Journal: Gut pathogens

Article Title: Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

doi: 10.1186/s13099-024-00628-6

Figure Lengend Snippet: Fig. 2 Role of prey (E. coli) in differential bile salt tolerance of T6SS-positive, T6SS-negative and Δhcp C. jejuni. (a-f) FESEM micrographs illustrate clear mor phological alterations in C. jejuni when exposed to either the presence or absence of prey (E. coli). Deflated sac-like morphology of C. jejuni was observed, but no such difference was noticed in the case of T6SS-negative C. jejuni and Δhcp C. jejuni (see insets). Moreover, prey cell damages were evident (red box) in the presence of C. jejuni but not in the case of T6SS-negative C. jejuni and Δhcp C. jejuni. Scale bar: 1 μm. (g-o) Epifluorescence images showing intracel lular ROS generation in C. jejuni. H2DCFDA-treated C. jejuni cells displaying higher fluorescence signals (green) in the case of T6SS-positive C. jejuni when the prey is present, indicating a high-level intracellular accumulation of ROS. Little or no fluorescence signal was detected in T6SS-negative cells as well as in the hcp-knock out C. jejuni (with or without the presence of prey). The fluorescence signal of T6SS-positive C. jejuni cells incubated with prey and bile salt exhibited a significantly higher mean fluorescence intensity (MFI) compared to conditions without prey (i). Regardless with or without the presence of prey no such elevated MFI was observed in T6SS-negative C. jejuni (l) and in Δhcp C. jejuni (o). Scale bar:10 μm. (p) Quantification of total ROS present in C. jejuni cells indicates enhanced MFI in T6SS-positive C. jejuni when prey bacteria is present. No such difference was noticed for T6SS-negative and Δhcp C. jejuni (n = 6). (q) When prey was present, the count of T6SS-positive C. jejuni colonies (CFU/mL) was notably lower compared to when C. jejuni was grown alone. The C. jejuni cells were cultured in the growth medium containing bile salt solutions (0.05% w/v). However, there was no alteration in CFU counts observed for T6SS-negative and Δhcp C. jejuni regardless with or without the presence of prey. Error bars depict standard deviation (mean ± SD) (n = 6)

Article Snippet: To this end, we used non-pathogenic Escherichia coli (E. coli-DH5α) as prey for the T6SS-positive reference strain of C. jejuni (ATCC 43,431-TGH 9011) and another reference strain of C. jejuni (NCTC 11,168; GenBank ID: AL111168.1), which lacks the complete T6SS sequence as a negative control to assess stress tolerance using chickens as an in vivo model host.

Techniques: Fluorescence, Knock-Out, Incubation, Bacteria, Cell Culture, Standard Deviation

Journal: Gut pathogens

Article Title: Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

doi: 10.1186/s13099-024-00628-6

Figure Lengend Snippet: Fig. 3 T6SS mediates the intracellular influx of fluorescently labeled bile salt. (a) Reaction scheme for synthesizing the iridium (Ir)-conjugated bile salt complex (Ir-TBS). Functionalized bile salt (BS) was reacted with 2,2’:6’,2’’-terpyridine (Tpy), followed by iridium [Ir(ppy)2 [Ir = iridium, ppy = 2-phenyl pyri dine)] conjugation to generate Ir-TBS complex. Yield: 31 mg (25.5%). (b) Photophysical profile of Ir-TBS complex at room temperature in acetonitrile. Spec tral data showed that Ir-TBS absorbs at 200–520 nm and emits at λmax (λex = 390 nm) = 430 and 600 nm with a shoulder at 436 nm. (c) Observed ESI-MS spectra ([M - Cl− + Na+]/2 = 1359.3811) of Ir-TBS complex (green) with simulated spectra ([M - Cl− + Na+]/2 = 1359.3589) (red) showing the M2+ spectral pattern. (d) Time-dependent 1H NMR showing kinetic stability of Ir-TBS in PBS (pH 7.4) having 0.5% DMSO for 4 days. (e) Comparative analysis of emission fluorescence intensity (EFI) of Ir-TBS complexes present in C. jejuni. Following incubation of C. jejuni cells with Ir-TBS complex for 7 h, the cells were washed and the pellets were processed for spectrophotometry and image analysis. The EFI values indicate higher internalization of Ir-TBS in C. jejuni when prey (E. coli) was present. No differences were recorded for T6SS-negative and Δhcp C. jejuni (n = 6). (f) Representative CLSM images of C. jejuni cells grown in the presence of prey showed a higher influx of Ir-TBS complex in T6SS-positive C. jejuni than in T6SS-negative C. jejuni (Scale bar: 2 μm). Further analysis reveals a significant difference in mean fluorescence intensity (MFI) between T6SS-positive C. jejuni and both Δhcp C. jejuni and T6SS-negative C. jejuni cells under conditions where prey is present. Standard deviations are represented as error bars (mean ± SD) (n = 40)

Article Snippet: To this end, we used non-pathogenic Escherichia coli (E. coli-DH5α) as prey for the T6SS-positive reference strain of C. jejuni (ATCC 43,431-TGH 9011) and another reference strain of C. jejuni (NCTC 11,168; GenBank ID: AL111168.1), which lacks the complete T6SS sequence as a negative control to assess stress tolerance using chickens as an in vivo model host.

Techniques: Labeling, Conjugation Assay, Fluorescence, Incubation, Spectrophotometry

Journal: Gut pathogens

Article Title: Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

doi: 10.1186/s13099-024-00628-6

Figure Lengend Snippet: Fig. 4 In vitro T6SS activity on C. jejuni load in primary chickens embryonic intestinal cells (CEICs). (a) Schematic of the in vitro gentamycin protection assay to investigate the effect of T6SS activity on C. jejuni adherence and invasion in CEICs. Confluent monolayers of CEICs were co-incubated for 7 h with T6SS-positive, -negative, or Δhcp C. jejuni in the presence of E. coli and 0.05% bile salt solution. Subsequently, the cells were washed to eliminate extracel lular bacteria and any remaining bile salts in the medium, followed by treatment with gentamycin to remove the adhered bacteria. The invading C. jejuni number was calculated (CFU/mL) after lysing the cells using TritonX100. The cells were imaged to visualize cellular changes. (b, c, d) The effect of prey on C. jejuni load on CEICs. Comparative data indicate a significant decrease in the number of T6SS-positive C. jejuni invasions in the presence of prey (E. coli) (b). Irrespective of the prey, no significant difference in C. jejuni population was observed when T6SS-negative (c) and Δhcp C. jejuni (d) strains were used for infection. (e, f, g) Images representing C. jejuni (stained with DAPI, shown in blue) invasion of CEICs (cell membrane stained with phalloidin, shown in red) (scale bar: 50 μm) suggest that when prey and bile salt stress (0.05%, w/v) are present, only a small number of T6SS-positive C. jejuni are detectable (shown in blue), contrasting with cells infected solely with C. jejuni (e). Comparison of the images reveals distinct alterations, notably rounded-off cells when prey and bile salt are absent (e). Regardless of the involvement of prey and bile salt, CEICs exhibited a rounded shape when infected with T6SS- negative C. jejuni (f) and Δhcp C. jejuni (g). All error bars represent standard deviations, denoted as mean ± SD (n = 6)

Article Snippet: To this end, we used non-pathogenic Escherichia coli (E. coli-DH5α) as prey for the T6SS-positive reference strain of C. jejuni (ATCC 43,431-TGH 9011) and another reference strain of C. jejuni (NCTC 11,168; GenBank ID: AL111168.1), which lacks the complete T6SS sequence as a negative control to assess stress tolerance using chickens as an in vivo model host.

Techniques: In Vitro, Activity Assay, Incubation, Bacteria, Infection, Staining, Membrane, Comparison

Journal: Gut pathogens

Article Title: Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

doi: 10.1186/s13099-024-00628-6

Figure Lengend Snippet: Fig. 5 Prey-dependent depletion of C. jejuni in chickens maintained on bile salt supplementation. (a) Schematic of in vivo chickens feeding trial. After 7 days of acclimatization, birds were maintained in a bile salt (0.2%, w/v) containing diet till day 30. Chickens fed with a normal diet without bile salt were kept in control. From day 14 onwards, experimental birds were fed with either C. jejuni or C. jejuni and E. coli at the indicated time points (black circle). On day 35 (open circle), birds were sacrificed, and cecum, its content, and intestinal lavages were collected for the analysis of bacterial load, anti-C. jejuni antibody response, transcriptional profiles of pro-inflammatory cytokines, and cecal tissue histopathological study. The experimental group details were as follows: Group A: C. jejuni only (no bile salt); Group B: C. jejuni + E. coli (no bile salt); Group C: C. jejuni + bile salt and Group D: C. jejuni + E. coli + bile salt. Parallel with this, in vivo feeding with T6SS-negative C. jejuni under similar experimental conditions was performed (n = 9 birds for each group). (b) Cecal load of C. jejuni showing effective clearance of C. jejuni in Group D birds compared to the other experimental groups. No such difference was observed when T6SS-negative C. jejuni was used. (c) Comparative analysis of local antibody (sIgA) responses in the intestinal lavage against C. jejuni indicates a significantly low level of sIgA titer in the birds belonging to Group D compared to the other experimental groups. Reduced sIgA titer in these birds sug gests prey-dependent clearance of T6SS-positive C. jejuni in the presence of bile salt. However, no such difference was noticed when the experiment was performed with T6SS-negative C. jejuni. (d) Pro-inflammatory cytokine gene expression profile of caecal tissue collected from different groups of birds indicates low-level expression of IL-1β, IL-8, IL-17A and IL-6 genes in Group D compared to the other experimental groups. (e) Histopathological changes in cecal tissue from different experiment groups. Panels i-iii show higher lesion scores characterized by necrotic lesions (arrow), lymphocytic infiltration (block arrow), disruption in the top layer of epithelium, and unorganized cell boundaries. Panel-iv displays perfectly oriented, continuous, and well-demarked surface epithelium

Article Snippet: To this end, we used non-pathogenic Escherichia coli (E. coli-DH5α) as prey for the T6SS-positive reference strain of C. jejuni (ATCC 43,431-TGH 9011) and another reference strain of C. jejuni (NCTC 11,168; GenBank ID: AL111168.1), which lacks the complete T6SS sequence as a negative control to assess stress tolerance using chickens as an in vivo model host.

Techniques: In Vivo, Control, Gene Expression, Expressing, Blocking Assay, Disruption