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A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression

BMC Microbiology volume 25, Article number: 22 (2025) Cite this article

Abstract

The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.

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Introduction

Hypervirulent Klebsiella pneumoniae (hvKP) is widespread worldwide and is an important cause of community-acquired and nosocomial infections. The hvKP may cause many infectious diseases in the clinic, including skin and soft tissue infection, wound infection, bacteremia, liver abscess, and central nervous system complications [1,2,3]. Increasing pan-drug and multi-drug resistant hvKP strains in recent years has brought a great challenge for treating the infectious induced by these strains, and MDR-hvKP may become the next “super-bacteria” [4, 5]. Effective antibiotics are the primary strategy for preventing and controlling hvKP infection. However, with increasing hvKP resistance, the number of effective drugs against these bacteria is slowly diminishing in clinics. Hence, researchers are urgently seeking to develop new drugs or strategies for controlling hvKP.

Bacteria have a variety of secretion systems, which can transport proteins with biological functions from the cytoplasm to the outside of the cell, and participate in many important biological processes such as bacterial pathogenicity, virulence, and environmental adaptability [6, 7]. The type VI secretion system (T6SS) is one of the vital bacteria secretion systems involved in several biological processes [8]. Recent studies have shown that T6SS is involved in the pathogenic process of K. pneumoniae, and it is essential to the pathogen’s high virulence [9, 10]. ClpV protein is one of the basic components of T6SS, and its function is to reassemble the sheath structure of T6SS to form a dynamic cycle [11]. It has been proved that the clpV gene is the stable expression in K. pneumoniae, and it is a necessary factor in driving T6SS function [12]. A competitive experiment with E. coli found that clpV gene deletion strains of K. pneumoniae had lower killing activity against E. coli than wild-type strains, suggesting that clpV plays an important role in the competition between K. pneumoniae and other bacteria [13]. However, the effect of clpV deletion on hvKP has not been fully reported. It is important to elucidate the influence of clpV on the biological characteristics of hvKP and provide a potential target for the clinician development of novel anti-hvKP drugs.

Lithocholic acid (LCA), a secondary bile acid formed by the metabolism of cholic acid, was a second endogenous agonist of vitamin D [14]. LCA has antibacterial activity and can affect the colonization resistance of some fungi and bacteria in the gastrointestinal tract [15,16,17]. The study of an alcohol exposure model established by our team has shown that low concentrations of LCA increase K. pneumoniae colonization in the intestinal tract of mice [18]. Therefore, we speculate that LCA supplementation may reduce or prevent K. pneumoniae colonization, which may be a potential strategy to decrease the risk of K. pneumoniae infection clinically.

In the present study, we constructed hvKP clpV gene deletion strains using CRISPR-Cas9 technology and assessed the effects of clpV deletion on the biofilm formation, interspecific and intraspecific competition, virulence, and transcriptome of hvKP. Then the effect of LCA supplementation on the virulence of hvKP and the expression of clpV have also been explored. This study provides a potential target for the clinical development of drugs to prevent and control hvKP infection.

Materials and methods

Bacterial strains and growth conditions

The hypervirulent K. pneumoniae strain hvKP13 used in this work was isolated from clinical pneumonia patients’ sputum by our laboratory. It possesses hypervirulent plasmid sequences or the associated virulence genes (peg-344, iroB, iucA, and rmpA), and has been identified as K1 serotype and sequence type ST23. The strains and plasmids used in the present study are shown in Table 1. All bacteria were inoculated and grown in Luria-Bertani (LB) broth or LB agar at 37 °C for 18 to 24 h. The following antibiotics were added to the mediums for selective bacterial culture, including Spectinomycin (100 µg/mL), Abramycin (50 µg/mL), and Kanamycin (50 µg/mL).

Table 1 Strains and plasmids used in this study
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Construction of clpV deletion and complemental strains

As described in a previous study, the clpV gene was excised utilizing a CRISPR-Cas9-mediated two-plasmid system involving pCasKP and pSGKP [19]. In brief, the pCasKP-hph plasmid was introduced into the hvKP13 strain via electroporation, followed by selection on agar plates containing hygromycin B (100 µg/ml). PCR verification subsequently yielded a hvKP13 strain harboring the pCasKP-hph plasmid. The 20nt base-pairing region of a single-guide RNA (sgRNA) was designed using the online tool available at http://www.rgenome.net/cas-designer. Two complementary 20-base pair spacers, designated clpV-spacer-1, and clpV-spacer-2, flanked by BsaI restriction sites, were synthesized and annealed in vitro. The resulting annealed product was then ligated into a BsaI-digested pSGKP-apr plasmid to create the pSGKP-apr construct harboring the targeted sgRNA (pSGKP-clpV-N20). Subsequently, 200 ng of the N20-containing pSGKP-clpV-N20 plasmids were co-transformed with 300 µM single-stranded DNA (donor repair template) into the hvKP13 strain carrying the pCasKP-hph plasmid, using electroporation. The transformed cells were then cultured on LB agar plates supplemented with 100 mg/L hygromycin B and 30 mg/L apramycin to isolate strains with the desired antibiotic resistance genes. The clpV gene’s successful deletion was confirmed using PCR amplifying and sequencing. For complemental strain acquisition, the full-length clpV gene was amplified from the hvKP13 strain and cloned into the pGEM-T-apr plasmid using a Gibson assembly cloning kit (Sangon BiotechCo., Ltd. Shanghai). The resultant plasmids, designated pGEM-T-apr:clpV, were subsequently introduced into the ΔclpV knockout strains via electroporation. The successful complementation of the knockout strains was verified by PCR amplifying and sequencing, ensuring the restoration of the clpV gene.

The growth dynamics and drug sensitivity testing of hvKP

To determine the growth curves, wild-type strains (WT), clpV deletion strains (ΔclpV), and complementary strains (C-ΔclpV) were inoculated in LB broth at 37℃, 200 rpm for 18 h. 200 µl of the above medium was transferred into 20 mL fresh sterile LB broth, and cultured at 37℃. Then 1 ml of bacterial culture was taken every hour and the absorbance at 600 nm (OD600) was measured. Every detection was repeated three times, and the average value was used to draw the growth curves of bacteria.

The sensitivity of WT, ΔclpV, and C-ΔclpV strains against ten antibiotics was measured using the disk diffusion method. Following antibiotics were used in the present study: Gentamicin (10 µg), Kanamycin (30 µg), Ciprofloxacin (5 µg), Piperacillin (10 µg), Imipenem (10 µg), Cefoxitin (30 µg), Piperacillin/Tazobactam (100 µg/10 µg), Meropenem (10 µg), Cefotaxime (30 µg), Aztreonam (30 µg). Drug-sensitivity results were determined according to CLSI 2021 criteria [20]. The strains resistant to more than three kinds of antibiotics are considered multidrug-resistant bacteria.

The mucous phenotype and biofilm formation assays

The mucinous phenotype of WT, ΔclpV, and C-ΔclpV strains was analyzed by the “string-forming test” according to the previous [21]. Briefly, the strains were transferred to Columbia blood plate for overnight culture at 37℃. Dip the colony with the inoculum loop, then lift the inoculum loop. If the viscous string formed was larger than 5 mm, it was judged as positive; otherwise, it was negative. The positives in the “String-forming test” are regarded as the hypermucoviscosity strains.

The biofilm-forming ability of WT, ΔclpV, and C-ΔclpV strains was measured by the determination of adhesion to flat-bottomed microtiter plates (96-well). Briefly, 200 µl sterile broth liquid medium was added to the 96-well microtitration plates. Bacterial cultures were added to each well according to 1:100 in a liquid medium. Only a liquid medium was used as a negative control. After incubation at 37 °C for 48 h, total cell mass was measured as absorbance at 570 nm (OD1) while the blank was OD10. Each well was washed three times with PBS, dried for 1 h at 60 °C, and stained for 20 min with 200 µl of 1% crystal violet. After the crystal violet solution was removed with water and air dried, each well was added 200 µl of 95% ethanol for 30 min, the absorbance was measured at 570 nm (OD2), while the blank was OD20. The biofilm formation capacity was calculated by BI=(OD2-OD20)/(OD1-OD10).

Galleria mellonella larvae killing assays

The virulence of target strains was determined using the Galleria mellonella (G. mellonella) larvae infection model. We selected larvae weighing about 250–300 mg and without color alteration. They were placed in the plates for freezing anesthesia. Three doses of 1 × 105, 1 × 106, and 1 × 107 CFU/mL each with 14 worms per group were tested, and the dose of 1 × 105 CFU/mL was selected for the final test. 10µL cell is injected into the left lower abdominal foot of the G. mellonella larvae using a microsyringe. The control group was injected with PBS buffer, and the group with acupuncture without injection was used to assess trauma. The treated larvae were incubated at 37 °C in sterile petri dishes at a darkroom. Mortality was monitored daily for 3 days, and larvae were considered dead when they were immobile, no longer responding to stimuli, and melanized. The number of larvae that died at various times was noted, and the survival rate was calculated by the Kaplan-Meier method in GraphPad Prism software (version 9.0).

Intraspecific and interspecific competitive growth assays

WT, ΔclpV, and C-ΔclpV strains were employed as predators, and K.pneumoniae (KP5-GFP) was used as the prey strains for the intraspecific competition tests. Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were the prey strains for the interspecific competition experiments. The predators and prey bacteria were cultured overnight in LB broth to OD600 = 1, and the culture was diluted with LB broth to OD600 = 0.4. Then the predators and prey bacteria were mixed at 10:1 (interspecific competition) and 4:1 (intraspecific competition), and 20 µL of the mixed bacterial culture were spotted onto LB agar for 5 h at 37 °C. Bacterial spots were harvested, and the colony-forming units (CFU) per milliliter of surviving prey or predator strains were measured by plating serial dilutions on selective agar. E.coli and P.aeruginosa were inoculated on MacConkey Inositol Adonitol Carbenicillin Agar, Salmonella was inoculated on Salmonella-Shigella Agar. S. aureus was inoculated on TMP Agar. The output/input ratio of the prey-to-predator strains was interpreted as survival rates.

RNA sequencing

WT, ΔclpV, and C-ΔclpV strains were inoculated in LB broth and cultured to OD600 = 0.5. Then, the bacteria were collected by centrifugation at 5000 rpm. Total RNA was extracted from bacteria using TRIzol® reagent (Dingguo Changsheng Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s protocol. RNA quantity and quality were determined using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA). The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to assess RNA integrity. RNA-seq libraries were prepared using VAHTS Universal V6 RNA-seq Library Prep Kit following the manufacturer’s protocol. Then the paired-end RNA-seq libraries were sequenced with the Illumina HiSeq X Ten platform (2 × 150 bp read length) by Major Biotech Co., Ltd (Shanghai, China). After the quality control, clean reads performed bioinformatics analysis.

Quantification of gene expression using RT-qPCR

Total RNA was extracted from WT、ΔclpV and C-ΔclpV strains using the Trigol kits (Dingguo Changsheng Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s protocol, and was reversed transcribed using the kit from All-in-One Script RTpremix (Kryptoner Mei Co., Ltd, Zhengzhou, China). Then RT-qPCR was performed with TB Green Premix Ex Taq II (TaKaRa Biotechnology, China) to evaluate the amount of mRNA expression according to the manufacturer’s recommendations. RT-qPCR amplified the following genes including iucA, iroB, entA, rmpA, peg-344, and fimA. 16s RNA of bacteria was used to normalize the RT-qPCR. Subsequently, PCR products were detected on a sequence detection system. The primer sequences were listed in Supplemental Table 1 in this study. The relative gene expression levels were calculated using the 2 −ΔΔCt methods. Three independent technical replicates were carried out for each target.

Effect of lithocholic acid on clpV expression

LCA was added into LB broth with four concentrations of 0.025 mg/mL, 1.5 mg/mL, 0.1 mg/mL, and 12.5 mg/mL respectively. Then, the hvKP13 strain was inoculated and cultured for 5 h at 37℃. The total RNA was extracted with Trigol kits, and the effects of different concentrations of LCA on clpV expression were detected by RT-qPCR.

Statistical analysis

GraphPad Prism software version 9.0 was used to conduct all statistical comparisons. A nonparametric t-test and one-way ANOVA were used to compare the different groups. p-value of 0.05 or less was considered to be statistically significant. A Tukey post-hoc test was used to determine pair wise differences where appropriate. All RNA-Seq raw FastQ files were trimmed and sorted by SolexQA. To obtain estimates of transcription levels, Burrows-Wheeler alignment was used to map the trimmed sequencing reads against the genome sequence of K.pneumoniae GCF-000009885.1. The bioinformatics analysis of transcriptional data was performed at the cloud platform of Major Biotech (https://cloud.majorbio.com/).

Results

clpV deletion did not affect the growth but changed the drug resistance of hvKP

clpV deletion strain (ΔclpV) and complemental strain (C-ΔclpV) were constructed using the CRISPR-Cas9 technique, and Fig. 1A shows the gel electrophoresis images of the successful construction of ΔclpV and C-ΔclpV (Fig. 1A). To validate the actual knock-out region, the amplicons of WT and ΔclpV strains were sequenced, and the multiple sequence alignment was performed. The results showed that the clpV gene was accurately deleted from the hvKP13 strain. The amplicon sequences of WT and ΔclpV strains were demonstrated at the supplemental material. Then we detected the growth curves of WT, ΔclpV, and C-ΔclpV strains. The results show that the three strains have similar growth curves with culture 18 h, which indicated that the clpV deletion did not affect the growing dynamics of hvKP (Fig. 1B). Then, the sensitivity of WT, ΔclpV, and C-ΔclpV strains against 10 antibiotics. The results showed that compared with the WT strain, the sensitivity of the ΔclpV strain against Cefotaxime, Gentamicin, Kanamycin, Piperacillin, and Aztreonam increased and C-ΔclpV strains showed similar sensitivity with ΔclpV strain against these antibiotics (Table 2). Interestingly, the clpV gene complementary strain (C-ΔclpV) restored the resistance against Piperacillin (Table 2). clpV gene knockout did not affect the resistance phenotypes of the other 5 antibiotics (Table 2). Meanwhile, the MIC of these three strains against polymyxin B also was investigated using the microdilution method. The results showed that the MIC values of WT, ΔclpV, and C-ΔclpV strains were 0.5 mg/L. It suggested that the deletion clpV gene did not alter the susceptibility of hvKP against polymyxin B. Taken together, these results indicated that clpV gene deletion did not affect the growth but changed the resistance against some antibiotics of hvKP.

Fig. 1
figure 1

Effect of clpV gene deletion on the growth of hypervirulent Klebsiella pneumoniae. (A) Gel electrophoresis of identification of clpV deletion strain. M: 2000 bp DNA marker. 1 lane: negative control with the primer of hcp-F/hcp-R. 2 lane: the PCR of WT strain with the primer of hcp-F/hcp-R. 3 lane: the PCR of ΔclpV strain with the primer of hcp-F/hcp-R. 4 lane: negative control with the primer of vgrG-F/vgrG-R. 5 lane: the PCR of WT strain with the primer of vgrG-F/vgrG-R. 6 lane: the PCR of ΔclpV strain with the primer of vgrG-F/vgrG-R. (B) The growth curves of WT, ΔclpV, and C-ΔclpV strains. WT: wild-type strain. ΔclpV: clpV gene deletion strain. C-ΔclpV: clpV gene complementary strain

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Table 2 Susceptibility of WT, ΔclpV, and C-ΔclpV strains against 10 antibiotics
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clpV deletion reduces the mucoviscous phenotype, biofilm-forming ability, and pathogenicity of hvKP

The “string-forming test” shows that the viscous string of ΔclpV and C-ΔclpV strains was significantly shortened compared with that of the WT strain (p < 0.001 Fig. 2A) and there was no significant difference in the viscous string between ΔclpV and C-ΔclpV strain (Fig. 2A). These results indicated that the deletion of clpV may dramatically decrease the microviscosity and complementing this gene did not restore the mucous phenotype of hvKP. Figure 2B shows the results of biofilm formation capacity. Compared with the WT strains, we found that clpV gene deletion significantly reduced the biofilm formation ability of hvKP (p < 0.001, Fig. 2B). When the clpV gene was recovered, the biofilm formation ability increased significantly, but there was a significant difference between C-ΔclpV and WT strains (Fig. 2B). Then, we detected the pathogenicity of WT, ΔclpV, and C-ΔclpV strains infecting the larvae of G. mellonella. The results showed that 72 h after infection with WT, ΔclpV, and C-ΔclpV strains, the larvae survival rates were 28.93%, 71.43%, and 47.29%, respectively (Fig. 2C). All these results indicated that deletion clpV gene decreased the mucoviscosity, the biofilm-forming ability, and the pathogenicity of hvKP.

Fig. 2
figure 2

Effect of deleting clpV gene on the mucinous, biofilm, and virulence of hypervirulent Klebsiella pneumoniae. (A) string test. (B) biofilm formation ability. (C) Survival of Galleria mellonella during 72 h of infection with different strains. Significance was assessed using a one-way ANOVA test followed by Tukey’s post-hoc test for multiple comparisons. The discrepancy of survival rates was compared with the Gehan-Breslow Wilcoxon test. WT: wild-type strain. ΔclpV: clpV gene deletion strain. C-ΔclpV: clpV gene complementary strain. ***P<0.001, ***P<0.0001

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Deletion of clpV weakens the intraspecific and interspecific competitiveness of hvKP

To explore the role of the clpV gene in hvKP’s participation in bacterial interspecies competition, Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi, and Staphylococcus aureus were selected as the prey bacteria. The interspecific competitiveness results show that the inhibition rate of ΔclpV strain against prey bacteria was significantly reduced after co-culture with prey strains compared with wild-type strains (Fig. 3A-D). The inhibitory rate of the C-ΔclpV strain against Pseudomonas aeruginosa was significantly increased than that of the ΔclpV strain (Fig. 3B), while the inhibitory rate of C-ΔclpV against Escherichia coli, Salmonella paratyphi and Staphylococcus aureus were not significantly different with that of the ΔclpV strain (Fig. 3A, C, D). Then, the hvKP strain of hvKP-KP13 and lower-virulent K. pneumoniae strain KP5 were used as the prey bacteria to assess the intraspecific competitiveness of hvKP. The results show that the inhibitory rate of the ΔclpV strain against both high-virulence strains and low-virulence strains was dramatically decreased compared to that of the WT strain (Fig. 3E, F). Altogether, the deletion of clpV significantly reduces the intraspecific and interspecific competitiveness of hvKP.

Fig. 3
figure 3

Deleting the clpV gene reduces the intraspecific and interspecific competitiveness of hypervirulent Klebsiella pneumoniae. The growth competitiveness of WT, ΔclpV, and C-ΔclpV strains with E. coli (A), P. Aeruginosa (B), S. aureus (C), S. paratyphi (D), K. pneumoniae KP13 (E) and K. pneumoniae KP5 (F), Significance was assessed using one-way ANOVA test followed by Tukey’s post-hoc test for multiple comparisons. WT: wild-type strain. ΔclpV: clpV gene deletion strain. C-ΔclpV: clpV gene complementary strain. **P<0.00 = 1, ***P<0.001, **** P<0.0001, ns: no significant

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Knocking out the clpV gene alters the transcriptome of hvKP and decreases the expression of virulence factors

To further assess how clpV gene deletion affects the biological characteristics of hvKP, transcriptome sequencing was performed on the WT and ΔclpV strains. The transcriptome data analysis showed 1259 differential expressed genes (DEGs) in the ΔclpV strain compared with the wild-type strain, of which 662 DEGs were up-regulated and 597 DEGs were down-regulated (FDR < 0.05, and FC > 1, Fig. 4A, ). Subsequently, GO and KEGG pathway enrichment analyses were performed to clarify the function of these DEGs. The top 20 GO enrichment pathways showed that these DEGs were mainly involved in biological programs and molecular functions, of which there were 6 biological programs, including the “small molecule catabolic process”, “glutamate metabolic process”, “organic substance catabolic process”, “amide catabolic process” and “alcohol catabolic process”. The enriched molecular functions of these DEGs involved the enzyme activity and transport protein function, mainly including “ATPase-coupled transmembrane transporter activity”, “ATP hydrolysis activity”, “transporter activity” and “transporter activity” (Fig. 4B).

Fig. 4
figure 4

The clpV gene deletion alters the transcriptome of hypervirulent Klebsiella pneumoniae. (A) The Volcano diagram of DEGs between WT and ΔclpV strains. (B) The top 20 GO enrichment analyses of DEGs. (C) The top 15 KEGG enrichment analyses of DEGs. (D) The expression levels of iucA, peg-344, rmpA, iroB, fimA, and entA with RT-qPCR and RNA-seq, Significance was assessed using an unpaired t-test. DEGs: differentially expressed genes. WT: wild-type strain. ΔclpV: clpV gene deletion strain. C-ΔclpV: clpV gene complementary strain. *P < 0.05, **P < 0.01, ***P < 0.001, **** P<0.0001

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The top 15 KEGG pathways enrichment analyses at classification level 1 indicated that these DEGs are mainly involved in environmental information processing, Genetic information processing, metabolism, and biofilm formation (Fig. 4C). At classification level 2 of the KEGG analysis, we found 9 pathways relating to metabolism, including nitrogen metabolism, photosynthesis, and inositol phosphate metabolism. Interestingly, there was a pathway involving the biofilm formation among the enrichment top 15 pathways. In addition, the pathways of “ABC transporters” and “two-component systems” are also enriched in these DEGs (Fig. 4C). Then, we performed an RT-qPCR to assess the validity of the transcriptome. The results revealed that the genes of iucA, peg-344, rmpA, iroB, fimA, and entA were significantly decreased in the ΔclpV strain compared with that in the WT strain (Fig. 4D), which indicated that the RNA-Seq data is reliable. In summary, our data revealed that clpV gene deletion changed the transcriptome profile and inhibited the virulent gene expression of hvKP.

LCA decreases the virulence of hvKP by downregulating clpV gene expression

Based on the above results, we hypothesize that selecting a substance that inhibits clpV gene expression may reduce the risk of hvKP infection and transmission. In previous studies, we have found that LCA can reduce hvKP’s adhesion in host cells and colonization in mice [18]. Therefore, hvKP was co-cultured with different concentrations of LCA, and the clpV expression was detected by RT-qPCR in the present study. The results showed that when the concentration of LCA was higher than 0.1 mg/mL, clpV expression was significantly inhibited, and this inhibition was dose-dependent. When the concentration of LCA exceeds 12.5 mg/mL, the clpV expression can be completely inhibited (Fig. 5A). Subsequently, we pretreated the hvKP for 4 h with different concentrations of LCA and infected the G. mellonella. The results of the survival rate of the G. mellonella are shown in Fig. 5B. Compared with the control group, the mortality of the G. mellonella infecting hvKP pretreating with LCA was significantly reduced in a dose-dependent manner. The 72-hour survival rate of G. mellonella was 41.67% at infection hvKP with 0.1 mg/mL LCA treatment. However, when the LCA concentration reached 12.5 mg/ mL, the survival rate of G. mellonella was 91.67%. All these results indicated that LCA treatment could inhibit the expression of the clpV gene and significantly reduce the pathogenicity of hvKP.

Fig. 5
figure 5

LCA inhibits clpV gene expression and reduces the virulence of hypervirulent Klebsiella pneumoniae. (A) The expression levels of clpV gene in WT strain pretreating with different concentration LCA. (B) The survival curve of Galleria mellonella infected with K. pneumoniae pretreated with the different concentrations of LCA. Significance was assessed using an unpaired t-test. LCA: Lithocholic acid. WT: wild-type strain. **P < 0.01, ***P < 0.001

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Discussion

In recent years, the infection caused by hvKP has been increasing, and its high fatality rate and drug resistance have caused widespread concern. In particular, the fusion phenotype of multi-drug resistance and high virulence may lead to the emergence of superbugs, and pose a great threat to global public health (22–23). With the increasing resistance of hvKP, it is urgent to find and develop new targets and drugs for the prevention and treatment of hvKP. T6SS is a secretory system widely existing in Gram-negative bacteria, which is closely related to the virulence and pathogenicity of bacteria [24]. As one of the core components of T6SS, clpV is necessary to participate in the secretion of specific effectors of T6SS. However, current studies of clpV’s role in bacteria mainly focus on Escherichia coli [25], and there were scarce documents on the clpV of hvKP. In the present study, we detected the effect of clpV deletion on the growth characteristics of hvKP. The results showed that clpV loss did not change the growth curve of hvKP, indicating that clpV was not a necessary factor for the growth of K. pneumoniae. Then, the relationship between the clpV gene deletion and drug resistance was explored. We found that compared with the WT strains, the sensitivity of the ΔclpV strains against Gentamicin, Kanamycin, and Aztreonam was significantly increased. Combined with RNA sequencing results, it was found that acrA, oqxB, and oqxA were obviously down-regulated in the ΔclpV strain, and these drug-resistance genes were often present in hvKP, mediating the resistance of bacteria against aminoglycosides. Therefore, we speculated that the clpV might regulate the expression of aminoglycoside antibiotic resistance genes in hvKP.

Biofilm is a protected growth mode of bacteria, which makes bacterial cells less susceptible to external conditions such as antimicrobials so that bacteria can survive in adverse environments. It also allows bacteria to disperse and occupy new niches (26–27). Bacterial biofilm formation is an important cause of many infectious diseases including device-associated infection and chronic infection in the absence of foreign bodies [28]. The biofilm was an important virulence factor of bacteria. It has been proved that biofilm formation can increase the probability of K. pneumoniae colonization in the respiratory tract, gastrointestinal tract, and urinary tract, and promote the occurrence and development of invasive infections [29]. Biofilm formation also promotes the colonization of K. pneumoniae in the urinary tract and indwelling medical devices, which is one of the important causes of nosocomial infection [30]. Many factors affect biofilm formation. Whether the clpV gene influences the biofilm development of hvKP is still unclear. Our data revealed that the biofilm-forming ability of hvKP after clpV gene knockout was significantly lower than that of the WT strain, which suggested that clpV gene plays an important role in the biofilm-forming process of hvKP. By what mechanism does clpV regulate hvKP biofilm formation, we hypothesize that the clpV deletion affects the function of T6SS, which plays an important role in the secretion of some bacterial adhesion factors. In addition, clpV gene is also involved in the regulation of other genes. Transcriptomic analysis in this study found that the efflux pump protein synthesis genes (acrA, oqxB, and oqxA) in the ΔclpV strain were significantly down-regulated compared with the WT strain. Previous documents showed that the efflux pump genes (such as acrA and oqxA) were inhibited, and the biofilm-formation capacity of K. pneumoniae was significantly decreased [31], which seemed to explain the decline in the biofilm-formation capacity of hvKP after the deletion of clpV.

It is generally believed that hypermucoviscous phenotype is one of the important characteristics of high virulence of K. pneumoniae [32]. In the present study, we observed that clpV deletion significantly reduced the forming muco ability of hvKP, which suggested that clpV plays an important role in the mucus phenotype formation of K. pneumoniae. Previous studies have shown that rmpA controls the expression of bacterial mucous phenotype, and most hvKP strains carry the rmpA gene, which regulates the production of hvKP capsules (32–33). The transcriptome results revealed that the expression of the rmpA gene significantly down-regulation in the ΔclpV strain compared with the WT strain, which indicated that clpV could regulate the expression of the rmpA gene and thus control the mucous phenotype of hvKP. It has been proved that the main virulent factors of hvKP include pilus, capsule, lipopolysaccharide, and ironophore [5]. The rmpA is a virulence factor located in the plasmid of K.pneumoniae and regulates the synthesis of polysaccharides in the capsule. K.pneumoniae strains carrying rmpA were strongly associated with high mucous phenotype and suppurative tissue infections such as liver abscesses of hvKP [34]. Similarly, the ability to use iron is crucial to the growth and reproduction of bacteria. A study has shown that hvKP strains can produce more activated iron-absorbing molecules vs. classical strains, which may contribute to their higher virulence and pathogenicity [35]. hvKP strains variably can produce 4 different siderophores: enterobactin, salmochelin, yersiniabactin, and aerobactin [36]. In the present study, we found that the expression of the rmpA and siderophores are decreased in the clpV deletion strains, which seems to explain the reason for the virulence weakening of hvKP ΔclpV strain.

One of the key indications of bacterial virulence is the competitiveness for survival between bacteria and other species in the environment. Research has demonstrated that the T6SS of Gram-negative bacteria is involved in bacterial competitiveness [9]. The absence of the clpV gene, an important component of T6SS, weakens interspecific and intraspecific competition in hvKP. To verify this issue, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were selected to perform the interspecific competition test with ΔclpV hvKP. The results showed that after deleting the clpV gene, the survival competitiveness of hvKP was dramatically weakened with gram-positive and gram-negative bacteria. Interestingly, the C-ΔclpV strain showed a similar inhibitory rate with the ΔclpV strain against Escherichia coli, Salmonella paratyphi, and Staphylococcus aureus. However, it showed a higher inhibitory rate against Pseudomonas aeruginosa than the ΔclpV strain. We speculate that this may be due to the different inhibition mechanisms of the C-ΔclpV strain against these bacteria, and more investigation is needed in the next research.

Similarly, the intraspecific competition test also showed that the loss of the clpV gene reduced the survival competitiveness of hvKP with other K. pneumoniae strains. The mechanism of competition among bacteria is complex and involves many factors. Previous studies have shown that bacterial survival competitiveness is related to bacterial biofilm formation and siderophores transport special metabolites 37. It may also be associated with the release of metabolites produced by bacteria through extracellular vesicles or the direct delivery of effector proteins such as metabolites to competitors by T6SS 37. As a conserved structure of T6SS, clpV plays an important role in the operation of T6SS, and the deletion of it may make T6SS malfunction. Our transcriptome data showed the siderophores-related genes were significantly down-regulated after knocking out the clpV gene. Based on the above views, we speculate that the survival competitiveness of hvKP weakened due to the decreased biofilm formation ability, down-regulation of gene expression of the siderophores-related genes, and dysfunction of T6SS after clpV deletion. Interestingly, we found that the biofilm-forming capability, hypermucoviscous phenotype, competition, and infectivity of the C-ΔclpV strain were significantly different compared with that of the WT strain. The possible reason for these discrepancies was that during the construction of the C-ΔclpV strain, the pGEM-T-apr:clpV plasmid was introduced into the ΔclpV knockout strains via electroporation, and the clpV gene may not be fully integrated into the same original location as the WT strain. Therefore, the expression of some genes related to these biological functions will be affected. More evidence is needed to prove this view in the future.

The deletion of the clpV gene significantly reduced the biofilm formation, bacterial virulence, and survival competitiveness of hvKP. We tried to find a substance to inhibit the expression of the clpV gene and change the biological characteristics of hvKP, thereby reducing the infection rate and mortality caused by hvKP. Our team has proved that LCA could reduce K.pneumoniae colonization in mice in a previous study [18]. In this study, we found that LCA can inhibit the expression of the hvKP clpV gene and this inhibition effect is dose-dependent. When the concentration of LCA exceeds 12.5 mg/mL, the clpV expression is almost completely inhibited. Simultaneously, G. mellonella larvae were infected with hvKP pretreated with LCA, and the results showed that the mortality rate of larvae had a negative correlation with the concentration of LCA. However, high concentrations of LCA have certain toxicity for the body, and it is crucial to select an LCA concentration that not only effectively inhibits the expression of the hvKP clpV gene, but also is harmless to the host. More research will be required to address this issue in the future.

Study limitations

This study has some limitations that need to be mentioned. First, due to the lack of clpV antibodies, we did not validate the complementation of clpV at the protein level with Western Blotting, which might confuse the role of clpV on the biological characteristics of hvKP. Second, this study lacks the general K.pneumoniae as a control group to verify whether the present results only happen in hvKP strains. Third, the clpV’s effect on hvKP lethality has only been surveyed in Galleria mellonella larvae, not in mice. It is also not clear whether supplementing LCA could inhibit or weaken hvKP colonization in vivo. Hence, future studies need to establish a K. pneumoniae infection animal model with LCA intervention to resolve these limitations.

Conclusions

The present study revealed that clpV deletion did not change the growth characteristics of hvKP, but significantly decreased its mucous phenotype and biofilm formation capacity. The clpV gene deletion weakened the intraspecific and interspecific competitiveness of hvKP with E. coli, Salmonella, P. aeruginosa, Staphylococcus aureus, and K. pneumoniae. Knocking out the clpV gene alters the transcriptome profile of hvKP and decreases its pathogenicity against G. mellonella larvae. LCA can reduce the virulence of hvKP by inhibiting the expression of clpV. The clpV gene is expected to be a potential target for decreasing hvKP infection and spread risk.

Data availability

The raw data of the transcriptome were submitted to the Genome Sequence Archive in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession PRJNA1183402 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1183402).

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Acknowledgements

The authors wish to acknowledge funding from the Science and Technology Research Project of Henan Province (grant 242102521045) and the Doctoral Scientific Research Foundation of Xinxiang Medical University (XYBSKYZZ202007).

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  1. The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China

    Wenke Liu, Qiangxing He, Junwei Cui & Fan Yang

  2. Department of Pathogenic Biology, School of Basic Medical Science, Xinxiang Medical University, Xinxiang, China

    Wenke Liu, Dan Wang, Shiwen Cao, Jiaxin Cao, Huajie Zhao & Fan Yang

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Contributions

LWK and WD, Investigation and writing. LWK, CSW, and CJX, experiment, and data analysis. CJW, editing, and review. HQX and ZHJ, software, and data analysis. YF, funding acquisition, and project administrate. All authors reviewed the final manuscript.

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Correspondence to Junwei Cui or Fan Yang.

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The collection of K. pneumoniae strains from clinical patients was approved by the Human Research Ethics Committee of the first affiliated hospital of Xinxiang Medical University (No. EC-023-089).

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Liu, W., Wang, D., He, Q. et al. A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression. BMC Microbiol 25, 22 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03748-4

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BMC Microbiology

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