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. 2023 Jan 27;4:100181. doi: 10.1016/j.crmicr.2023.100181

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

Wan-Hua Yang a, Po-Hsiang Chen b, Hung-Hsin Chang c, Hong Luen Kwok c, Arnold Stern d, Po-Chi Soo e, Jiun-Han Chen c, Hung-Chi Yang c,⁎
PMCID: PMC9926097  PMID: 36798906

Highlights

  • •

    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.

Keywords: Klebsiella pneumoniae, Bacterial colonization, Tight junction, GSPD-1 C. elegans

Abbreviations: GSPD-1, Glucose Six (6) Phosphate Dehydrogenase; NADPH, Nicotinamide adenine dinucleotide phosphate

Abstract

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.

Graphical abstract

Image, graphical abstract

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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), CB1370 [daf-2(e1370)], CF1038 [daf-16(mu86)], VC3875 [clc-1(gk3754)], RB2582 [tsp-1(ok3594)], and CB6738 [lys-7(ok1384)] 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 h 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 20 s, 40 cycles of 95 °C for 3 s, 60 °C for 30 s and 95 °C for 30 s. 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% β-mercaptoethanol, 1 mM ε-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 12,000 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 a red fluorescent protein (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 the Mock control or Gi C. elegans were transferred to a red fluorescent protein (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 using a worm picker made of an 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 s homogenizations followed by a quick spin down with a desktop mini-centrifuge (Thermo). The worm lysates were centrifuged at 12,000 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 and 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) (Fig. 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) (Fig. 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 (Fig. 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 (Fig. 1d). No difference of pharyngeal pumping between the Mock control and gspd-1-knockdown C. elegans infected with KP was found (Fig. S2).

Fig. 1.

Fig 1:

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Effects of bacterial diet on C. elegans. (a) Lifespan of the Mock control and gspd-1 (RNAi) C. elegans infected with KP, (b) Lifespan of C. elegans with E. coli OP50 (Ec) and KP, (c) Bacterial burden of the Mock control and gspd-1 (RNAi) C. elegans infected with KP. (d) GSPD-1 activity of the Mock control and gspd-1 (RNAi) C. elegans infected with KP for 2 days.

The gspd-1-knockdown C. elegans infected with KP labeled with a red fluorescent protein was visualized by microscopy to examine how GSPD-1 deficiency affects bacterial colonization (Fig. 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 (Fig. 2).

Fig. 2.

Fig 2:

Fig 2:

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Fluorescent microscopy of the Mock control and gspd-1 (RNAi) C. elegans infected with KP. Infected worms were harvested at the indicated time. Red fluorescence represents the accumulation of KP. The white triangle mark indicates Pharynx (Ph) of C. elegans. The red arrowhead mark indicates intestine (Int) of C. elegans. * indicates KP leakage.

3.2. Genome-wide screening and validation of differentially expressed genes modulated by gspd-1 knockdown

The transcriptome of mock and gspd-1-knockdown C. elegans was analyzed by RNAseq to investigate the gene regulation network in GSPD-1 deficiency (Fig. 3). High-throughput sequencing revealed that using the selection criteria, 493 DEGs were modulated by GSPD-1 deficiency. Specifically, 202 up-regulated DEGs and 291 down-regulated DEGs were obtained. An enrichment analysis of gene ontology (GO) based on the DEGs showed that among the top 10 biological processes, the most significant one was the innate immune response (Fig. 3a and Table 1). A related process, the defense response to gram-negative bacteria, was also listed. This indicated that innate immunity is the most critical biological process modulated by gspd-1 in C. elegans. qPCR was employed to validate the DEGs in the innate immune response. The expression level of selected DEGs was comparable with the RNAseq analysis (Fig. 3b).

Fig. 3.

Fig 3:

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RNAseq analysis of the Mock control and gspd-1 (RNAi) C. elegans. (a) The top 10 enriched GO biological process terms were plotted on the y-axis versus a measure of significance (negative logarithm of the P-value or Q-value) on the x-axis. The Q-value was calculated by Benjamini. (b) The DEGs between the Mock control and gspd-1 (RNAi) C. elegans.

Table 1.

List of DEGs in the innate immune response.

GO:0,045,087∼innate immune response; Q-value: 3.74367E-13
Gene symbol Description Ensembl Gene ID Fold change log2 fold change p value q value
clc-1 CLaudin-like in Caenorhabditis
[Source:UniProtKB/TrEMBL;Acc:Q93198]		 WBGene00000522 0.4257 -1.231932653 0.04295 0.904228
gst-5 Probable glutathione S-transferase 5
[Source:UniProtKB/Swiss-Prot;Acc:Q09596]		 WBGene00001753 8.1208 3.021614541 0.00005 0.0186075
gst-6 Probable glutathione S-transferase 6
[Source:UniProtKB/Swiss-Prot;Acc:P91252]		 WBGene00001754 4.8036 2.264104877 0.00265 0.234262
gst-13 Glutathione S-Transferase
[Source:UniProtKB/TrEMBL;Acc:Q22814]		 WBGene00001761 2.6450 1.403288469 0.0105 0.464447
gst-38 Glutathione S-Transferase
[Source:UniProtKB/TrEMBL;Acc:O45451]		 WBGene00001786 5.2102 2.381347459 0.00545 0.339089
pgp-1 Multidrug resistance protein pgp-1
[Source:UniProtKB/Swiss-Prot;Acc:P34712]		 WBGene00003995 3.3795 1.756796853 0.00135 0.167657
prx-11 PeRoXisome assembly factor
[Source:UniProtKB/TrEMBL;Acc:O62103]		 WBGene00004196 3.3645 1.750389138 0.04155 0.89061
spp-3 SaPosin-like Protein family
[Source:UniProtKB/TrEMBL;Acc:Q22336]		 WBGene00004988 2.4755 1.307696008 0.00555 0.341466
spp-18 SaPosin-like Protein family
[Source:UniProtKB/TrEMBL;Acc:A0A061AKY5]		 WBGene00005003 2.3583 1.237750421 0.0069 0.377793
tsp-1 Tetraspanin-1
[Source:UniProtKB/Swiss-Prot;Acc:P34285]		 WBGene00006627 0.1542 -2.696842482 0.03025 0.757338
dod-24 Downstream Of DAF-16 (Regulated by DAF-16)
[Source:UniProtKB/TrEMBL;Acc:Q9XUH3]		 WBGene00007875 3.4515 1.787225622 0.00005 0.0186075
ugt-44 UDP-GlucuronosylTransferase
[Source:UniProtKB/TrEMBL;Acc:O17757]		 WBGene00008486 2.3475 1.23110459 0.0111 0.477571
clec-62 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:O45441]		 WBGene00009393 2.1624 1.112619742 0.0052 0.333147
clec-66 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:O45445]		 WBGene00009397 2.3773 1.249337185 0.01825 0.594303
irg-5 Infection Response protein
[Source:UniProtKB/TrEMBL;Acc:O02357]		 WBGene00009429 2.7378 1.453033477 0.02305 0.651854
clec-169 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:A4F316]		 WBGene00009526 4.8700 2.283912396 0.00945 0.445075
clec-258 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:Q9XUC9]		 WBGene00010928 4.1181 2.041973834 0.0366 0.833835
acdh-1 Acyl CoA DeHydrogenase
[Source:UniProtKB/TrEMBL;Acc:Q8IAB6]		 WBGene00016943 0.0388 -4.688114218 0.02425 0.667781
asp-12 ASpartyl Protease
[Source:UniProtKB/TrEMBL;Acc:O01531]		 WBGene00017678 19.3370 4.273292678 0.00005 0.0186075
irg-3 Infection Response protein
[Source:UniProtKB/TrEMBL;Acc:A0A0M7RF66]		 WBGene00018760 7.7878 2.961216301 0.00005 0.0186075
cyp-35A5 CYtochrome P450 family
[Source:UniProtKB/TrEMBL;Acc:O44649]		 WBGene00019473 9.2017 3.201892871 0.01645 0.56268
asp-14 ASpartyl Protease
[Source:UniProtKB/TrEMBL;Acc:Q94271]		 WBGene00019619 11.4504 3.517320461 0.0007 0.116195
K12H4.7 Putative serine protease K12H4.7
[Source:UniProtKB/Swiss-Prot;Acc:P34528]		 WBGene00019682 3.2259 1.689708186 0.00005 0.0186075
endu-2 ENDonuclease, poly(U) specific
[Source:UniProtKB/TrEMBL;Acc:Q9GYM9]		 WBGene00019779 2.4527 1.294400091 0.0046 0.31666
clec-84 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:Q8WTL2]		 WBGene00021895 2.4709 1.305050903 0.01945 0.611471
clec-210 C-type LECtin
[Source:UniProtKB/TrEMBL;Acc:Q9TXW7]		 WBGene00022261 32.1417 5.006376442 0.00745 0.395024
dod-19 Downstream Of DAF-16 (Regulated by DAF-16)
[Source:UniProtKB/TrEMBL;Acc:A0A0K3ASK2]		 WBGene00022644 0.4043 -1.306647425 0.0007 0.116195
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3.3. Reduced tight junction gene expression in gspd-1-knockdown C. elegans and a G6PD-deficient human colon cell line

The RNAseq analysis revealed that among the DEGs in the innate immunity response, down-regulation of clc-1 (0.43 fold) and tsp-1 (0.15 fold) were detected in gspd-1-knockdown C. elegans (Table 1). To examine whether or not gspd-1 deficiency affects the expression of TJ and immune-related genes in C. elegans, zoo-1, clc-1, and tsp-1 were analyzed by qPCR. The transcript level of these genes was reduced in gspd-1-knockdown C. elegans (Fig. 4a). The down-regulation of clc-1 and zoo-1 indicates that gspd-1 plays a role in the maintenance of barrier integrity of the digestive tract in C. elegans. The colon epithelium HCT116 cell line treated with G6PD inhibitors (6-AN and DHEA), was analyzed by qPCR to investigate whether or not G6PD deficiency modulates TJ genes in human cells. 6-AN treatment down-regulated ZO-1 and ATF-3, while DHEA treatment down-regulated ZO-1, ZO-2, Claudin-4 and ATF-3 (Fig. 4b). The findings in the human colon cells were consistent with that of C. elegans, indicating that the modulation of the TJ genes by G6PD and gspd-1 is conserved in humans and nematodes, respectively. During infection, KP down-regulated clc-1 and zoo-1 genes in C. elegans, while gspd-1-knockdown further down-regulated TJ genes compared to the Mock control at 2, 6, and 11 dpi (Fig. 5). Reduced expression of TJ genes upon infection with KP indicated that gspd-1 deficiency is prone to severe tissue damage.

Fig. 4.

Fig 4:

Fig 4:

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Gene expression of TJ and the innate immune response. (a) the Mock control and gspd-1 (RNAi) C. elegans, (b) human colon epithelial HCT116 cells with and without the G6PD inhibitor. Bars represents the mean±SD. *P<0.05; ***P<0.001.

Fig. 5.

Fig 5:

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Gene expression of TJ and the innate immune response of the Mock control and gspd-1 (RNAi) C. elegans infected with KP. Bars represents the mean±SD. *P<0.05; **P<0.01, ***P<0.001.

3.4. Genetic interaction of gspd-1 with daf-2 and down-stream effectors upon infection with KP

The survival and GSPD-1 activity of the gspd-1-knockdown daf-2 mutant infected with KP was evaluated to understand the relationship between GSPD-1 and DAF-2 during bacterial pathogenesis (Fig. 6). Inactivation of daf-2 restored the shorten lifespan caused by KP in gspd-1-knockdown C. elegans infected with KP (Fig. 6a). Likewise, KP-derived shorten lifespan in gspd-1-knockdown C. elegans was not observed in the daf-16 mutant (Fig. 6b). daf-2 mutation increased GSPD-1 activity in both basal condition (Fig. 6c) and gspd-1 deficiency (Fig. 6d), indicating that DAF-2 acts up-stream of GSPD-1.

Fig. 6.

Fig 6:

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Survival and GSPD-1 status of the gspd-1 and IIS signaling mutant background infected with KP. (a) Lifespan of the Mock control and gspd-1 (RNAi) C. elegans in the daf-2 mutant, (b) Lifespan of the Mock control and gspd-1 (RNAi) C. elegans in the daf-16 mutant. (c) GSPD-1 activity with OP50. (d) GSPD-1 activity with HT115 and KP. Bars represents the mean±SD. *P<0.05; ***P<0.001.

Under basal conditions, where E. coli was the food source, the transcript level of lys-7 did not change between the Mock control and gspd-1-knockdown C. elegans (Fig. 7a). However, lys-7 was reduced (P<0.001) as early as 2 days and up to 11 days in gspd-1-knockdown C. elegans infected with KP (Fig. 7b).

Fig. 7.

Fig 7:

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Gene expression of lysozymes in C. elegans and a human cell line HCT116. (a) the Mock control and gspd-1 (RNAi) C. elegans without KP. (b) the Mock control and gspd-1 (RNAi) C. elegans infected with KP. (c) HCT116 cell treated with G6PD inhibitors. Bars represents the mean±SD. *P<0.05; **P<0.01, ***P<0.001.

3 types of lys genes and 2 types of ilys genes were examined by qPCR to confirm whether or not the expression of C. elegans lysozymes was affected by gspd-1. At 2 or 3 dpi, all lysozyme genes were down-regulated in gspd-1-knockdown C. elegans infected with KP (Figure S3). The reduced LYZ (lysozyme) gene expression in HCT116 cells treated with G6PD inhibitors was consistent with that of C. elegans, suggesting that G6PD/gspd-1 is required for the expression of lysozymes (Fig. 7c).

3.5. Role of daf-2 in gspd-1 knockdown C. elegans induced by KP

Upon infection with KP, lys-7 expression continued to increase at 6 and 11 dpi compared with 2 dpi in the daf-2 mutant. In gspd-1-knockdown, lys-7 expression was suppressed compared to the daf-2 mutant (Fig. 8). There was a down-hill trend of gene expression in the daf-2 mutant, suggesting that daf-2 inactivation is still sensitive to KP-induced TJ damage and immune dysfunction (Fig. 8). Gene expression in the double deficiency of gspd-1 and daf-2 did not differentiate that from the daf-2 mutant (Fig. 8). This is consistent with the fact that the lifespan of the gspd-1-knockdown daf-2 mutant was identical to that of the daf-2 mutant (Fig. 6). Epigenetic analysis showed that deletion of clc-1, tsp-1, or lys-7 abolished the lethality of gspd-1-deficient C. elegans infected with KP (Fig. 9), indicating that clc-1, tsp-1, and lys-7 were down-stream targets of gspd-1, and involved in anti-microbial defense.

Fig. 8.

Fig 8:

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Gene expression of lysozymes and TJ in the Mock control and gspd-1 (RNAi) daf-2 mutant C. elegans infected with KP. Bars represents the mean±SD. *P<0.05; **P<0.01, ***P<0.001.

Fig. 9.

Fig 9:

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Lifespan of the Mock control and gspd-1 (RNAi) clc-1, tsp-1 and lys-7 mutant C. elegans infected with KP.

4. Discussion

Traditional G6PD studies mainly have focused on red cell-related pathology. Plasmodium infection causes hemolytic anemia in G6PD deficient individuals. Clostridium difficile infection precipitates hemolysis in G6PD-deficient premature neonates leading to severe neonatal jaundice (Kaplan et al., 2008). Neutrophil dysfunction and viral infection contribute to G6PD deficiency-induced cellular pathophysiology. The current study is the first report showing that G6PD deficiency is an immune-compromised risk factor in an animal model. Impaired immune effector lysozymes and TJ genes were found in GSPD-1-deficient C. elegans infected with KP. The dysregulated innate immunity and intestinal barrier dysfunction caused increased bacterial colonization and translocation, leading to a shorten lifespan.

Lack of G6PD is associated with an impaired immune response. LPS or PMA-stimulated nitric oxide, superoxide, and hydrogen peroxide are abrogated in G6PD-deficient granulocytes (Tsai et al., 1998). G6PD-deficient epithelial cells are sensitive to Staphylococcus aureus infection (Hsieh et al., 2013). Impaired inflammasome activation and bacterial clearance have been observed in G6PD-deficient peripheral blood mononuclear cells from patients and human monocytic THP-1 cells (Yen et al., 2020). These studies highlight an essential role of G6PD in the cellular immune response, however, a need for a comprehensive understanding of this biological process at the organismal level cannot be met without a proper animal model. A simple invertebrate organism, such as C. elegans, has become an attractive animal model for studying the mechanism of the innate immune response. In particular, it has facilitated the identification of new virulence factors, host immune pathways, and the mechanism of host-pathogen interactions. Despite the evolutionary distance between C. elegans and humans, their host-pathogen interactions are surprisingly similar (Ermolaeva and Schumacher, 2014). Hence, it is a valuable tool for elucidating host-pathogen interactions and identifying the link between modulators and effectors. Such mechanistic studies can also shed light on the discovery of potential anti-microbial strategies.

C. elegans as a model has been used for the study of common human intestinal pathogens, including P. aeruginosa and S. typhimurium (Tan and Ausubel, 2000; Vega et al., 2013). Short-term infection with KP in C. elegans causes a distended intestine and intestinal atrophy (Kamaladevi and Balamurugan, 2017). Proteomic analysis reveals that infection with KP in C. elegans induces autophagy due to inhibition of mTOR, resulting in dauer formation. In the current study, long-term KP infection was consistent with previous findings that KP infection mainly disrupts intestinal physiology. GSPD-1 deficiency is prone to increasing the KP burden, as colonization is an essential strategy in bacterial pathogenesis. Unregulated intestinal bacterial proliferation or dysbiosis reduces C. elegans lifespan (Portal-Celhay et al., 2012). Extensive bacterial colonization is consistent with the increased lethality caused by KP, indicating that an immune-compromised condition induced by GSPD-1 deficiency increases disease severity.

RNAseq is a powerful platform for screening differential gene expression under genetic manipulation. Transcriptomic analysis by RNAseq revealed several “downstream targets of DAF-16″ (dod) genes in the innate immune response, suggesting that daf-16 is involved in the gspd-1-mediated innate immune response. Inactivation of the insulin/IGF-1 signaling (IIS) pathway during adulthood enhances C. elegans lifespan and innate immunity (Garsin et al., 2003). Mutation of daf-2 is associated with reduced colonization and enhanced clearance of bacterial pathogens in C. elegans (EA Evans et al., 2008). DAF-16, the forkhead box O (FOXO) transcription factor, is activated in the absence of DAF-2. Nuclear translocation of DAF-16 triggers gene expression, including longevity and the stress response. A proteomic study in C. elegans has revealed that several glycolytic enzymes, including GSPD-1, are up-regulated in daf-2 mutants (Depuydt et al., 2014). Since G6PD is associated with the cellular immune response against human bacterial pathogens (Yen et al., 2020; Hsieh et al., 2013), GSPD-1 may directly or indirectly interact with the IIS pathway and contribute to cellular anti-microbial defense.

Transcriptional profiling has identified target genes whose expression is up-regulated by DAF-16 and DAF-2 either directly or indirectly, for example, antimicrobial effector molecules, such as lysozymes (EA Evans et al., 2008). Resistance to bacterial infections is associated with increased immunity gene expression in daf-2 mutant (EA Evans et al., 2008). lys-7 expressed in the C. elegans intestine, is necessary for restricting bacteria colonization in the daf-2 mutant (EA Evans et al., 2008). Phylogenetic sequence analysis has revealed that 16 lysozymes with varied sequence divergence are found in C. elegans (Schulenburg and Boehnisch, 2008). One class is related to protists (lys) and the other belongs to invertebrate types (ilys). Pseudomonas aeruginosa inhibits the C. elegans immune response by down-regulating lys-7, while lys-7 expression is restored in the daf-2 mutant (EA Evans et al., 2008; Fatin et al., 2017). lys-7 was increased in the daf-2 mutant, while lys-7 expression was suppressed in gspd-1-deficient C. elegans infected with KP. These findings indicate that the modulation of lysozymes is critical in host-pathogen interactions. Even so, the attenuated expression of lysozymes in GSPD-1 deficiency was not reflected in the lifespan assay. Perhaps an alternative pathway under daf-2 inactivation plays an important role in pathogen defense and the maintenance of organismal survival.

GSPD-1 is required during reproduction and embryonic development (Yang et al., 2020; Yang et al., 2013). Although GSPD-1 deficiency does not affect growth during neither the larval stage nor the lifespan in adults, GSPD-1 knockdown at the larval stage results in enhanced germ cell apoptosis and embryonic pathologies. Reduced GSPD-1 activity at the larval stage is sufficient to alter the innate immune response and induce long-term pathologies upon pathogenic infection, suggesting that a temporal requirement of GSPD-1 activity is involved in the immune response.

During embryonic development, one of the main embryonic pathologies in GSPD-1-deficient embryos is the disruption of the permeability barrier in the egg shell, which causes increased permeability and embryonic lethality (Chen et al., 2017). Among the DEGs in the innate immune response, clc-1 is a TJ gene. clc-1 (Claudin-like in Caenorhabditis), the orthologue of human claudin, is involved in epithelial cell-cell adhesion and anti-bacterial defense (Asano et al., 2003). The barrier integrity of the pharynx requires CLC-1. Knockdown of clc-1 causes dextran diffusion to the outside of the lumen of the digestive tract, indicating a disruption of barrier function in the pharyngeal portion of the intestine (Asano et al., 2003). tsp-1 encodes a tetraspanin and is involved in the pathogen response in C. elegans (Kudlow et al., 2012). ZO-1, a biomarker of disrupted barrier function in humans, is involved in the regulation of intestinal TJ permeability (Power et al., 2021). zoo-1, an orthologue of zonula occluden (ZO-1), is located in the adherens junction and the bi-cellular TJ in C. elegans. As defective TJ in C. elegans contributed to the bacterial virulence, remodeling and repair of TJ is important for preventing microbial pathogenesis (Gonzalez et al., 2009). It is hypothesized that increased sensitivity to pathogen-derived toxins, instead of dissemination of bacteria across the intestine, is capable of increasing bacteria susceptibility in C. elegans (Sim and Hibberd, 2016). Consistent with the dysfunctional embryonic barrier, altered TJ expression was associated with GSPD-1 deficiency. These findings suggest that G6PD activity is tightly connected to membrane integrity and function. Transketolase (TKT), the key enzyme in the non-oxidative branch of the PPP, is closely associated with inflammatory bowel disease (IBD) (Tian et al., 2021). The mouse model lacking TKT in intestinal epithelial cells shows growth retardation and colitis. Specifically, TKT deficiency causes mucosal erosion, impaired TJ and barrier function, and elevated inflammatory cell penetration, indicating that the PPP is required for barrier function. Ablation of key enzymes in the PPP can lead to severe immune dysfunction in the digestive tract.

Translocation of bacteria or bacterial components across intestinal cells can be achieved through different pathways (Martel et al., 2022). For example, bacterial lipopolysaccharides (LPS) and microbe-associated molecular patterns (MAMPs) can be taken up by endocytosis via a trans-cellular pathway. Bacteria and MAMPs can also be absorbed through a paracellular pathway by immune cells and TJ rearrangement. Unrestricted passage of pathogens can occur in the presence of ulcers, erosion or intestinal cell death. The localization of KP outside the foregut (pharynx) was observed in C. elegans infected with KP, indicating a leak. Active bacterial invasion through the transcellular pathway contributes to the intestinal translocation of KP in human enterocyte-like cell lines (Hsu et al., 2015).

The RNAseq analysis showed that tsp-1 is a target of gspd-1. Tetraspanin is a protein family that serves as an organizer or scaffold for other proteins (Perot and Menager, 2020). TSP-15 is required for the activation of BLI-3 (Dual Oxidase) coupled with the dual oxidase maturation factor (DUOXA). Deficiency of BLI-3, DUOXA and peroxidase MLT-7 phenocopies the exoskeletal defect of the tsp-15 mutant in C. elegans. The protein complex formed by TSP-15, BLI-3, and DOXA-1 is required for ROS production and for the cross-linking of collagen during cuticle synthesis (Moribe et al., 2012). C. elegans produces ROS when infected with Entercoccus faecalis infection, however, knockdown of bli-3 in the intestine and hypodermis reduces ROS production, leading to increased susceptibility to E. faecalis (Chavez et al., 2009).

NADPH oxidase 1 (NOX1)-deficient mice display low levels of ROS production in the intestine, a mucus layer defect with bacterial infiltration into crypts and susceptibility to colitis, leading to mortality (Aviello et al., 2019). In addition to causing colon damage, NOX variants induce IBD (Makhezer et al., 2019; Hayes et al., 2015; Dhillon et al., 2014). The colitis observed in IBD is similar to chronic granulomatous disease (CGD), which is an immunodeficiency due to defective NOX (Dhillon et al., 2014). Clinical studies indicate that severe or complete deficiency of G6PD activity is associated with neutrophil dysfunction, including low H2O2 production and inefficient bactericidal activity, resembling CGD (Vives Corrons et al., 1982; Cooper et al., 1972; Baehner et al., 1972; Gray et al., 1973). Since NOX activity is determined by the availability of NADPH, G6PD is indispensable for NOX because G6PD is the major source for NADPH in the cell. Reduction of H2O2-derived from NOX is detected in G6PD-deficient granulocytes (Tsai et al., 1998). In addition to the intestine and hypodermis, BLI-3 also localizes in the pharynx (van der Hoeven et al., 2015). The alteration of clc-1, tsp-1, and lys-7 in GSPD-1-deficient C. elegans infected with KP indicates that GSPD-1 is a master regulator in the anti-microbial defense (Fig. 10).

Fig. 10.

Fig 10:

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Schematic diagram of how GSPD-1 status modulates innate immunity and survival in C. elegans infected with KP through the maintenance of barrier function (clc-1 and zoo-1), innate immunity (tsp-1), and anti-microbial peptides (lys-7).

In conclusion, GSPD-1 maintains barrier integrity and boosts immunity through maintaining barrier junction and lysozymal activity in the intestine. In GSPD-1-deficient C. elegans infected with KP, an impaired tight junction rendered translocation of bacteria and/or toxins, while defective bacteriolytic ability failed to restrict bacterial colonization, leading to their demise.

Funding

This work is supported by a grant from the Ministry of Science and Technology of Taiwan (MOST-109-2320-B-264-001-MY2 to HCY). Supports also from En Chu Kong Hospital (106-COMP6012-10 to HCY and D02-03-005-03 to JHC)

CRediT authorship contribution statement

Wan-Hua Yang: Investigation, Validation. Po-Hsiang Chen: Investigation, Validation. Hung-Hsin Chang: Investigation, Software, Visualization. Hong Luen Kwok: Investigation, Software, Visualization. Arnold Stern: Writing – review & editing. Po-Chi Soo: Investigation, Methodology. Jiun-Han Chen: Conceptualization, Supervision. Hung-Chi Yang: Conceptualization, Writing – review & editing.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Hung-Chi Yang reports financial support was provided by Ministry of Science and Technology of Taiwan. Hung-Chi Yang reports financial support was provided by En Chu Kong Hospital. Jiun-Han Chen reports financial support was provided by En Chu Kong Hospital. The corresponding author has served in the Editorial Board of "Current Research in Microbial Sciences".

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.crmicr.2023.100181.

Appendix. Supplementary materials

mmc1.docx (15.8KB, docx)
mmc2.docx (261.2KB, docx)

Data availability

  • Data will be made available on request.

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Associated Data

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Supplementary Materials

mmc1.docx (15.8KB, docx)
mmc2.docx (261.2KB, docx)

Data Availability Statement

  • Data will be made available on request.

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