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Impaired immune response & barrier function in GSPD-1-deficient C. elegans infected with Klebsiella


gspd-1-RNAi knockdown Caenorhabditis elegans was used as an immune-compromised model to investigate the role of G6PD in host-pathogen interactions. A shorted lifespan, increased bacterial burden and bacterial translocation were observed in gspd-1-knockdown C. elegans infected with Klebsiella pneumoniae (KP). RNAseq revealed that the innate immune pathway, including clc-1 and tsp-1, was affected by gspd-1 knockdown. qPCR confirmed that tight junction (zoo-1, clc-1) and immune-associated genes (tsp-1) were down-regulated in gspd-1-knockdown C. elegans and following infection with KP. The down-regulation of antimicrobial effector lysozymes, including lys-1, lys-2, lys-7, lys-8, ilys-2 and ilys-3, was found in gspd-1-knockdown C. elegans infected with KP. Deletion of clc-1, tsp-1, lys-7, and daf-2 in gspd-1-knockdown C. elegans infected with KP abolished the shorten lifespan seen in the Mock control. GSPD-1 deficiency in C. elegans resulted in bacterial accumulation and lethality, possibly due to a defective immune response. These findings indicate that GSPD-1 has a protective role in microbial defense in C. elegans by preventing bacterial colonization through bacterial clearance.


•Lack of GSPD-1 increased sensitivity to the infection of K. pneumoniae in C. elegans•The innate immune response was the most important pathway affected by GSPD-1 revealed by RNAseq•GSPD-1 maintained tight junction and intestinal barrier integrity•GSPD-1 enhanced innate immunity though regulating the antimicrobial effector, lysozymes•G6PD-deficient human cell culture recapitulated the defective immune response

1. Introduction

Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping gene from bacteria to human. As the rate-limiting enzyme in the pentose phosphate pathway (PPP), G6PD produces ribose-5-phosphate and NADPH for nucleic acid synthesis and reductive biosynthesis, respectively. The biological function of G6PD is essential for cell proliferation and organismal development (Yang et al., 2019). Insufficient G6PD due to mutations causes red cell-related clinical symptoms, including neonatal jaundice, favism, and drug or infection-induced hemolysis (Luzzatto and Seneca, 2014). Severe G6PD deficiency disrupts lipid metabolism and the permeability barrier in nematode embryos leading to embryonic lethality (Yang et al., 2020, Chen et al., 2017).

Caenorhabditis elegans is a free-living soil nematode and widely used for biomedical research because of many advantages, such as ease of culture, a transparent body for microscopy and tractable genetics (Jorgensen and Mango, 2002). The G6PD homologue of C. elegans, GSPD-1, shares high sequence similarity with the human counterpart (Yang et al., 2013). gspd-1-knockdown C. elegans exhibit several embryonic impairments, including a hatching defect, abnormal eggshell structure, enhanced permeability, defective polarity and cytokinesis (Chen et al., 2017). Despite the fact that G6PD is associated with the immune response and microbial infections (Yen et al., 2020, Yang et al., 2021), the role of G6PD in the host-pathogen interaction is largely unknown. Klebsiella pneumoniae (KP), a Gram-negative, facultative anaerobic bacillus, causes different types of healthcare-associated disorders (Panjaitan et al., 2021). KP is commonly found and harmless in the human intestine. While healthy individuals are rarely infected with KP, immuno-compromised individuals are highly susceptible to the infections (Paczosa and Mecsas, 2016).

The current study involved the investigation of the role of GSPD-1 during infection with KP. Specifically, shorten lifespan, increased bacterial burden and translocation were observed in gspd-1-knockdown C. elegans infected with KP. Several innate immune response and tight-junction (TJ)-related genes modulated by gspd-1 status have been identified by transcriptomic analysis and PCR validation. The epistatic genetic studies showed that daf-2 acts up-stream and lys-7/clc-1/tsp-1 act down-stream of gspd-1. Mechanistically, gspd-1 deficiency down-regulated lysozyme gene lys-7, causing defective bacterial clearance. gspd-1 deficiency also down-regulated the TJ-related genes clc-1 and zoo-1, leading to compromised barrier integrity of the digestive tract. The current work not only identified the key innate immune gene regulatory network modulated by gspd-1 during KP infection, but also provided an immuno-compromised model for studying the mechanism of host-pathogen interactions.

2. Materials and methods

2.1. Bacteria and nematode culture

Bacterial strains, Klebsiella pneumoniae type strain (ATCC 13883) and Escherichia coli (HT115, OP50), were cultured on a rotatory shaker at 200 rpm and 37°C in LB medium supplemented with appropriate antibiotics at the following final concentration: ampicillin (200 μg/mL). C. elegans strains N2 (wild type), daf-2(e1370), daf-16(mu86), clc-1(VC3875), tsp-1(RB2582), and lys-7(CB6738) were acquired from Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA). All C. elegans strains were maintained on Nematode growth medium (NGM) agar plates seeded with bacterial lawn (Incubator DBL120, DENG YNG) according to standard protocols (Stiernagle, 2006).

2.2. gspd-1 RNAi Knockdown

RNAi knockdown was carried out by feeding dsRNA-expressed bacteria based on a recent report (Yang et al., 2020). Briefly, gravid hermaphrodites fed on E. coli OP50 were treated with 1% hypochlorite bleach and a 0.5 M sodium hydroxide solution. Eggs were washed and incubated in M9 buffer for 16 hr to obtain synchronized L1 larvae, followed by culturing on NGM agar consisting of 1 mM IPTG, ampicillin (Sigma-Aldrich, St. Louis, MO, USA), and seeded with E. coli HT115 expressing a L4440 vector control (Mock) or a gspd-1 RNAi (Gi) (Yang et al., 2013).

2.3. Cell culture

The human intestinal epithelial cell line, HCT116, was grown in RPMI supplemented with 10% FCS, antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) and 5% CO2 at 37°C. The generation of the G6PD deficient cells was performed by using the G6PD inhibitors, 6-AN and DHEA (Thermo). The control cells were treated with DMSO.

2.4. Hiseq sequencing and data analysis

Total RNA was extracted from the Mock control and Gi C. elegans without KP infection at the 5th day of the adult stage by using TRIzol (Thermo). The quality of RNA was examined by nanodrop, Agilent Bioanalyzer 2100 and 1.5% agarose gel electrophoresis to guarantee purity and integrity of the RNA samples. The mRNA was enriched and used for the construction of a double-stranded library. RNA sequencing was performed by Illumina HiSeq. Raw reads were trimmed to remove low quality bases. Spliced transcript alignment was performed followed by transcripts reconstruction and estimation of transcripts abundance by Cuffquant. To determine the expression level of the genes, read sequences were compared with the C. elegans reference genome (Ensembl WBcel235). Normalized gene expression was performed by calculating the number of RNA-Seq Fragments Per Kilobase of transcript per total Million fragments mapped. Cuffdiff and CummeRbund were employed to identify the differentially expressed genes (DEGs) and to plot expression, respectively. The significantly expressed genes were selected based on the log2 fold change ≥ 1 and a P value < 0.05.

2.5. Lifespan assay

A C. elegans lifespan assay was performed at 20°C based on standard protocols. In brief, ∼40 L4 stage Mock or Gi C. elegans were fed with E. coli HT115 and were transferred to KP-seeded NGM agar plates supplemented with 50 μM 5-fluoro-2’-deoxyuridine (FUDR) (Thermo Scientific). This was considered as day 0. Live adult worms were transferred to fresh plates daily and their death was scored until no worms survived. The experiment was performed in triplicate and survival data was calculated by the Kaplan–Meir survival analysis in Prism (version 8.4) (GraphPad).

2.6. Reverse Transcription and Quantitative PCR (qPCR)

Total RNA of C. elegans or HCT116 cells was extracted using TRIzol (Thermo). cDNA was synthesized by the use of SuperScript III Reverse Transcriptase (Thermo) with 0.5 μg of oligo (dT)18 primer (Bioman Scientific, Taipei, Taiwan). Quantitative PCR was performed by using a StepOne (ABI) and a SYBR green reagent (KAPA, SIGMA). The thermal cycle program was as follows: 95°C for 20s, 40 cycles of 95°C for 3s, 60°C for 30s and 95°C for 30s. Primers were listed in the supplementary section (Table S1 and S2). The gene expression level was normalized to threshold cycle (Ct) values of the housekeeping gene (ama-1, for C. elegans; β-actin for HCT116 cells). The relative index (2−ΔΔCt) was calculated by comparing the average expression levels for controls with the index defined as 1.0.

2.7. G6PD activity assay

The GSPD-1 activity of adult C. elegans was assayed spectrophotometrically at 340 nm by the reduction of NADP+ as previously described (Yang et al., 2013). In brief, staged day 1 adults were harvested from an NGM agar plate by washing with PBS to remove bacteria. The worms were resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 3 mM magnesium chloride, 1 mM EDTA, 0.02% b-mercaptoethanol, 1 mM e-aminocaproic acid and 0.1% Triton X-100). The worm suspension was chilled immediately on ice and homogenized by a pellet pestle motor (Kontes). The crude lysates were centrifuged at 12000 r.p.m. for 15 min at 4°C (Z 233 MK-2, Hermle) and the supernatants (protein-containing lysate) were obtained. The Protein concentration of the lysate was determined by the Bradford method (Bio-Rad, Hercules, CA, USA). A typical assay mixture consisted of 100 mg of protein lysate in 0.2 ml of assay buffer (50 mM Tris-HCl pH 8, 50 mM MgCl2, 4 mM NADP+, 4 mM glucose 6-phosphate). The change of absorbance at 340 nm in each sample was measured spectrophotometrically for 15 min at 37°C (SPECTROstar Nano, BMG Labtech).

2.8. Microscopy

The bacterial accumulation in C. elegans was visualized based on the detection of red fluorescent (pBSK::Km::dsRED) labelled KP. In brief, staged adult hermaphrodites grown at 20°C were harvested from NGM agar plates seeded with red fluorescent KP. These worms were washed with PBS and anesthetized with 2% levamisole followed by mounting on 2% agarose pads on glass slides. Fluorescent images were taken by using an epifluorescence microscope (Nikon Eclipse E400) coupled with an LED lamp (BioPioneer) and CMOS digital camera (FL-20, BioPioneer) and analyzed by imaging software (Image-Pro Plus, Media Cybernetics).

2.9. Bacterial load assay

The bacterial load assay, modified from a protocol (Ayala et al., 2017), was measured based on the colony forming unit (CFU) of bacteria colonized in the C. elegans gut. In brief, staged L4 larvae of Mock or Gi C. elegans were transferred to red fluorescent (pBSK::Km::dsRED) labelled KP-seeded NGM agar plates supplemented with 50 μg/ml kanamycin. After 2 days of infection, 50 live adult worms of each group were transferred to an Eppendorf tube containing 50 μl of PBS by a worm picker made of eyebrow hair. The samples were washed thrice with PBS to remove outside bacteria. The samples were homogenized by a pellet pestle motor (Kontes) trice. Each round consisted of 30 sec homogenizations followed by a quick spin down with a desktop mini-centrifuge (Thermo). The worm lysates were centrifuged at 12000 rpm for 15 min. Serial dilution (2X) of the worm lysates were prepared with sterilized PBS. 50 μl of each dilution was spread on an LB agar plate supplemented with 50 μg/ml kanamycin. After overnight incubation at 37°C, bacterial colonies between 30-300 on a plate were counted and recorded. The number of CFU/worm was calculated as follows: CFU/worm in a given plate = (number of colonies X dilution factor)/50.

2.10. Statistical Analysis

All statistical analyses were conducted using the Prism 8.4. version (GraphPad, San Diego, CA, USA). Data of three independent experiments were presented as the mean ± SD. The statistical difference between the control and the experimental groups was analyzed by the independent student's t-test. P-values below 0.05 were considered statistically significant.

3. Results

3.1. Effect of gspd-1 knockdown in C. elegans with KP infection

The lifespan of gspd-1-knockdown C. elegans infected with KP was significantly reduced compared with the Mock control infected with KP (P=0.0006) (Figure 1a). The shorten lifespan caused by gspd-1-knockdown was not observed with E. coli (P=0.5007). Compared with E. coli OP50, KP significantly reduced the C. elegans lifespan (P<0.0001) (Figure 1b). No difference in bacterial lawn avoidance between E. coli and KP was found in C. elegans (Figure S1). The bacterial load assay revealed that increased KP colonization was detected in gspd-1-knockdown C. elegans compared to the Mock control (Figure 1c). The G6PD activity assay showed that reduced GSPD-1 activity was detected in gspd-1-knockdown C. elegans infected with KP compared to the Mock control (Figure 1d). No difference of pharyngeal pumping between the Mock control and gspd-1-knockdown C. elegans infected with KP was found (Figure S2). The gspd-1-knockdown C. elegans infected with KP labeled with red fluorescence was visualized by microscopy to examine how GSPD-1 deficiency affects bacterial colonization (Figure 2). The difference in KP accumulation between the Mock control and gspd-1-knockdown C. elegans was not obvious upon short-term infection (less than 6 days-post-infection (dpi)). However, the distended foregut and increased KP accumulation were observed in both the Mock control and gspd-1-knockdown C. elegans upon long-term infection (8 - 13 dpi). Notably, from 10 dpi, KP was detected outside the foregut in both mock and gspd-1-knockdown C. elegans, indicating that KP disrupts barrier function of the alimentary tract (Figure 2). Free article. Read more at:

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