Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

Jones, Robert T.; Sánchez-Contreras, María; Vlisidou, Isabella; Amos, Matthew R.; Yang, Guowei; Muñoz-Berbel, Xavier; Upadhyay, Abhishek; Potter, Ursula; Joyce, Susan A.; Ciche, Todd A.; Jenkins, A. Toby A.; Bagby, Stefan; ffrench-Constant, Richard H.; Waterfield, Nicholas R.

doi:10.1186/1471-2180-10-141

Cited by 11 publications

(9 citation statements)

References 39 publications

(50 reference statements)

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“…The initial attachment of the mutant strains to an abiotic surface was quantified by using surface plasmon resonance (SPR), a technique previously used to measure cellular and molecular binding to an abiotic gold surface (28). The majority of mutants showed surface attachment similar to that of parental strain TT01 rif (confirmed by Kruskal-Wallis statistical analysis).…”

Section: Resultsmentioning

confidence: 92%

Influence of thePhotorhabdus luminescensPhosphomannose Isomerase Gene,manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

Amos

Sánchez-Contreras

Jackson

et al. 2011

Appl Environ Microbiol

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Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.Photorhabdus luminescens is a Gram-negative, entomopathogenic gammaproteobacterium belonging to the family Enterobacteriaceae. Photorhabdus has an unusual life-style, displaying a switch from a symbiotic relationship with a heterorhabditid nematode to a virulent infection of an insect host (51). The bacteria colonize the gut of a free-living, host-seeking, infective juvenile (IJ) Heterorhabditis bacteriophora nematode (19). The IJ invades an insect larva, regurgitating P. luminescens into the hemolymph, wherein the bacteria rapidly multiply and kill the insect by using a plethora of virulence factors, including proteases, lipases, and toxin complexes (18, 51). The bacteria proliferate in insect tissues, providing a food source for the symbiotic nematode, which then replicates. After several nematode generations, the IJs reacquire P. luminescens and leave the host in search of new insects to continue the cycle.Biofilms are likely to be important in Photorhabdus biology, as they allow the establishment of surface-associated populations in order to colonize habitats such as the gut of the nematode vector (13) and the midgut epithelium in the insect host (42). Biofilm formation involves the attachment of bacterial cells to a solid surface, utilizing an extensive matrix of polymers, predominantly extracellular polysaccharides (EPSs), to develop more mature community structures (50). EPSs are secreted by most bacterial species, both Gram positive and Gram negative, as capsular polysaccharides (CPSs) closely associated with cells and/or polysaccharides, which are more diffuse and readily released into the surrounding environment.

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Section: Resultsmentioning

confidence: 92%

Influence of thePhotorhabdus luminescensPhosphomannose Isomerase Gene,manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

Amos

Sánchez-Contreras

Jackson

et al. 2011

Appl Environ Microbiol

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“…A previous proteomic analysis comparing the secretome of Pa ATCC43949 at 28°C and 37°C revealed a significant decrease in the abundance of the plasmid-encoded protein pPAU_0028 at 37°C. This protein has an unknown function however it does contain a bacterial Ig-like domain also found in invasins [ 34 ]. Nevertheless we could detect no significant difference in the transcription of any of the plasmid genes between the two temperatures using DESeq ( S1 Data ).…”

Section: Resultsmentioning

confidence: 99%

From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity

et al. 2015

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Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called “nutritional virulence” strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

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“…mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C 12,13 , allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap.…”

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confidence: 99%

The Insect <em>Galleria mellonella</em> as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Ramarao¹,

Nielsen-LeRoux²,

Lereclus³

2012

JoVE

239

231

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The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system 1 . Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides 2,3 . In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals 1 . Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis [4][5][6][7] . Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model [8][9][10][11] . Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C 12,13 , allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD 50 14 , measurement of bacterial survival 15,16 and examination of the infection process 17 . Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial...

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Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

Cited by 11 publications

References 39 publications

Influence of thePhotorhabdus luminescensPhosphomannose Isomerase Gene,manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

Influence of thePhotorhabdus luminescensPhosphomannose Isomerase Gene,manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity

The Insect <em>Galleria mellonella</em> as a Powerful Infection Model to Investigate Bacterial Pathogenesis

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