Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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

Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. 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

The Lantibiotic Lacticin 3147 Prevents Systemic Spread of Staphylococcus aureus in a Murine Infection Model

The objective of this study was to investigate the in vivo activity of the lantibiotic lacticin 3147 against the luminescent Staphylococcus aureus strain Xen 29 using a murine model. Female BALB/c mice (7 weeks old, 17 g) were divided into groups (n = 5) and infected with the Xen 29 strain via the intraperitoneal route at a dose of 1 × 106 cfu/animal. After 1.5 hr, the animals were treated subcutaneously with doses of phosphate-buffered saline (PBS; negative control) or lacticin 3147. Luminescent imaging was carried 3 and 5 hours postinfection. Mice were then sacrificed, and the levels of S. aureus Xen 29 in the liver, spleen, and kidneys were quantified. Notably, photoluminescence and culture-based analysis both revealed that lacticin 3147 successfully controlled the systemic spread of S. aureus in mice thus indicating that lacticin 3147 has potential as a chemotherapeutic agent for in vivo applications

Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

<p>Abstract</p> <p>Background</p> <p>The foodborne, gram-positive pathogen, <it>Listeria monocytogenes</it>, is capable of causing lethal infections in compromised individuals. In the post genomic era of <it>L. monocytogenes </it>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 <it>L. monocytogenes </it>strains, with a particular emphasis on the development of multiple strain competitive index assays.</p> <p>Results</p> <p>We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of <it>L. monocytogenes </it>from complex samples. 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 sample. 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 <it>L. monocytogenes </it>commonly used in laboratory studies.</p> <p>Conclusion</p> <p>This study has developed a competitive index assay that can be broadly applied to all <it>L. monocytogenes </it>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 <it>L. monocytogenes </it>in complex biological samples.</p

Anti-Salmonella lacatic acid bacteria from porcine intestinal sources

The aim of this study was to isolate lactic acid bacteria (LAB) with anti-Salmonella activity from the porcine gastrointestinal tract (GIT) and to characterise these for potentially probiotic properties using in vitro assays. Porcine caecal and faecal samples were screened for the presence of anti-Salmonella LAB; the ten most promising isolates belonged to the genera Lactobacillus and Pediococcus. The LAB exhibited large variation in their ability to survive in simulated gastric juice at pH 1.85. While Lactobacillus acidophilus species survived at up to 80% for 30 min, Lb. pentosus species declined to less than 0.001%. All isolates tolerated porcine bile at a concentration of 0.3%, with some capable of growth in the presence of up to 5% bile. The ability of the LAB isolates to prevent Salmonella invasion of intestinal epithelial cells varied, with reductions of 55% (Lb. acidophilus spp.) to 82% (Lb. salivarius spp.) observed. The data demonstrates that some porcine intestinal LAB isolates may offer potential as probiotics for the reduction of Salmonella carriage in pigs

Development of a click beetle luciferase reporter system for enhanced bioluminescence imaging of Listeria monocytogenes: analysis in cell culture and murine infection models

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-luc) based vector (pPL2CBRopt) expressing codon optimized CBR-luc under the control of a highly expressed Listerial promoter (PHELP) for L. monocytogenes and have compared this to a lux-based system expressing bacterial luciferase for BLI of the pathogen using in vitro growth experiments and in vivo models. The CBR-luc plasmid stably integrates into the L. monocytogenes chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR-luc labeled L. monocytogenes emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, lux-labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR-luc labeled bacteria was more efficient than the signal from lux-labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR-luc is suited to BLI approaches and demonstrated enhanced sensitivity relative to lux in the context of Listeria infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to lux and may have significant applications in the analysis of L. monocytogenes pathogenesi

Carbohydrate Syntrophy enhances the establishment of Bifidobacterium breve UCC2003 in the neonatal gut

The non-digestible oligosaccharide fraction of maternal milk represents an important of carbohydrate and energy source for saccharolytic bifidobacteria in the gastrointestinal tract during early life. However, not all neonatal bifidobacteria isolates can directly metabolise the complex sialylated, fucosylated, sulphated and/or N-acetylglucosamine-containing oligosaccharide structures present in mothers milk. For some bifidobacterial strains, efficient carbohydrate syntrophy or crossfeeding is key to their establishment in the gut. In this study, we have adopted advanced functional genomic approaches to create single and double in-frame deletions of the N-acetyl glucosamine 6-phosphate deacetylase encoding genes, nagA1 and nagA2, of B. breve UCC2003. In vitro phenotypic analysis followed by in vivo studies on co-colonisation, mother to infant transmission, and evaluation of the relative co-establishment of B. bifidum and B. breve UCC2003 or UCC2003 Delta nagA1 Delta nagA2 in dam-reared neonatal mice demonstrates the importance of crossfeeding on sialic acid, fucose and N-acetylglucosamine-containing oligosaccharides for the establishment of B. breve UCC2003 in the neonatal gut. Furthermore, transcriptomic analysis of in vivo gene expression shows upregulation of genes associated with the utilisation of lactose, sialic acid, GlcNAc-6-S and fucose in B. breve UCC2003, while for UCC2003 Delta nagA1 Delta nagA2 only genes for lactose metabolism were upregulated

A long and abundant non-coding RNA in Lactobacillus salivarius

Lactobacillus salivarius, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq) transcriptomic analysis of L. salivarius strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75 % of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in L. salivarius UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the L. salivarius species; however, among 45 L. salivarius genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested L. salivarius strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon

Program evaluation with high-dimensional data

Background It has become increasingly apparent that establishing and maintaining a complex and diverse gut microbiome is fundamental to human health. There are growing efforts to identify methods that can modulate and influence the microbiome, especially in individuals who due to disease or circumstance have experienced a disruption in their native microbiome. Faecal microbial transplantation (FMT) is one method that restores diversity to the microbiome of an individual by introducing microbes from a healthy donor. FMT introduces a complete microbiome into the recipient, including the bacteriome, archaeome, mycome and virome. In this study we investigated whether transplanting an autochthonous faecal virome consisting primarily of bacteriophages could impact a bacteriome disrupted by antibiotic treatment (Faecal Virome Transplantation; FVT).Results Following disruption of the bacteriome by penicillin and streptomycin, test mice (n=8) received a bacteria free, faecal transplant, while Control mice (n=8) received a heated and nuclease treated control. The bacteriomes (as determined via 16S rRNA sequencing) of mice that received an FVT, in which bacteriophages predominate, separated from those of the Control mice as determined by principle co-ordinate analysis (PCoA), and contained differentially abundant taxa that reshaped the bacteriome profile such that it more closely resembled that of the pre-treatment mice. Similarly, metagenomic sequencing of the virome confirmed that the bacteriophages present in the gut of treatment and Control mice differed over time in both abundance and diversity, with transplanted phages seen to colonise the FVT mice.Conclusions An autochthonous virome transplant impacts on the bacteriome and virome of mice following antibiotic treatment. The virome, consisting mainly of bacteriophages, reshapes the bacteriome such that it more closely resembles the pre-antibiotic state. To date, faecal transplants have largely focussed on transferring living microbes, but given that bacteriophage are inert biological entities incapable of colonising in the absence of a sensitive host they could form a viable alternative that may have fewer safety implications and that could be delivered as a robust formulation

Autochthonous faecal viral transfer (FVT) impacts the murine microbiome after antibiotic perturbation

Background: It has become increasingly accepted that establishing and maintaining a complex and diverse gut microbiota is fundamental to human health. There are growing efforts to identify means of modulating and influencing the microbiota, especially in individuals who have experienced a disruption in their native microbiota. Faecal microbiota transplantation (FMT) is one method that restores diversity to the microbiota of an individual by introducing microbes from a healthy donor. FMT introduces the total microbial load into the recipient, including the bacteria, archaea, yeasts, protists and viruses. In this study, we investigated whether an autochthonous faecal viral transfer (FVT), in the form of a sterile faecal filtrate, could impact the recovery of a bacteriome disrupted by antibiotic treatment. Results: Following antibiotic disruption of the bacteriome, test mice received an FVT harvested prior to antibiotic treatment, while control mice received a heat- and nuclease-treated FVT. In both groups of mice, the perturbed microbiome reverted over time to one more similar to the pre-treatment one. However, the bacteriomes of mice that received an FVT, in which bacteriophages predominate, separated from those of the control mice as determined by principal co-ordinate analysis (PCoA). Moreover, analysis of the differentially abundant taxa indicated a closer resemblance to the pre-treatment bacteriome in the test mice that had received an FVT. Similarly, metagenomic sequencing of the virome confirmed that faecal bacteriophages of FVT and control mice differed over time in both abundance and diversity, with the phages constituting the FVT persisting in mice that received them. Conclusions: An autochthonous virome transfer reshaped the bacteriomes of mice post-antibiotic treatment such that they more closely resembled the pre-antibiotic microbiota profile compared to mice that received non-viable phages. Thus, FVT may have a role in addressing antibiotic-associated microbiota alterations and potentially prevent the establishment of post-antibiotic infection. Given that bacteriophages are biologically inert in the absence of their host bacteria, they could form a safe and effective alternative to whole microbiota transplants that could be delivered during/following perturbation of the gut flora