Host Resistance to Bacterial Infection Varies Over Time, but Is Not Affected by a Previous Exposure to the Same Pathogen

Abstract

Immune priming describes the phenomenon whereby after a primary pathogen exposure, a host more effectively fights a lethal secondary exposure (challenge) to the same pathogen. Conflicting evidence exists for immune priming in invertebrates, potentially due to heterogeneity across studies in the pathogen species tested, the antigen preparation for the primary exposure, and the phenotypic trait used to test for priming. To explore these factors, we injected Drosophila melanogaster with one of two bacterial species, Lactococcus lactis or Providencia burhodogranariea, which had either been heat-killed or inactivated with formaldehyde, or we injected a 1:1 mixture of the two inactivation methods. Survival and resistance (the inverse of bacterial load) were assessed after a live bacterial challenge. In contrast to our predictions, none of the primary exposure treatments provided a survival benefit after challenge compared to the controls. Resistance in the acute phase, i.e., 1 day post-challenge, separated into a lower- and higher-load group, however, neither group varied according to the primary exposure. In the chronic phase, i.e., 7 days post-challenge, resistance did not separate into two groups, and it was also unaffected by the primary exposure. Our multi-angled study supports the view that immune priming may require specific circumstances to occur, rather than it being a ubiquitous aspect of insect immunity.

Keywords: Drosophila melanogaster; bacterial pathogen; formaldehyde inactivated bacteria; heat-killed bacteria; immune priming; innate immunity; resistance; survival.

FIGURE 1

Experimental design. (A) Timeline of the experiment with essential steps and assaying timepoints. Emergence refers to the time at which the adults eclosed. (B) Previous exposure and challenge treatment combinations used in the experiment. The primary exposure was either to Drosophila Ringer’s solution (R), Lactococcus lactis (Ll) or Providencia burhodogranariea (Pb). The primary bacterial exposure was either formaldehyde inactivated (F), a mixture of formaldehyde-inactivated and heat-killed bacteria (F/HK), or heat-killed bacteria (HK). For each primary exposure-challenge combination treatment flies were challenged with live bacteria; either 92 colony forming units (CFUs) of L. lactis or 920 CFUs of P. burhodogranariea. Figure adapted from Kutzer et al. (2019).

FIGURE 2

Effect of the previous exposure on survival 7 days post-challenge. Flies were challenged with either (A) 92 colony forming units (CFUs) of Lactococcus lactis, or (B) 920 of Providencia burhodogranariea. Previous exposure treatments are Drosophila Ringer’s solution (R), formaldehyde-inactivated bacteria (F), a mixture of formaldehyde-inactivated and heat-killed bacteria (F + HK), and heat-killed bacteria (HK). Survival did not differ significantly according to previous exposure treatment. For statistics, see Table 1.

FIGURE 3

Bacterial load of individual flies 1 and 7 days after a homologous challenge with (A,C) 92 colony forming units (CFUs) of Lactococcus lactis, or (B,D) 920 CFUs of Providencia burhodogranariea. Bacterial load on the y-axis was quantified as the number of colony-forming units per fly. Here, we present a log transformation of the CFU (+1) for ease of interpretation. On the x-axis, previous exposure treatments are presented as Drosophila Ringer’s solution (R), formaldehyde-inactivated bacteria (F), a mixture of formaldehyde-inactivated and heat-killed bacteria (F+HK), and heat-killed bacteria (HK). Bacterial load at day one is in the left-hand column, and the load at day seven is in the right-hand column. Black lines show the geometric mean of the bacterial load per treatment, and per subset for bacterial load 1-day post-challenge. The grey dotted lines represent the cut-off points dividing the low and high bacterial load subsets, which were analysed separately. We did not find any effect of the previous exposure on bacterial load for either of the 2 days assayed. For statistics, see Table 2.