Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism

Abstract

Background: Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide; however, actual clinical infections have so far only been identified in North America, Australia, and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells.

Methods: In this study, we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. Pathogenicity was analysed using infection assays with both cultured cell lines (THP-1, CHO, and HEK cells) and primary immune cells, and peripheral blood mononuclear cells (PBMCs) isolated from human blood.

Results: Here, we present the findings from the Australian (Kingscliff) and North American (ATCC43949) clinical isolates, and non-clinical soilborne nematode isolates from Thailand (PB68) and Northern Europe (HIT and JUN) of P. asymbiotica. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells.

Conclusion: Here for the first time, we show how P. asymbiotica selectively infects certain immune cells while avoiding others and that infectivity varies depending on growth temperature. We also show that the tropism varies depending on the geographic location a strain is isolated from, with only the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

Keywords: Photorhabdus; emerging pathogen; geographical localisation; human lymphocytes; tropism.

Figure 1

Subclades of the various Photorhabdus species. Tree lengths are not drawn to scale and for illustrative purposes only. Displayed are the approximate thermotolerance and known potential hosts of archetypal strains of Photorhabdus within each species. Data on thermotolerance are from Mulley et al. (2015) and our own observations from working with our strain collection. While published data on the HIT and JUN strains showed inability to grow at 37°C, during this project it was found that given extended growth times, of 48 h compared to 24 h, they would regularly reach higher ODs and eventually reach stationary phase.

Figure 2

Global distribution of Photorhabdus strains used in this study. Table shows the Photorhabdus isolates used for the experiments in this study. (Clinical) indicates the isolate used was isolated directly from a human infection; otherwise, the isolate was isolated from soil dwelling nematodes in the region. A question mark indicates that not enough research has been done previously to confirm or deny the attribute.

Figure 3

Invasion and survival of P. asymbiotica species inside mammalian cells. Gentamycin-based invasion assays were performed on several mammalian cell lines using the different bacterial isolates. Bacteria were grown at either 28°C or 37°C prior to assay. During the assay, the bacteria were allowed to interact with the mammalian cell lines for 2 h, after which gentamycin was added to kill any extracellular bacteria. The mammalian cells were subsequently lysed, and surviving (presumably intracellular) bacterial numbers assessed using colony-forming count plating assays. The infection assays were done with (A) HEK 293 T, (B) CHO cells, and (C) THP-1 cells that had been differentiated into macrophage-like cells using PMA, prior to infection. (two-way ANOVA, **<0.01, ****<0.0001, n = 3 biological replicates). HIT and JUN were not tested in CHO and HEK 293 T cell lines.

Figure 4

Phase contrast images of Photorhabdus asymbiotica subsp. strains; HIT, and JUN and fluorescent imaging of Kingscliff and PB68 with THP-1 cells. Before the experiment the THP-1 cells were seeded into 24 well plates on glass coverslips, and differentiated into macrophage like cells using PMA. Bacteria were grown O/N at either 28°C or 37°C prior to infection and allowed to infect for 2 h, at 37°C, at a MOI of 1:50. After infection the THP-1 cells were washed to remove non-internalised/attached bacteria prior to imaging. THP-1 cell nuclei were stained with DAPI (blue) while the Kingscliff and PB68 strains were constitutively expressing GFP (green).

Figure 5

Photorhabdus asymbiotica subsp. Australis strains, Kingscliff, and PB68, with phagocytosis inhibited THP-1 cells. THP-1 cells were activated by PMA 48 h prior to experiment. Bacteria were grown at either 28°C or 37°C prior to infection and allowed to interact for 2 h. Prior to the infection assay, phagocytosis inhibitor cytochalasin-D was added to the THP-1 cells (t-test, *<0.05, ****<0.0001, n = 3 biological replicates).

Figure 6

Flow cytometry strategy for identification of Photorhabdus-infected PBMCs. (A) Human-derived PBMCs were exposed to various strains of Photorhabdus expressing GFP for 2 h, before analysis by flow cytometry to calculate the percentage of PBMCs infected. In theory, a GFP signal should be detected from PBMCs which have attached or internalised bacteria. The Photorhabdus strains were cultured first at either 28°C (simulated ambient insect body temperature) or 37°C (human core body temperature) prior to the infection. As controls, Photorhabdus cultures of each strain were killed 1 h before exposure to the PBMCs. (B) Gating strategy for identifying the different PBMC cell types using flow cytometry. Antibody and fluorophore panel can be found in the methods section. (C) Alongside detecting signals for each of the antibodies, once the PBMC types were gated out, GFP signal from any internal or attached bacteria was also detected. A distinct population of the cells (in this case monocytes) can be seen to have a GFP signal (+TT01_GFP), which is not seen when PBMCs are infected with non-GFP expressing bacteria (TT01). Using this, a percentage of each PBMC cell type that exhibited a GFP signal, indicating infection with bacteria, could be calculated.

Figure 7

Flow cytometry analysis of varying infection rates of different strains of Photorhabdus. Human PBMCs were infected with different strains of GFP +ve Photorhabdus and analysed by flow cytometry as described in figure (three experimental replicates for each sample). Four strains of Photorhabdus were tested: (A) Texas the clinical P. luminescens isolate from a human neonate infection in the USA. (B) TT01-DJC the P. luminescens lab strain originally isolated from a soil nematode. For this strain data could not be obtained for 38°C due to TT01_DJC’s inability to grow at this higher temperature. (C) Kingscliff—a P. asymbiotica subsp. Australis isolate from a human infection in Australia, and (D) PB68—a P. asymbiotica subsp. Australis nematode isolate from Thailand (n = 2–4, two-way ANOVA, **<0.01). Only relevant p-values have been shown due to the number of statistical comparisons.