Publications by authors named "Trevor Lithgow"

182 Publications

Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry.

Biochim Biophys Acta Biomembr 2021 Feb 24:183587. Epub 2021 Feb 24.

Department of Materials Science & Engineering, Monash University, Clayton, VIC 3800, Australia; Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex catalyses the assembly of β-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - β-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential β-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.
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http://dx.doi.org/10.1016/j.bbamem.2021.183587DOI Listing
February 2021

Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.

Nat Microbiol 2021 Feb 11;6(2):157-161. Epub 2021 Jan 11.

School of Biological Sciences, Monash University, Clayton, Victoria, Australia.

We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.
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http://dx.doi.org/10.1038/s41564-020-00830-7DOI Listing
February 2021

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

Elife 2020 12 14;9. Epub 2020 Dec 14.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, United Kingdom.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
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http://dx.doi.org/10.7554/eLife.62614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806268PMC
December 2020

DeepBL: a deep learning-based approach for in silico discovery of beta-lactamases.

Brief Bioinform 2020 Nov 20. Epub 2020 Nov 20.

Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

Beta-lactamases (BLs) are enzymes localized in the periplasmic space of bacterial pathogens, where they confer resistance to beta-lactam antibiotics. Experimental identification of BLs is costly yet crucial to understand beta-lactam resistance mechanisms. To address this issue, we present DeepBL, a deep learning-based approach by incorporating sequence-derived features to enable high-throughput prediction of BLs. Specifically, DeepBL is implemented based on the Small VGGNet architecture and the TensorFlow deep learning library. Furthermore, the performance of DeepBL models is investigated in relation to the sequence redundancy level and negative sample selection in the benchmark dataset. The models are trained on datasets of varying sequence redundancy thresholds, and the model performance is evaluated by extensive benchmarking tests. Using the optimized DeepBL model, we perform proteome-wide screening for all reviewed bacterium protein sequences available from the UniProt database. These results are freely accessible at the DeepBL webserver at http://deepbl.erc.monash.edu.au/.
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http://dx.doi.org/10.1093/bib/bbaa301DOI Listing
November 2020

AcrHub: an integrative hub for investigating, predicting and mapping anti-CRISPR proteins.

Nucleic Acids Res 2021 01;49(D1):D630-D638

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, VIC 3800, Australia.

Anti-CRISPR (Acr) proteins naturally inhibit CRISPR-Cas adaptive immune systems across bacterial and archaeal domains of life. This emerging field has caused a paradigm shift in the way we think about the CRISPR-Cas system, and promises a number of useful applications from gene editing to phage therapy. As the number of verified and predicted Acrs rapidly expands, few online resources have been developed to deal with this wealth of information. To overcome this shortcoming, we developed AcrHub, an integrative database to provide an all-in-one solution for investigating, predicting and mapping Acr proteins. AcrHub catalogs 339 non-redundant experimentally validated Acrs and over 70 000 predicted Acrs extracted from genome sequence data from a diverse range of prokaryotic organisms and their viruses. It integrates state-of-the-art predictors to predict potential Acrs, and incorporates three analytical modules: similarity analysis, phylogenetic analysis and homology network analysis, to analyze their relationships with known Acrs. By interconnecting all modules as a platform, AcrHub presents enriched and in-depth analysis of known and potential Acrs and therefore provides new and exciting insights into the future of Acr discovery and validation. AcrHub is freely available at http://pacrispr.erc.monash.edu/AcrHub/.
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http://dx.doi.org/10.1093/nar/gkaa951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779044PMC
January 2021

BastionHub: a universal platform for integrating and analyzing substrates secreted by Gram-negative bacteria.

Nucleic Acids Res 2021 01;49(D1):D651-D659

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, VIC 3800, Australia.

Gram-negative bacteria utilize secretion systems to export substrates into their surrounding environment or directly into neighboring cells. These substrates are proteins that function to promote bacterial survival: by facilitating nutrient collection, disabling competitor species or, for pathogens, to disable host defenses. Following a rapid development of computational techniques, a growing number of substrates have been discovered and subsequently validated by wet lab experiments. To date, several online databases have been developed to catalogue these substrates but they have limited user options for in-depth analysis, and typically focus on a single type of secreted substrate. We therefore developed a universal platform, BastionHub, that incorporates extensive functional modules to facilitate substrate analysis and integrates the five major Gram-negative secreted substrate types (i.e. from types I-IV and VI secretion systems). To our knowledge, BastionHub is not only the most comprehensive online database available, it is also the first to incorporate substrates secreted by type I or type II secretion systems. By providing the most up-to-date details of secreted substrates and state-of-the-art prediction and visualized relationship analysis tools, BastionHub will be an important platform that can assist biologists in uncovering novel substrates and formulating new hypotheses. BastionHub is freely available at http://bastionhub.erc.monash.edu/.
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http://dx.doi.org/10.1093/nar/gkaa899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778982PMC
January 2021

AcrDB: a database of anti-CRISPR operons in prokaryotes and viruses.

Nucleic Acids Res 2021 01;49(D1):D622-D629

Nebraska Food for Health Center, Department of Food Science and Technology, University of Nebraska - Lincoln, Lincoln, NE 68588, USA.

CRISPR-Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR-Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR-Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.
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http://dx.doi.org/10.1093/nar/gkaa857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778997PMC
January 2021

Forensic genomics of a novel type from a neonatal intensive care unit in China reveals patterns of colonization, evolution and epidemiology.

Microb Genom 2020 10;6(10)

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia.

During March 2017, a neonatal patient with severe diarrhoea subsequently developed septicaemia and died, with isolated as the causative microorganism. In keeping with infection control protocols, the coincident illness of an attending staff member and three other neonates with infection triggered an outbreak response, leading to microbiological assessment of isolates collected from the staff member and all 21 co-housed neonates. Multilocus sequence typing and genomic sequencing identified that the isolates from the 21 neonates were of a new sequence type, ST2727, and taxonomically belonged to subsp. (formerly referred to as KpIIB). Genomic characterization showed that the isolated ST2727 strains had diverged from other subsp. strains at least 90 years ago, whereas the neonatal samples were highly similar with a genomic divergence of 3.6 months. There was no relationship to the isolate from the staff member. This demonstrates that no transmission occurred from staff to patient or between patients. Rather, the data suggest that ST2727 colonized each neonate from a common hospital source. Sequence-based analysis of the genomes revealed several genes for antimicrobial resistance and some virulence features, but suggest that ST2727 is neither extremely-drug resistant nor hypervirulent. Our results highlight the clinical significance and genomic properties of ST2727 and urge genome-based measures be implemented for diagnostics and surveillance within hospital environments. Additionally, the present study demonstrates the need to scale the power of genomic analysis in retrospective studies where relatively few samples are available.
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http://dx.doi.org/10.1099/mgen.0.000433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660260PMC
October 2020

Polymyxins Bind to the Cell Surface of Unculturable and Cause Unique Dependent Resistance.

Adv Sci (Weinh) 2020 Aug 8;7(15):2000704. Epub 2020 Jun 8.

Infection & Immunity Program Biomedicine Discovery Institute and Department of Microbiology Monash University Melbourne 3800 Australia.

Multidrug-resistant is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent strain possesses mutations in both (lipopolysaccharide biosynthesis) and (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance.
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http://dx.doi.org/10.1002/advs.202000704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403960PMC
August 2020

The architecture and stabilisation of flagellotropic tailed bacteriophages.

Nat Commun 2020 07 27;11(1):3748. Epub 2020 Jul 27.

Infection & Immunity Program, Biomedicine Discovery Institute & Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a β-hairpin loop, and interconnected along the tail by the splayed β-hairpins. By contrast, we show that the β-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.
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http://dx.doi.org/10.1038/s41467-020-17505-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385642PMC
July 2020

Formation and function of bacterial organelles.

Nat Rev Microbiol 2020 Jul 24. Epub 2020 Jul 24.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia.

Advances in imaging technologies have revealed that many bacteria possess organelles with a proteomically defined lumen and a macromolecular boundary. Some are bound by a lipid bilayer (such as thylakoids, magnetosomes and anammoxosomes), whereas others are defined by a lipid monolayer (such as lipid bodies), a proteinaceous coat (such as carboxysomes) or have a phase-defined boundary (such as nucleolus-like compartments). These diverse organelles have various metabolic and physiological functions, facilitating adaptation to different environments and driving the evolution of cellular complexity. This Review highlights that, despite the diversity of reported organelles, some unifying concepts underlie their formation, structure and function. Bacteria have fundamental mechanisms of organelle formation, through which conserved processes can form distinct organelles in different species depending on the proteins recruited to the luminal space and the boundary of the organelle. These complex subcellular compartments provide evolutionary advantages as well as enabling metabolic specialization, biogeochemical processes and biotechnological advances. Growing evidence suggests that the presence of organelles is the rule, rather than the exception, in bacterial cells.
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http://dx.doi.org/10.1038/s41579-020-0413-0DOI Listing
July 2020

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.

BMC Microbiol 2020 07 20;20(1):218. Epub 2020 Jul 20.

Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China.

Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment.

Results: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored bla and bla. The bla and bla genes were not detected in FK-2723 and FK-2820. bla, qnrB4, aac (6')-IIc, and bla, EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes bla-1, qnrB4, aac (6')-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate.

Conclusion: This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.
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http://dx.doi.org/10.1186/s12866-020-01898-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372807PMC
July 2020

DeepVF: a deep learning-based hybrid framework for identifying virulence factors using the stacking strategy.

Brief Bioinform 2020 Jun 29. Epub 2020 Jun 29.

Leiden Institute of Advanced Computer Science, Leiden University.

Virulence factors (VFs) enable pathogens to infect their hosts. A wealth of individual, disease-focused studies has identified a wide variety of VFs, and the growing mass of bacterial genome sequence data provides an opportunity for computational methods aimed at predicting VFs. Despite their attractive advantages and performance improvements, the existing methods have some limitations and drawbacks. Firstly, as the characteristics and mechanisms of VFs are continually evolving with the emergence of antibiotic resistance, it is more and more difficult to identify novel VFs using existing tools that were previously developed based on the outdated data sets; secondly, few systematic feature engineering efforts have been made to examine the utility of different types of features for model performances, as the majority of tools only focused on extracting very few types of features. By addressing the aforementioned issues, the accuracy of VF predictors can likely be significantly improved. This, in turn, would be particularly useful in the context of genome wide predictions of VFs. In this work, we present a deep learning (DL)-based hybrid framework (termed DeepVF) that is utilizing the stacking strategy to achieve more accurate identification of VFs. Using an enlarged, up-to-date dataset, DeepVF comprehensively explores a wide range of heterogeneous features with popular machine learning algorithms. Specifically, four classical algorithms, including random forest, support vector machines, extreme gradient boosting and multilayer perceptron, and three DL algorithms, including convolutional neural networks, long short-term memory networks and deep neural networks are employed to train 62 baseline models using these features. In order to integrate their individual strengths, DeepVF effectively combines these baseline models to construct the final meta model using the stacking strategy. Extensive benchmarking experiments demonstrate the effectiveness of DeepVF: it achieves a more accurate and stable performance compared with baseline models on the benchmark dataset and clearly outperforms state-of-the-art VF predictors on the independent test. Using the proposed hybrid ensemble model, a user-friendly online predictor of DeepVF (http://deepvf.erc.monash.edu/) is implemented. Furthermore, its utility, from the user's viewpoint, is compared with that of existing toolkits. We believe that DeepVF will be exploited as a useful tool for screening and identifying potential VFs from protein-coding gene sequences in bacterial genomes.
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http://dx.doi.org/10.1093/bib/bbaa125DOI Listing
June 2020

Characterization of the Core Ribosomal Binding Region for the Oxazolidone Family of Antibiotics Using Cryo-EM.

ACS Pharmacol Transl Sci 2020 Jun 13;3(3):425-432. Epub 2020 May 13.

Drug and Development Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, 3052 Victoria, Australia.

Linezolid and tedizolid are oxazolidinones with established clinical utility for the treatment of Gram-positive pathogens. Over time it has become apparent that even modest structural changes to the core phenyl oxazolidinone leads to drastic changes in biological activity. Consequently, the structure-activity relationship around the core oxazolidinone is constantly evolving, often reflected with new structural motifs present in nascent oxazolidinones. Herein we describe the use of cryo-electron microscopy to examine the differences in binding of several functionally different oxazolidinones in the hopes of enhanced understanding of their SAR. Tedizolid, radezolid, T145, and contezolid have been examined within the peptidyl transferase center (PTC) of the 50S ribosomal subunit from methicillin resistant . The ribosome-antibiotic complexes were resolved to a resolution of around 3 Å enabling unambiguous assignment of how each antibiotic interacts with the PTC.
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http://dx.doi.org/10.1021/acsptsci.0c00041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296538PMC
June 2020

Mapping bacterial effector arsenals: in vivo and in silico approaches to defining the protein features dictating effector secretion by bacteria.

Curr Opin Microbiol 2020 10 5;57:13-21. Epub 2020 Jun 5.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, 3800 Victoria, Australia. Electronic address:

Many bacterial pathogens rely on dedicated secretion systems to translocate virulence proteins termed 'effectors' into host cells. These effectors engage and manipulate host cellular functions to support bacterial colonization and propagation. The secretion systems are molecular machines that recognize targeting 'features' in these effector proteins in vivo to selectively and efficiently secrete them. The joint analysis of whole genome sequencing data and computational predictions of amino acid characteristics of effector proteins has made available extensive lists of candidate effectors for many bacterial pathogens, among which Dot/Icm type IVB secretion system in Legionella pneumophila reigns with the largest number of effectors identified to-date. This system is also used by the causative agent of Q fever, Coxiella burnetii, to secrete a large pool of distinct effectors. By comparing these two pathogens, we provide an understanding of the rationale behind effector repertoire expansion. We will also discuss recent bioinformatic advances facilitating high-throughput discovery of secreted effectors through in silico 'feature' recognition, and the current challenge to substantiate the biological relevance and bona fide nature of effectors identified in silico.
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http://dx.doi.org/10.1016/j.mib.2020.04.002DOI Listing
October 2020

Hyperosmotic Infusion and Oxidized Surfaces Are Essential for Biofilm Formation of From the Neonatal Intensive Care Unit.

Front Microbiol 2020 13;11:920. Epub 2020 May 13.

Infection and Immunity Theme, Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

is an opportunistic pathogen often implicated in bloodstream infections in the neonatal intensive care unit (NICU). This is assisted by its ability to form biofilms on indwelling central venous catheters (CVC), which are highly resistant to antibiotics and the immune system. We sought to understand the fundamentals of biofilm formation by in the NICU, using seventeen clinical isolates including the endemic NRCS-A clone and assessing nine commercial and two modified polystyrene surfaces. clinical isolates from the NICU initiated biofilm formation only in response to hyperosmotic conditions, followed by a developmental progression driven by expression to establish mature biofilms, with polysaccharide being their major extracellular polymer substance (EPS) matrix component. Physicochemical features of the biomaterial surface, and in particular the level of the element oxygen present on the surface, significantly influenced biofilm development of . A lack of highly oxidized carbon species on the surface prevented the immobilization of EPS and the formation of mature biofilms. This information provides guidance in regard to the preparation of hyperosmolar total parenteral nutrition and the engineering of CVC surfaces that can minimize the risk of catheter-related bloodstream infections caused by in the NICU.
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http://dx.doi.org/10.3389/fmicb.2020.00920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237634PMC
May 2020

PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins.

Nucleic Acids Res 2020 07;48(W1):W348-W357

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, VIC 3800, Australia.

Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR-Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR-Cas machineries for the development of new CRISPR-Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.
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http://dx.doi.org/10.1093/nar/gkaa432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319593PMC
July 2020

Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane.

Biochim Biophys Acta Biomembr 2020 09 4;1862(9):183317. Epub 2020 May 4.

Department of Materials Science and Engineering, Monash University, Clayton, VIC 3800, Australia; Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia; Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, Australia. Electronic address:

In Gram-negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.
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http://dx.doi.org/10.1016/j.bbamem.2020.183317DOI Listing
September 2020

The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

Acta Crystallogr D Struct Biol 2020 May 27;76(Pt 5):484-495. Epub 2020 Apr 27.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia.

The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.
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http://dx.doi.org/10.1107/S2059798320004398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193533PMC
May 2020

Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae.

mBio 2020 04 14;11(2). Epub 2020 Apr 14.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, Australia

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like β-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for β-lactam permeation. Sequence similarity network aalysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant (CRKP). The outer membrane of forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of , demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and β-lactamase inhibitors could be effective on porin-deficient Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for infections, given the way porin expression will be modulated to confer phage-resistant-and collateral drug-resistant-phenotypes in .
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http://dx.doi.org/10.1128/mBio.00603-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157821PMC
April 2020

Dynamic Epidemiology and Virulence Characteristics of Carbapenem-Resistant in Wenzhou, China from 2003 to 2016.

Infect Drug Resist 2020 31;13:931-940. Epub 2020 Mar 31.

Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People's Republic of China.

Purpose: To investigate transitions in resistance mechanisms, virulence characteristics and molecular epidemiology of carbapenem-resistant (CRKP) during 2003-2016 in a major Eastern Chinese medical center.

Patients And Methods: From a total of 2299 clinical strains collected from 2003 to 2016, 214 were found to be CRKP isolates and were selected for further study. Characterization of these was conducted by molecular detection of antibiotic resistance markers and virulence determinants, modified carbapenem inactivation method and multilocus sequence typing (MLST).

Results: In this study, the prevalence of CRKP was increasing over the 14-year period, mirroring a national trend. These CRKP strains were resistant to most of the tested, clinically relevant drugs. The majority of these CRKP strains were positive for carbapenemases, with the carbapenemase (KPC) found to be the dominant type (207/210, 98.6%). The carrier rates of virulence genes and increased in 2016, while the  and showed a relatively constant trend. From MLST data, ST11 (88.8%, 190/214) was the preponderant sequence type (ST), followed by ST15 (1.9%, 4/214) and ST656 (1.4%, 3/214). Several strains with less common STs (ST690, ST895, ST1823 and ST1384) were also detected, and these too showed high levels of antimicrobial resistance.

Conclusion: The average national rise in CRKP across China is mirrored in this in-depth analysis of a single hospital, while the prevalence of hypervirulent CRKP (such as ST15) was relatively low as of 2016. Continuous monitoring is necessary to keep track of CRKP and should include the prospect of newly emerging strains with less common STs and the prospect of detecting carbapenem-resistant, carbapenemase-negative .
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http://dx.doi.org/10.2147/IDR.S243032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128075PMC
March 2020

Investigation of LuxS-mediated quorum sensing in .

J Med Microbiol 2020 Mar;69(3):402-413

Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, PR China.

Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation. To investigate the role of in biofilm formation and gene expression in the nosocomial pathogen . A gene deletion was made in KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and strains grown in media supplemented with different carbohydrates. Potential roles of in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide ( and ), polysaccharide (), and type 3 fimbriae () synthesis genes in wild-type and mutant biofilm extracts. AI-2 production was dependent on the presence of . AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and mutant using a microtiter plate assay were observed. In mutant biofilm extracts, the expression of was down-regulated, and the expression of , which encodes a porin for poly-β-1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in . These inter-connected processes could be important for bacterial group behaviour and persistence.
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http://dx.doi.org/10.1099/jmm.0.001148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377169PMC
March 2020

The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism.

J Biol Chem 2019 12 11;294(51):19523-19534. Epub 2019 Nov 11.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, 3800 Victoria, Australia.

The ferric iron uptake (Fiu) transporter from functions in the transport of iron-catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1-2.9 Å and analyzed its function in Through analysis of the Fiuo crystal structure, in combination with docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.
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http://dx.doi.org/10.1074/jbc.RA119.011018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926462PMC
December 2019

The multifunctional enzyme S-adenosylhomocysteine/methylthioadenosine nucleosidase is a key metabolic enzyme in the virulence of Salmonella enterica var Typhimurium.

Biochem J 2019 11;476(22):3435-3453

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.
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http://dx.doi.org/10.1042/BCJ20190297DOI Listing
November 2019

Protease-associated import systems are widespread in Gram-negative bacteria.

PLoS Genet 2019 10 15;15(10):e1008435. Epub 2019 Oct 15.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.
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http://dx.doi.org/10.1371/journal.pgen.1008435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6793856PMC
October 2019

Structure of the mitochondrial import gate reveals distinct preprotein paths.

Nature 2019 11 10;575(7782):395-401. Epub 2019 Oct 10.

Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes. Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.
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http://dx.doi.org/10.1038/s41586-019-1680-7DOI Listing
November 2019

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi-like protein tail fibre.

Mol Microbiol 2019 12 9;112(6):1831-1846. Epub 2019 Oct 9.

Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, 3800, Australia.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.
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http://dx.doi.org/10.1111/mmi.14396DOI Listing
December 2019

An Outbreak of Carbapenem-Resistant and Hypervirulent in an Intensive Care Unit of a Major Teaching Hospital in Wenzhou, China.

Front Public Health 2019 19;7:229. Epub 2019 Aug 19.

School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, China.

Carbapenem-resistant, hypervirulent (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), -macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% ( = 8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase . Most isolates had hypervirulent genotypes being positive for 19 virulence-associated genes, including (25/29, 86.2%), (27/29, 93.1%), (27/29, 93.1%), (29/29, 100%), (29/29, 100%), and (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the model with the highest LD values for three isolates (<10 CFU/mL) demonstrating the degree of hypervirulence of these CR-hvKP isolates, and is discussed relative to previous outbreaks of CR-hvKP.
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http://dx.doi.org/10.3389/fpubh.2019.00229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736603PMC
August 2019

PeNGaRoo, a combined gradient boosting and ensemble learning framework for predicting non-classical secreted proteins.

Bioinformatics 2020 02;36(3):704-712

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, VIC 3800, Australia.

Motivation: Gram-positive bacteria have developed secretion systems to transport proteins across their cell wall, a process that plays an important role during host infection. These secretion mechanisms have also been harnessed for therapeutic purposes in many biotechnology applications. Accordingly, the identification of features that select a protein for efficient secretion from these microorganisms has become an important task. Among all the secreted proteins, 'non-classical' secreted proteins are difficult to identify as they lack discernable signal peptide sequences and can make use of diverse secretion pathways. Currently, several computational methods have been developed to facilitate the discovery of such non-classical secreted proteins; however, the existing methods are based on either simulated or limited experimental datasets. In addition, they often employ basic features to train the models in a simple and coarse-grained manner. The availability of more experimentally validated datasets, advanced feature engineering techniques and novel machine learning approaches creates new opportunities for the development of improved predictors of 'non-classical' secreted proteins from sequence data.

Results: In this work, we first constructed a high-quality dataset of experimentally verified 'non-classical' secreted proteins, which we then used to create benchmark datasets. Using these benchmark datasets, we comprehensively analyzed a wide range of features and assessed their individual performance. Subsequently, we developed a two-layer Light Gradient Boosting Machine (LightGBM) ensemble model that integrates several single feature-based models into an overall prediction framework. At this stage, LightGBM, a gradient boosting machine, was used as a machine learning approach and the necessary parameter optimization was performed by a particle swarm optimization strategy. All single feature-based LightGBM models were then integrated into a unified ensemble model to further improve the predictive performance. Consequently, the final ensemble model achieved a superior performance with an accuracy of 0.900, an F-value of 0.903, Matthew's correlation coefficient of 0.803 and an area under the curve value of 0.963, and outperforming previous state-of-the-art predictors on the independent test. Based on our proposed optimal ensemble model, we further developed an accessible online predictor, PeNGaRoo, to serve users' demands. We believe this online web server, together with our proposed methodology, will expedite the discovery of non-classically secreted effector proteins in Gram-positive bacteria and further inspire the development of next-generation predictors.

Availability And Implementation: http://pengaroo.erc.monash.edu/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz629DOI Listing
February 2020