention method in clinical practice. This study investigated the protective effect of the exosomes derived from urine of premature infants on cisplatin-induced acute kidney injury. MethodsIsolation of exosomes from fresh urine of premature infants: The characteristics of exosomes were determined by flow cytometry, transmission electron microscopy and Western blotting. A C57BL/6 mice model of cisplatin-induced acute kidney injury was established. The mice in the experimental group were given 100ug exosomes dissolved in 200ul solution. The mice in the control group were given normal saline (200ul). These treatments were performed 24 hours after AKI was induced by intraperitoneal injection of cisplatin. To evaluate renal function, blood was drawn 24 hours after AKI model was established and serum creatinine (sCr) was measured. The mice were euthanized 72 hours after exosome treatment. The kidneys were collected for pathological examination, RNA and protein extraction, and the evaluation of renal tubular damage and apoptosis. In the in-vitro experiment, human renal cortex/proximal tubular cells (HK2) was induced by cisplatin to assess the protective ability of the exosomes derived from urine of premature infants. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blotting were used to evaluate the effect of exosomes treatment on the apoptosis of HK2 cells induced by cisplatin. Exosome microRNA sequencing technology and bioinformatics analysis method were applied to investigate the miRNAs enriched in exosomes and their target genes. The dual luciferase gene reporter system was used to detect the interaction of target genes. ResultsTreatment of exosomes derived from urine of premature infants could decrease the level of serum creatinine and the apoptosis of renal tubular cell, inhibit the infiltration of inflammatory cell, protect mice from acute kidney injury induced by cisplatin and reduce mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes derived from urine of premature infants. It protected HK2 cells from cisplatin-induced apoptosis by targeting and down-regulating the 3UTR of mitogen-activated protein kinases (MAPK8) mRNA. ConclusionsAccording to our results, the exosomes derived from urine of premature infants alleviated cisplatin-induced acute kidney injury in mice and inhibited the apoptosis of human proximal tubular cells (HK2) induced by cisplatin in vitro. MiR-30a-5p in exosomes inhibited cisplatin-induced MAPK activation, ameliorated apoptosis, and protected renal function. The exosomes derived from urine of premature infants provided a promising acellular therapy for AKI.

molecular biology↗

bioRxiv2021

Programmable Sequences of Reflectin Proteins Provides Evidence for Their Evolution Processes, Tunable Intracellular Localization and Interaction with Cytoskeleton

Reflectin proteins are nature copolymers consist of repeated canonical domains, locating in a biophotonic system called Bragg lamellae and manipulating the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. In this study, reflectin A1, A2, B1 and C were found to present distinguished cyto-/nucleoplasmic localization preferences. Comparable intracellular localization can be reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. Secondly, the size-dependent access of reflectin variants into nucleus demonstrate a potential model of how reflectins get into Bragg lamellae. Thirdly, RfA1 was found to extensively interact with cytoskeleton, including its binding to actin and microtubule minus-end-directed movement. This implies cytoskeleton system plays fundamental role during the organization and transportation of reflectin proteins. Findings presented here recommend reflectins as programmable biomaterials which can be used to decipher their evolution processes, to delineate their biological mechanism, and to achieve tunable intracellular targeting as editable tags.

cell biology↗

bioRxiv2021

In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as minimized or expanded based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer. SIGNIFICANCEBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

microbiology↗

bioRxiv2021

EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle

Candidate phyla radiations (CPR), accounting for a major microbial supergroup with remarkably small genomes and reduced sizes, are widely distributed yet mostly uncultured. Limited culture and its obligate reliance upon other bacteria hindered investigation of their lifestyles. In this work we isolated a CPR bacterium, TM7i, with its host Leucobacter aridocollis J1, by combination of Emulsion, Paired Isolation and Concatenation PCR (epicPCR) detection and filtrate co-culture. Genomic profiling of TM7 genomes and microscopic investigation of TM7i-J1 symbiosis suggest the conservation of type IV pili and a pili-dependent lifestyle of TM7. Further, we observed twitching motility of TM7i mediated by pili and its role played in the interaction with its host. Our results shed a light on the lifestyle about this enigmatic bacterial radiation, which may also be adopted by other CPR organisms. The epicPCR-directed isolation method underlines high efficiency of CPR bacteria isolation and thus may be used in other symbiotic or epibiotic microorganisms.

microbiology↗

bioRxiv2021

In vitro Evaluation of Lytic Phages for Carbapenem-Resistant Klebsiella pneumoniae Isolates

Klebsiella pneumoniae is a dominant cause of community-acquired and nosocomial infections, specifically among immunocompromised individuals. The increasing occurrence of multidrug-resistant (MDR) isolates has significantly impacted the effectiveness of antimicrobial agents. As antibiotic resistance is becoming prevalent, the use of bacteriophages to treat pathogenic bacterial infections is becoming revitalized. Elucidating the details of phage-bacteria interactions will provide insights into phage biology and the better development of phage therapies. In this study, a total of 22 K. pneumoniae isolates were assessed for their genetic and phenotypic relatedness by multi-locus sequence typing (MLST), endonuclease S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and in vitro antibiotic susceptibility testing. In addition, the beta-lactamase gene (blaKPC) was characterized to determine the spread and outbreak of K. pneumoniae carbapenemase (KPC)-producing enterobacterial pathogens. Using these K. pneumoniae isolates, three phages were isolated and characterized to evaluate the application of lytic phages against these 22 ST11 K. pneumoniae isolates. In vitro inhibition assays with three phages and K. pneumoniae strain ZS15 demonstrated the strong lytic potential of the phages, however, followed by the rapid growth of phage-resistant mutants. Together, this data adds more comprehensive knowledge to known phage biology and further emphasizes their complexity and future challenges to overcome prior to using phages for controlling this important MDR bacterium.

microbiology↗

bioRxiv2021

In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as minimized or expanded based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical minimized systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as minimized or expanded: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels. SignificanceBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and firmly support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate substrate entry into and passage through T4SS channels.

microbiology↗

bioRxiv2021

Influence of age on functional memory T cell diversity

Memory T cells exhibit considerable diversity that determines their ability to be protective and their durability. Here, we examined whether changes in T cell heterogeneity contribute to the age-associated failure of immune memory. By screening for age-dependent T cell surface markers, we have identified CD4 and CD8 memory T cell subsets that are unrelated to previously defined subsets of central and effector memory cells. Memory T cells expressing the ecto-5-nucleotidase CD73 constitute a functionally distinct subset of memory T cells that declines with age. They exhibit many features favorable for immune protection, including longevity and polyfunctionality. They have a low turnover, but are poised to display effector functions and to develop into cells resembling tissue-resident memory T cells (TRM). Upstream regulators of differential chromatin accessibility and transcriptomes include transcription factors that are characteristic for conferring these superior memory features as well as facilitating CD73 expression. CD73 is not just a surrogate marker of these regulatory networks but is directly involved in T cell survival and TRM differentiation Interventions preventing the decline of this T cell subset or increasing CD73 expression have the potential to improve immune memory in older adults.

immunology↗