ORCID Profile
0000-0001-8032-2154
Current Organisation
University of Melbourne
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Publisher: Frontiers Media SA
Date: 19-10-2020
Publisher: American Society for Microbiology
Date: 19-05-2022
DOI: 10.1128/IAI.00614-21
Abstract: The modulation of programmed cell death (PCD) processes during bacterial infections is an evolving arms race between pathogens and their hosts. The initiation of apoptosis, necroptosis, and pyroptosis pathways are essential to immunity against many intracellular and extracellular bacteria.
Publisher: Frontiers Media SA
Date: 17-12-2019
Publisher: eLife Sciences Publications, Ltd
Date: 14-06-2022
DOI: 10.7554/ELIFE.77195
Abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues and is exposed to new selective pressures, thus, potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation however, a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2590 S . aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr -mediated adaptation, we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1 . The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
Publisher: Microbiology Society
Date: 31-10-2022
Abstract: Bacterial type VI secretion systems (T6SSs) are contractile nanomachines that deliver proteinic substrates into target prokaryotic or eukaryotic cells and the surrounding milieu. The genus C ylobacter encompasses 39 recognized species and 13 subspecies, with many belonging to a group known as ‘emerging C ylobacter pathogens’. Within C ylobacter , seven species have been identified to harbour a complete T6SS cluster but have yet to be comparatively assessed. In this study, using systematic bioinformatics approaches and the T6SS-positive C ylobacter jejuni 488 strain as a reference, we explored the genus-wide prevalence, similarity and make-up of the T6SS amongst 372 publicly available ‘complete’ C ylobacter genomes. Our analyses predict that approximately one-third of C ylobacter species possess a T6SS. We also putatively report the first identification of a T6SS in four species: C ylobacter cuniculorum, C ylobacter helveticus, C ylobacter armoricus and C ylobacter ornithocola . The C ylobacter T6SSs cluster into three distinct organizations (I–III), of which two break down into further variants. Thirty T6SS-containing genomes were found to harbour more than one vgrG gene, with C ylobacter lari strain NCTC 11845 possessing five. Analysis of the C. jejuni Pathogenicity Island-1 confirmed its conservation amongst T6SS-positive C. jejuni strains, as well as highlighting its erse genetic composition, including additional putative effector–immunity pairs (e.g. PoNe and DUF1911 domains). Effector–immunity pairs were also observed neighbouring vgrG s in several other C ylobacter species, in addition to putative genes encoding nucleases, lysozymes, ATPases and a ferric ATP-binding cassette uptake system. These observations highlight the erse genetic make-up of the T6SS within C ylobacter and provide further evidence of its role in pathogenesis.
Publisher: eLife Sciences Publications, Ltd
Date: 13-04-2023
Publisher: American Society for Microbiology
Date: 16-10-2023
Publisher: Wiley
Date: 04-2010
Publisher: Elsevier BV
Date: 03-2008
DOI: 10.1016/J.MICINF.2007.11.011
Abstract: Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.
Publisher: eLife Sciences Publications, Ltd
Date: 17-05-2022
Publisher: American Society for Microbiology
Date: 07-2002
DOI: 10.1128/JB.184.13.3433-3441.2002
Abstract: Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK . The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.
Publisher: Oxford University Press (OUP)
Date: 26-03-2010
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that affects immunocompromised in iduals and causes life-threatening infections in cystic fibrosis (CF) patients. Colonization of CF lung by P. aeruginosa involves a biofilm mode of growth, which is promoted by the production of exopolysaccharides. These polymers are essential components of the extracellular biofilm matrix. P. aeruginosa possesses several clusters contributing to the formation of the matrix, including the pel or psl genes. In the present study, we identified anionic cyclic glucans produced by P. aeruginosa, which are associated with the matrix of strains PAKDeltaretS and PA14. Their structure has been elucidated using chemical analysis, 1- and 2D nuclear magnetic resonance techniques and mass spectrometry. They belong to a family of cyclic beta-(1-->3)-linked glucans of 12-16 glucose residues with 30-50% of glucose units substituted by 1-phosphoglycerol at O-6. These glucans were also recovered in pel mutant strains, which indicated that their biosynthesis was pel independent. In an effort to understand the biogenesis of these glucans, we analyzed the matrix components of a previously characterized P. aeruginosa PA14 mutant, the PA14::ndvB mutant strain. The ndvB gene was predicted to be involved in the synthesis of perisplasmic glucans, capable of physically interacting with aminoglycoside antibiotics. We revealed that the highly glycerol-phosphorylated beta-(1-->3)-glucans are lacking in the ndvB mutant, and we showed that these glucans are capable of direct binding with the aminoglycoside antibiotic kanamycin. This observation fills a gap in our understanding of the relationship between biofilm, cyclic glucans and high-level antibiotic resistance.
Publisher: Elsevier BV
Date: 06-2014
Publisher: American Society for Microbiology
Date: 20-05-2020
DOI: 10.1128/IAI.00913-19
Abstract: The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication.
Publisher: MDPI AG
Date: 13-01-2022
DOI: 10.3390/IJMS23020835
Abstract: The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue s les.
Publisher: Springer Science and Business Media LLC
Date: 27-01-2023
Publisher: Frontiers Media SA
Date: 29-06-2021
DOI: 10.3389/FMICB.2021.694824
Abstract: The Type VI Secretion System (T6SS) has important roles relating to bacterial antagonism, subversion of host cells, and niche colonisation. C ylobacter jejuni is one of the leading bacterial causes of human gastroenteritis worldwide and is a commensal coloniser of birds. Although recently discovered, the T6SS biological functions and identities of its effectors are still poorly defined in C. jejuni . Here, we perform a comprehensive bioinformatic analysis of the C. jejuni T6SS by investigating the prevalence and genetic architecture of the T6SS in 513 publicly available genomes using C. jejuni 488 strain as reference. A unique and conserved T6SS cluster associated with the C ylobacter jejuni Integrated Element 3 (CJIE3) was identified in the genomes of 117 strains. Analyses of the T6SS-positive 488 strain against the T6SS-negative C. jejuni RM1221 strain and the T6SS-positive plasmid pCJDM202 carried by C. jejuni WP2-202 strain defined the “T6SS-containing CJIE3” as a pathogenicity island, thus renamed as C ylobacter jejuni Pathogenicity Island-1 (CJPI-1). Analysis of CJPI-1 revealed two canonical VgrG homologues, CJ488_0978 and CJ488_0998, harbouring distinct C-termini in a genetically variable region downstream of the T6SS operon. CJPI-1 was also found to carry a putative DinJ-YafQ Type II toxin-antitoxin (TA) module, conserved across pCJDM202 and the genomic island CJIE3, as well as several open reading frames functionally predicted to encode for nucleases, lipases, and peptidoglycan hydrolases. This comprehensive in silico study provides a framework for experimental characterisation of T6SS-related effectors and TA modules in C. jejuni .
Publisher: Elsevier BV
Date: 10-2019
Publisher: Elsevier BV
Date: 08-2012
Publisher: MDPI AG
Date: 02-03-2022
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which was classified as a pandemic in March 2020. As of 22 January 2022, globally more than 347 million cases of COVID-19 have been diagnosed, with 5.6 million deaths, making it the deadliest pandemic since the influenza pandemic in 1918. The clinical presentation of COVID-19-related illness spans from asymptomatic to mild respiratory symptoms akin to influenza infection to acute symptoms, including pneumonia necessitating hospitalisation and admission to intensive care units. COVID-19 starts in the upper respiratory tract and lungs but in severe cases can also involve the heart, blood vessels, brain, liver, kidneys and intestine. The increasing global health and economic burden of COVID-19 necessitates an urgent and global response. Understanding the functional characteristics and cellular tropism of SARS-CoV-2, and the pathogenesis that leads to multi-organ failure and death, has prompted an unprecedented adoption of organoid models. Successful drug discovery and vaccine development rely on pre-clinical models that faithfully recapitulate the viral life cycle and the host cell response to infection. Human stem cell-derived organoids fulfill these criteria. Here we highlight the role of organoids in the study of SARS-CoV-2 infection and modelling of COVID-19 pathogenesis.
Publisher: American Society for Microbiology
Date: 07-2015
Abstract: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first ex le of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa . We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first ex le of a bacterial protein interacting with the γTuRC.
Publisher: Rockefeller University Press
Date: 21-09-2022
Abstract: MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B–related metabolite antigens from erse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.
Publisher: Elsevier BV
Date: 07-2022
Publisher: Microbiology Society
Date: 2014
Abstract: The type III secretion apparatus (T3SA) is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri T3SA spans the bacterial envelope and its assembly requires the products of ~20 mxi and spa genes. Despite progress made in understanding how the T3SA is assembled, the role of several predicted soluble components, such as Spa13, remains elusive. Here, we show that the secretion defect of the spa13 mutant is associated with lack of T3SA assembly which is partly due to the instability of the needle component MxiH. In contrast to its Yersinia counterpart, Spa13 is not a secreted protein. We identified a network of interactions between Spa13 and the ATPase Spa47, the C-ring protein Spa33, and the inner-membrane protein Spa40. Moreover, we revealed a Spa13 interaction with the inner-membrane MxiA and showed that overexpression of the large cytoplasmic domain of MxiA in the WT background shuts off secretion. Lastly, we demonstrated that Spa13 interacts with the cleaved form of Spa40 and with the translocator chaperone IpgC, suggesting that Spa13 intervenes during the secretion hierarchy switch process. Collectively, our results support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch.
Publisher: Elsevier BV
Date: 05-2022
Publisher: Springer Science and Business Media LLC
Date: 19-07-2013
DOI: 10.1038/NI0813-877D
Publisher: Hindawi Limited
Date: 24-02-2021
DOI: 10.1111/CMI.13317
Abstract: Staphylococcus aureus is a major opportunistic human pathogen that is globally prevalent. Although S. aureus and humans may have co-evolved to the point of commensalism, the bacterium is equipped with virulence factors causing devastating infections. The adoption of an intracellular lifestyle by S. aureus is an important facet of its pathogenesis. Occupying a privileged intracellular compartment permits evasion from the bactericidal actions of host immunity and antibiotics. However, this localization exposes S. aureus to cell-intrinsic processes comprising autophagy, metabolic challenges and clearance mechanisms orchestrated by host programmed cell death pathways (PCDs), including apoptosis, pyroptosis and necroptosis. Mounting evidence suggests that S. aureus deploys pathoadaptive mechanisms that modulate the expression of its virulence factors to prevent elimination through PCD pathways. In this review, we critically analyse the current literature on the interplay between S. aureus virulence factors with the key, intertwined nodes of PCD. We discuss how S. aureus adaptation to the human host plays an essential role in the evasion of PCD, and we consider future directions to study S. aureus-PCD interactions.
Publisher: Elsevier BV
Date: 04-2010
DOI: 10.1016/J.BIOCHI.2009.12.013
Abstract: Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2023
DOI: 10.1101/2023.02.27.530350
Abstract: Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell ision. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ) translation (rplK ) DNA compaction ( hup ) initiation of DNA replication ( dnaA, hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.
Publisher: Elsevier BV
Date: 07-2014
Publisher: eLife Sciences Publications, Ltd
Date: 08-06-2023
DOI: 10.7554/ELIFE.84778
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model , we have developed a platform called InToxSa ( in tracellular tox icity of S. a ureus ) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S . aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA , encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: Elsevier BV
Date: 04-2011
Publisher: Public Library of Science (PLoS)
Date: 10-01-2022
DOI: 10.1371/JOURNAL.PPAT.1010166
Abstract: A hallmark of Listeria (L . ) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L . monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L . monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria -containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L . monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L . monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L . monocytogenes in macrophages is not inevitably tied to bacterial persistence.
Publisher: American Society for Microbiology
Date: 20-02-2019
DOI: 10.1128/IAI.00534-19
Abstract: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella -containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes.
Publisher: Cold Spring Harbor Laboratory
Date: 12-02-2022
DOI: 10.1101/2022.02.11.480068
Abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr -mediated adaptation we identified non-canonical, genome-wide significant loci including sucA - sucB and stp1 . The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
Publisher: Springer Science and Business Media LLC
Date: 18-03-2023
DOI: 10.1038/S41467-023-37200-W
Abstract: Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli , Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes . Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
Publisher: American Society for Microbiology
Date: 15-02-2014
DOI: 10.1128/JB.00863-13
Abstract: The type VI secretion system (T6SS) of Gram-negative bacteria has been involved in various processes, notably bacterial competition and eukaryotic cell subversion. Most Pseudomonas aeruginosa strains possess three T6SS gene clusters, but only the function of the first T6SS (H1-T6SS) has been clearly elucidated. It is involved in the secretion of three toxins (Tse1 to -3) that target bacterial competitors. In the case of the H2- and H3-T6SS, no clear function has been assigned, and only one effector has been associated with these systems. Yet the H2-T6SS was proposed to promote P. aeruginosa internalization in nonphagocytic epithelial cells. Although the H2-T6SS genetic organization is conserved across P. aeruginosa isolates, one feature is the presence of an additional transcriptional unit in the PA14 strain H2-T6SS cluster, which is ergent from the core H2-T6SS genes. A specific set of four genes encodes an Hcp protein (Hcp2), a VgrG protein (VgrG14), an Rhs element (PA14_43100 or RhsP2), and a protein with no homologies with previously characterized proteins (PA14_43090). In this study, we engineered a P. aeruginosa PA14 strain carrying an arabinose-inducible H2-T6SS on the chromosome. We showed that arabinose induction readily promotes assembly of the H2-T6SS, as seen by monitoring Hcp2 secretion. We further studied the secretion fate of VgrG14 and RhsP2, but these were not detectable in the extracellular medium. We finally investigated whether activation of the PA14 H2-T6SS gene cluster could influence phenotypic traits such as internalization in eukaryotic cells, and we reported noteworthy differences compared to strain PAO1, which may be accounted for by the described genetic differences.
Publisher: Wiley
Date: 24-10-2006
DOI: 10.1111/J.1365-2958.2006.05456.X
Abstract: During transcription, series of approximately 9 As or Ts can direct RNA polymerase to incorporate into the mRNA nucleotides not encoded by the DNA, changing the reading frame downstream from the slippage site. We detected series of 9 or 10 As in spa13, spa33 and mxiA encoding type III secretion apparatus components. Analysis of cDNAs indicated that transcriptional slippage occurs in spa13, mxiA and spa33. Changes in the reading frame were confirmed by using plasmids carrying slippage sites in the 5' part of lacZ. Slippage is required for production of Spa13 from two overlapping reading frames and should lead to production of truncated MxiA and Spa33 proteins. Complementation of spa13 and mxiA mutants with plasmids carrying altered sites indicated that slippage in spa13 is required for assembly of the secretion apparatus and that slippage sites in spa13 and mxiA have not been selected to encode Lys residues or to produce two proteins endowed with different activities. The presence of slippage sites decreases production of Spa13 by 70%, of MxiA and Spa33 by 15% and of Spa32 (encoded downstream from spa13) by 50%. These results suggest that transcriptional slippage controls protein production by reducing the proportion of mRNA translated into functional proteins.
Publisher: Frontiers Media SA
Date: 16-11-2021
Publisher: MyJove Corporation
Date: 20-03-2013
DOI: 10.3791/50103
Publisher: Cold Spring Harbor Laboratory
Date: 22-04-2022
DOI: 10.1101/2022.04.21.22273941
Abstract: Outcomes for patients with severe bacterial infections are determined by the interplay between host, pathogen, and treatments. Most notably, patient age and antibiotic resistance contributes significantly to poor outcomes. While human genomics studies have provided insights into the host genetic factors impacting outcomes of Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the idea that bacterial pathoadaptation is a key driver of clinical outcomes, we develop a new genome-wide association study (GWAS) framework to identify adaptive bacterial mutations associated with clinical treatment failure and mortality in three large and independent S. aureus bacteraemia cohorts, comprising 1358 episodes. We discovered S. aureus loci with previously undescribed convergent mutations linked to both poorer infection outcomes and reduced susceptibility to vancomycin. Our research highlights the potential of vancomycin-selected mutations and vancomycin MIC as key explanatory variables to predict SAB severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability 5%), however, GWAS allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine-learning, we were able to quantify the predictive potential of these adaptive S. aureus signatures, along with host determinants of bacteraemia outcomes. The statistical genomics framework we have developed is a powerful means to capture adaptive mutations and find bacterial factors that influence and predict severe infections. Our findings underscore the importance of systematically collected, rich clinical and microbiological data to understand bacterial mechanisms promoting treatment failure.
Publisher: Cold Spring Harbor Laboratory
Date: 13-12-2022
DOI: 10.1101/2022.12.11.519971
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa ( In tracellular Tox icity of S. a ureus ) to quantify intracellula cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: Elsevier BV
Date: 02-2016
DOI: 10.1016/J.MIB.2015.11.006
Abstract: Secretion systems play a central role in infectious diseases by enabling pathogenic bacteria to deliver virulence factors into target cells. The type VI secretion system (T6SS) mediates bacterial antagonism in various environments including eukaryotic niches, such as the gut. This molecular machine injects lethal toxins directly in target bacterial cells. It provides an advantage to pathogens encountering the commensal flora of the host and indirectly contributes to colonization and persistence. Yet, the T6SS is not employed for the sole purpose of bacterial killing and several T6SS effectors are dedicated to the subversion of eukaryotic cells. As described for type III and type IV secretion systems, these effectors impede host cell functions and promote immune evasion, thereby enabling successful infection.
Publisher: Springer Science and Business Media LLC
Date: 30-09-2012
DOI: 10.1038/NI.2426
Publisher: Proceedings of the National Academy of Sciences
Date: 16-09-2019
Abstract: Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, h ered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rif icin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read licon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.
Publisher: Microbiology Society
Date: 06-2008
DOI: 10.1099/MIC.0.2008/016840-0
Abstract: Several secretion systems have evolved that are widespread among Gram-negative bacteria. Recently, a new secretion system was recognized, which is named the type VI secretion system (T6SS). The T6SS components are encoded within clusters of genes initially identified as IAHP for IcmF-associated homologous proteins, since they were all found to contain a gene encoding an IcmF-like component. IcmF was previously reported as a component of the type IV secretion system (T4SS). However, with the exception of DotU, other T4SS components are not encoded within T6SS loci. Thus, the T6SS is probably a novel kind of complex multi-component secretion machine, which is often involved in interaction with eukaryotic hosts, be it a pathogenic or a symbiotic relationship. The expression of T6SS genes has been reported to be mostly induced in vivo. Interestingly, expression and assembly of T6SSs are tightly controlled at both the transcriptional and the post-translational level. This may allow a timely control of T6SS assembly and function. Two types of proteins, generically named Hcp and VgrG, are secreted via these systems, but it is not entirely clear whether they are truly secreted effector proteins or are actually components of the T6SS. The precise role and mode of action of the T6SS is still unknown. This review describes current knowledge about the T6SS and summarizes its hallmarks and its differences from other secretion systems.
Publisher: American Society for Microbiology
Date: 15-04-2007
DOI: 10.1128/JB.01677-06
Abstract: Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (Δ fliC ) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (Δ motAB ) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.
Location: No location found
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2022
End Date: 2023
Funder: Cass Foundation
View Funded ActivityStart Date: 2021
End Date: 2022
Funder: University of Melbourne
View Funded ActivityStart Date: 2015
End Date: 2018
Funder: European Commission
View Funded Activity