Microbiome-derived acidity protects against microbial invasion in Drosophila

Abstract

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

Keywords: CP: Microbiology; host-microbe interactions; lactic acid bacteria; microbe-microbe interactions; microbial colonization; pH.

Figure 1.. Presence of microbiome members reduces host susceptibility to P. entomophila (Pe)

(A) Scheme describing the generation of gnotobiotic flies. (B) Kaplan-Meier survival analysis of female flies infected with Pe via feeding (feeding occurred from t = 0 to t = 1 day); fly microbiome treatments included no bacteria (axenic), L. plantarum NAB1 (Lp), A. tropicalis (At), or a combination of Lp and At (LpAt). The mock-infected group shows survival of axenic flies fed LB media; survival of all gnotobiotic flies fed LB was also recorded but was not significantly different from axenic control and is not shown. Log-rank statistical analyses of each infection condition are compared to the other conditions. Significance is expressed as follows: NS, not significant; **p ≤ 0.01 and ****p ≤ 0.0001. n = 60 flies per control and 110–160 flies per Peinfected treatment over 3 independent replicates. (C) Number of colony-forming units (CFUs) of bacteria per fly infected with Pe screened in axenic flies and gnotobiotic flies colonized with Lp, At, and LpAt. Flies were sacrificed to determine Pe load at 24 h, 72 h, and 7 days post-feeding infection. Each point represents bacterial load from an individual fly; bars and error bars represent the median and 95% confidence intervals; limit of detection is 2 × 10² CFUs/fly. n = 9 flies per control and 10–15 flies (depending on availability of living flies) per infected treatment per time point over 3 biological replicates. Statistical significance was determined between flies containing different microbiomes by the Mann-Whitney method. p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 2.. Microbiome composition alters the microbial load of invasive organisms during infection

Number of CFUs of bacteria per fly infected with Ecc15 (A) and EcN (B). Each invasive organism was screened in axenic flies and gnotobiotic flies colonized with Lp, At, and LpAt. Bacterial load was determined at 3 h, 24 h, and 48 h post-feeding infection. Each point represents bacterial load from an individual fly; bars and error bars represent the median and 95% confidence intervals; limit of detection is 2 × 10² CFUs/fly. n = 15 flies per treatment per time point over 3 biological replicates. Statistical significance was determined between flies containing different microbiomes by the Mann-Whitney method. p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 3.. In vitro growth assays reveal inhibition of gram-negative invasive organisms by Lp

(A) Scheme describing the multi-organism interaction assay procedure. (B) Multi-organism interaction assays display growth effects of gut microbes Lp and At on invasive organisms Pe, Ecc15, and EcN. Microbiome members were grown in perpendicular streaks for 3 days, and invasive organisms were added to the adjacent quadrants and allowed to grow for an additional day. Zones of inhibition are indicated by dashed lines. (C) Co-culture analysis of invasive organisms with microbiome members. Pe, Ecc15, and EcN were grown in mono-culture or in media previously inoculated with Lp, At, or LpAt. Bars and error bars represent the mean concentration of invasive organisms in CFUs/mL ± SEM. N = 3 biological replicates per condition.

Figure 4.. Microbiome-derived shifts in pH contribute to inhibition by Lp

(A) Multi-organism interaction assay showing the effects of Lp and At on EcN growth on media containing the pH indicator bromophenol blue. After 3 days of growth, a yellow acidic zone appears around Lp, but it tapers off near the At interaction point. When EcN is added, the zone of inhibition overlaps with the acidic region. (B) pH measurement of microbiome mono-cultures and co-cultures reveals a sharp acidification of culture media by Lp. (C) Density of invasive organisms in media adjusted to pH 4.0 with lactic acid with or without 24 h of prior growth with At. Each bar represents mean CFUs/mL ± SEM of 3 biological replicates. (D) Microbial concentration of invasive organisms in media buffered to pH 6.0 with phosphate buffer with or without 24 h of prior growth with Lp^NAB1. Each bar represents mean CFUs/mL ± SEM for 3 biological replicates. (E) Ratio of Ecc15 microbial load in flies infected on buffered vs. standard fly diets, n = 15 flies per treatment per time point over three biological replicates. Statistical significance was determined for flies of each microbiome status between standard and buffered diets using the Mann-Whitney method. p values are represented as follows: NS, p > 0.05, *p ≤ 0.05, and **p ≤ 0.01. (F) The pH of the copper cell region of the intestine was approximated by feeding axenic and gnotobiotic flies food soaked with 2% bromophenol blue. Guts were dissected and imaged immediately. A yellow/green color in the copper cell region indicates an acidified environment (pH < 4.6).

Figure 5.. Lp lactate dehydrogenase (LDH) is implicated in its antimicrobial properties and host protection

(A) Multi-organism interaction assay showing the effects of Lp^WCFS1 Lp^TF103 (ΔldhL ldhD::cat) and At on EcN growth. Neither an acidic zone nor a zone of inhibition appears around Lp^TF103. (B) pH measurement of Lp^WCFS1 and Lp^TF103 cultures. (C) Analysis of the effect of Lp LDH activity on EcN growth levels during co-culture. EcN was grown in mono-culture or in medium previously inoculated with Lp^WCFS1 or Lp^TF103. Bars and error bars represent the mean concentration of invasive organisms in CFUs/mL ± SEM. N = 3 biological replicates per condition. (D) Number of CFUs of Ecc15 per fly. Ecc15 was screened in axenic flies and gnotobiotic flies colonized with Lp^WCFS1 or Lp^TF103. Bacterial load was determined at 3 h, 24 h, and 48 h post-feeding infection. Each point represents bacterial load from an individual fly; bars and error bars represent the median and 95% confidence intervals; limit of detection is 2 × 10² CFU/fly. n = 15 flies per treatment per time point over 3 biological replicates. Statistical significance was determined between flies containing different microbiomes by the Mann-Whitney method. p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 6.. Microbiome composition alters the chemical environment of fly food

(A) pH analysis of fly food after 3 days of growth of Lp, At, or 1:1 LpAt, with culture medium added as a control. Bars represent the mean pH of three biological replicates ± SEM. (B) pH analysis of fly food 3 days after the addition of 40 male flies of different microbiome statuses (axenic, Lp, At, LpAt). Bars represent the mean pH ± SEM of three biological replicates. (C) Bacterial load of Pe, Ecc15, and EcN present on fly food initially inoculated with microbiome members Lp, At, or 1:1 LpAt, with culture medium as a control. Each bar represents the mean CFUs/g fly food ± SEM for three biological replicates. (D) Pictorial representation of microbe-microbe interactions between microbiome members and gram-negative invasive bacteria on fly food and during consumption by the host.