Fig 1.
Antibacterial effect of Bac5 against gram-negative and Acid-Fast bacilli.
The active fragment of Bac5 was synthesised to high purity and incubated at different concentrations with mid-log phase bacteria of different species in a sterile solution of RPMI:Water (1:4, v:v). Metabolic activity (A) was calculated using the colorimetric Alamar Blue assay, while bactericidal activity was recorded by colony forming unit (CFU) assay (B). All data were collected at 24 hours, except for M. bovis BCG, where metabolic activity was recorded after 72 hours. Stars denote statistical (p< 0.01) significance from the untreated (zero) control for each species as calculated using Student’s T-test.
Fig 2.
Effect of Bac5 on THP-1 cell cytokine transcription during infection.
Fold change of IL-1β, TNF-α and MMP-9 mRNA levels in uninfected (A) and M. marinum-infected (B) resting THP-1 cells following incubation with 15 ng/μl AMPs for 24 h. Fold changes of mRNA levels were assessed by qRT-PCR, normalised to 18S and expressed relative to one control (no treatment) sample. Data pooled from two independent experiments is shown. Each data point represents two treatment wells (n = 12). Error bars represent S.E.M. Kruskal-Wallis with Dunn’s post-test. **p<0.01, *p<0.05.
Fig 3.
Injection of Bac5 to the hindbrain ventricle (HBV) increases macrophage recruitment.
Two days post-fertilisation Tg(mpeg-1:mCherry) zebrafish embryos were injected into their HBV with Mock, BAC, Mock/M. marinum (Mmar) or BAC/Mmar. 10 ng Bac5 was injected for both Bac5 treatment groups and approximately 190 CFU Mmar expressing GFP for both infection groups. Z-stack images of the HBV region were acquired at 3 hours post-injection (hpi) and 24 hpi. (A-H) Representative fluorescence overlay images of fish at 3 hpi (A-D) and 24 hpi (E-H) from a single experiment are shown. Scale bar 100 μm. (I-J) Macrophages in the HBV region were quantified from fluorescence images of zebrafish taken at 3 hpi (I) and 24 hpi (J) using Icy Spot Detector plugin. Data pooled from three independent experiments is shown. Sample size (n): 40, 36, 61, 53. Error bars represent S.E.M. One-way ANOVA with Bonferroni’s post-test. *p<0.05, ***p<0.001.
Fig 4.
Injection of Bac5 to the HBV induces early but not sustained neutrophil recruitment.
Two days post-fertilisation casper Tg(mpx:GFP) zebrafish embryos were injected into their HBV with Mock, Bac5, Mock/M. marinum (Mmar) or Bac5/Mmar. 10 ng BAC was injected for both BAC treatment groups and approximately 250 CFU Mmar expressing DsRed2 for both infection groups. Z-stack images of the HBV region were acquired at 6 hpi, 24 hpi and 72 hpi. (A-D) Representative fluorescence overlay images of fish from a single experiment are shown at 6 hpi. Scale bar 100 μm. (E-H) Representative fluorescence images of fish from the same single experiment are shown at 72 hpi. Images of the bacterial fluorescence channel are shown only for infected zebrafish groups, separated by white dashed line from the neutrophil channel images. Scale bar 100 μm. (I-K) Neutrophils in the HBV region were quantified from fluorescence images of zebrafish embryos using Icy Spot Detector plugin. From L to R: 6 hpi, 24 hpi and 72 hpi. Data was also analysed using Icy FPC protocol with the same outcome (S5 Fig). Sample size (n) = 39, 37, 38, 43. Data pooled from three independent experiments is shown. Error bars represent S.E.M. One-way ANOVA with Bonferroni’s post-test. *p<0.05, **p<0.01, ***p<0.001.
Fig 5.
Exogenous injected Bac5 slows rate of infection with m. marinum in zebrafish embryos.
Two days post-fertilisation casper Tg(mpx:GFP) zebrafish embryos were injected into their HBV with 10 ng Bac5 or mock control along with approximately 250 CFU Mmar expressing DsRed2 for both infection groups. Zebrafish embryos were live-imaged using a fluorescence stereomicroscope and z-stack images of the HBV region acquired at 6 hpi, 24 hpi and 72 hpi. Bacterial burden in the HBV region of M. marinum–infected zebrafish embryos was quantified from fluorescence images using Icy FPC protocol. Sample size (n): 38, 43. Data pooled from three independent experiments is shown. Error bars represent S.E.M. Mann-Whitney test. *p<0.05.