ORCID Profile
0000-0003-2385-8192
Current Organisation
University College Cork
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Publisher: Wiley
Date: 18-02-2009
DOI: 10.1111/J.1365-2958.2008.06589.X
Abstract: The Listeria monocytogenes Agr peptide-sensing system has been analysed by creating a deletion mutant in agrD, the structural gene for the putative quorum-sensing peptide. The DeltaagrD mutant displayed significantly reduced biofilm formation, a defect which could be restored by genetic or physical complementation. A reduced invasion of Caco-2 intestinal epithelial cells was observed for the DeltaagrD mutant while phagocytosis by THP-1 macrophages was unaffected. Additionally, the level of internalin A (InlA) in the cell wall was decreased in the DeltaagrD mutant. Expression profiling of virulence genes (hlyA, actA, plcA, prfA and inlA) identified a finely tuned regulation which resulted in an impaired virulence response in the DeltaagrD mutant. The mutant is also significantly attenuated for virulence in mice, as revealed by bioluminescent in vivo imaging. On day 3 post infection, systemic dissemination to livers and spleens had occurred for the wild type, whereas the DeltaagrD mutant remained localized to the liver. Microarray analysis identified 126 and 670 genes as significantly regulated in exponential and stationary phase respectively. The results presented here suggest that peptide sensing plays an important role in the biology of L. monocytogenes, with relevant phenotypes in both the saprophytic and parasitic lifecycles.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Springer Science and Business Media LLC
Date: 23-11-2022
Publisher: American Society for Microbiology
Date: 07-2008
DOI: 10.1128/AEM.00314-08
Abstract: We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogen Listeria monocytogenes . pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β- d -thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of “IPTG-dependent” mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essential secA gene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes . A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 × 10 6 and 6.7 × 10 6 CFU per μg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 × 10 7 CFU per μg is the highest efficiency reported thus far for L. monocytogenes F2365.
Publisher: Mary Ann Liebert Inc
Date: 04-2010
DOI: 10.1089/HUM.2009.022
Abstract: Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to icillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor s les ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic icillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and icillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.
Publisher: Springer Science and Business Media LLC
Date: 18-01-2019
Publisher: Informa UK Limited
Date: 24-11-2022
Publisher: American Society for Microbiology
Date: 11-2007
DOI: 10.1128/AEM.01394-07
Abstract: A novel vector has been constructed for the constitutive luminescent tagging of gram-negative bacteria by site-specific integration into the 16S locus of the bacterial chromosome. A number of gram-negative pathogens were successfully tagged using this vector, and the system was validated during murine infections of living animals.
Publisher: Microbiology Society
Date: 07-2012
Abstract: Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.
Publisher: Public Library of Science (PLoS)
Date: 12-05-2011
Publisher: BMJ
Date: 11-02-2019
DOI: 10.1136/GUTJNL-2018-317842
Abstract: Faecal microbiota transplant (FMT) effectively treats recurrent Clostridioides difficile infection (rCDI), but its mechanisms of action remain poorly defined. Certain bile acids affect C. difficile germination or vegetative growth. We hypothesised that loss of gut microbiota-derived bile salt hydrolases (BSHs) predisposes to CDI by perturbing gut bile metabolism, and that BSH restitution is a key mediator of FMT’s efficacy in treating the condition. Using stool collected from patients and donors pre-FMT ost-FMT for rCDI, we performed 16S rRNA gene sequencing, ultra performance liquid chromatography mass spectrometry (UPLC-MS) bile acid profiling, BSH activity measurement, and qPCR of bsh / bai CD genes involved in bile metabolism. Human data were validated in C. difficile batch cultures and a C57BL/6 mouse model of rCDI. From metataxonomics, pre-FMT stool demonstrated a reduced proportion of BSH-producing bacterial species compared with donors ost-FMT. Pre-FMT stool was enriched in taurocholic acid (TCA, a potent C. difficile germinant) TCA levels negatively correlated with key bacterial genera containing BSH-producing organisms. Post-FMT s les demonstrated recovered BSH activity and bsh / bai CD gene copy number compared with pretreatment (p .05). In batch cultures, supernatant from engineered bsh -expressing E. coli and naturally BSH-producing organisms ( Bacteroides ovatus, Collinsella aerofaciens, Bacteroides vulgatus and Blautia obeum ) reduced TCA-mediated C. difficile germination relative to culture supernatant of wild-type (BSH-negative) E. coli. C. difficile total viable counts were ~70% reduced in an rCDI mouse model after administration of E. coli expressing highly active BSH relative to mice administered BSH-negative E. coli (p .05). Restoration of gut BSH functionality contributes to the efficacy of FMT in treating rCDI.
Publisher: Springer Science and Business Media LLC
Date: 13-06-2008
Abstract: The foodborne, gram-positive pathogen, Listeria monocytogenes , is capable of causing lethal infections in compromised in iduals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays. We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex s les. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single s le. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies. This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological s les.
Publisher: American Society for Microbiology
Date: 30-06-2016
Abstract: Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis among humans and animals. The draft genome sequences of L. monocytogenes serotype 4b strains 944 and 2993 and serotype 1/2c strains 198 and 2932 are reported here.
Publisher: Springer Science and Business Media LLC
Date: 08-02-2019
Publisher: American Society for Microbiology
Date: 04-2006
DOI: 10.1128/AEM.72.4.2876-2884.2006
Abstract: In this paper we describe construction of a luciferase-based vector, pPL2 lux , and use of this vector to study gene expression in Listeria monocytogenes . pPL2 lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2 lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2 lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes .
Publisher: Springer Science and Business Media LLC
Date: 12-2010
Abstract: Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlA m murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m * . The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria -optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m * yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlA m strain. We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. This murinized L. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.JHEP.2014.06.025
Abstract: Despite the mortality associated with liver disease observed in patients with short bowel syndrome (SBS), mechanisms underlying the development of SBS-associated liver disease (SBS-ALD) are poorly understood. This study examines the impact of bacterially-mediated bile acid (BA) dysmetabolism on farnesoid X receptor (FXR) signalling pathways and clinical outcome in a piglet model of SBS-ALD. 4-week old piglets underwent 75% small bowel resection (SBR) or sham operation. Liver histology and hepatic inflammatory gene expression were examined. Abundance of BA biotransforming bacteria was determined and metabolomic studies detailed the alterations in BA composition of stool, portal serum and bile s les. Gene expression of intestinal and hepatic FXR target genes and small heterodimer partner (SHP) transrepression targets were assessed. Histological evidence of SBS-ALD included liver bile duct proliferation, hepatocyte ballooning and fibrosis. Inflammatory gene expression was increased. Microbiota changes included a 10-fold decrease in Clostridium and a two-fold decrease in Bacteroides in SBS-ALD piglets. BA composition was altered and reflected a primary BA dominant composition. Intestinal and hepatic regulation of BA synthesis was characterised by a blunted intestinal FXR activation response and a failure of SHP to repress key hepatic targets. We propose a pathological scenario in which microbial dysbiosis following SBR results in significant BA dysmetabolism and consequent outcomes including steatorrhoea, persistent diarrhoea and liver damage. Furthermore alterations in BA composition may have contributed to the observed disturbance in FXR-mediated signalling pathways. These findings provide an insight into the complex mechanisms mediating the development of liver disease in patients with SBS.
Publisher: American Society for Microbiology
Date: 05-2007
DOI: 10.1128/AEM.02940-06
Abstract: An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux -tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.
Publisher: Elsevier BV
Date: 07-2017
Publisher: Microbiology Society
Date: 04-2012
Abstract: Sensitive sensory mechanisms are instrumental in affording Pseudomonas aeruginosa the capacity to establish erse yet severe human infections, which can manifest themselves in long-term untreatable disease. The ability of P. aeruginosa to tightly regulate gene expression and virulence factor production, in response to activation of these sensory components, enables the pathogen to sustain infection despite the host immune response and aggressive antibiotic treatment. Although a number of factors are recognized as playing a role in early infection, very little is known regarding the sensors involved in this process. In this study, we identified P. aeruginosa PA3191 as a novel host-responsive sensor that plays a key role during P. aeruginosa-host interactions and is required for optimum colonization and dissemination in a mouse model of infection. We demonstrated that PA3191 contributed to modulation of the type III secretion system (T3SS) in response to host cells and T3SS-inducing conditions in vitro. PA3191 (designated GtrS) acted in concert with the response regulator GltR to regulate the OprB transport system and subsequently carbon metabolism. Through this signal transduction pathway, T3SS activation was mediated via the RsmAYZ regulatory cascade and involved the global anaerobic response regulator Anr.
Publisher: American Society for Microbiology
Date: 02-2008
DOI: 10.1128/IAI.01132-07
Abstract: The ability of Pseudomonas aeruginosa to cause a broad range of infections in humans is due, at least in part, to its adaptability and its capacity to regulate the expression of key virulence genes in response to specific environmental conditions. Multiple two-component response regulators have been shown to facilitate rapid responses to these environmental conditions, including the coordinated expression of specific virulence determinants. RsmA is a posttranscriptional regulatory protein which controls the expression of a number of virulence-related genes with relevance for acute and chronic infections. Many membrane-bound sensors, including RetS, LadS, and GacS, are responsible for the reciprocal regulation of genes associated with acute infection and chronic persistence. In P. aeruginosa this is due to sensors influencing the expression of the regulatory RNA RsmZ, with subsequent effects on the level of free RsmA. While interactions between an rsmA mutant and human airway epithelial cells have been examined in vitro, the role of RsmA during infection in vivo has not been determined yet. Here the function of RsmA in both acute and chronic models of infection was examined. The results demonstrate that RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia. Furthermore, while loss of RsmA results in reduced colonization during the initial stages of acute infection, the data show that mutation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice.
No related grants have been discovered for Cormac Gahan.