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

Abstract

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.

Fig 1. The genus Photorhabdus contains three predominant species.

A stylized representation of a previous six gene MLST phylogeny (adk, ghd, mdk, ndh, pgm and recA) of Photorhabdus (adapted from [5]) is shown. The grey areas indicate species that consist of multiple strains, the majority of which are unable to grow above 34°C, with only a few P. luminescens strains capable of growth at temperatures up to 37°C. Example strains are P. luminescens ^TT01 and P. temperata ^K122. The clinical strains adapted to 37°C are boxed. The stars and circles indicate the potential historical timing of temperature adaptation, which could have occurred ancestrally (star) or independently (circles) in different geographical isolates.

Fig 2. Clinical Photorhabdus isolates are able to survive exposure to higher temperatures than most non-clinical isolates.

The optical density achieved by representative strains after overnight growth in static conditions (at 28°C in LB medium) after prior 18 h exposure to a range of temperatures. A range of clinical (N. American and Australian) and non-clinical (European) strains of P. asymbiotica (Pa) were tested, and the well-studied P. luminescens strain (Pl ^TT01) was included for comparison. Green stars and red diamonds indicate thermal tolerance and intolerance respectively. Pa strain designations are indicated as superscripts.

Fig 3. The secreted metalloprotease PrtA is one of the most highly up regulated genes at 37°C.

An Artemis view of mapped RNA-seq data showing higher transcription of the prtA gene at 37°C compared to 28°C. A slight increase is also seen in the associated ABC transporter genes, prtBCD, and the predicted inhibitor gene inh.

Fig 4. The expression and function of the Photorhabdus natural product rhabduscin.

(A) Artemis views of the RNA-seq reads of the three replicates mapped onto the Pa ^ATCC43949 operons responsible for rhabduscin synthesis. The isnAB genes are responsible for synthesis of the aglycon precursor shown above the left panel. The PAU_02755–7 genes encode glycosidase enzymes that add the sugar groups to produce the final rhabduscin molecule. Note PAU_02756 is unique to the P. asymbiotica (replaced by a transposase in Pl ^TT01) and so the final Pa ^ATCC43949 rhabduscin structure from Pa ^ATCC43949 may not be the same as that shown from Pl ^TT01 (above the right panel). (B) The purified aglycon precursor of rhabduscin (shown above the key) is able to completely inhibit the human alternative complement pathway. (C) Cell free supernatants from Pa ^ATCC43949 (PaATCC43949), Pa ^Kingscliff (Pa Kc) and Pl ^TT01 (Pl TT01) can all inhibit the human alternative complement pathway (AP). Note the classical (CP) is only partially inhibited, while LB alone also inhibits the Maltose binding lectin (MBLP) pathway to some extent.

Fig 5. A schematic summarising some key differences in metabolism at 37°C compared to 28°C, centred on glutamate/asparagine metabolism and the TCA cycle.

This model is predicted by integrating data from the RNA-seq, proteomics and phenotype microarray studies. Intermediates (boxes) and pathways (arrows) predicted to be down regulated at 37°C are in red while those up regulated are in green. Data suggests TCA cycle intermediates (back boxes) would be relatively isolated from glutamate/asparagine metabolism and could be maintained via the conversion of L-serine into citrate via pyruvate. Black arrows indicate certain potential enzyme pathways that are present and predicted to be unchanged at 37°C. The data suggests a central role for imported peptides and amino acids in metabolism at 37°C. Opp/Dpp represent oligo- and di-peptide importers, TCT represents tricarboxylic acid and PEP is Phosphoenolpyruvate.