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Unveiling the anti-adhesive potential of tetraspanin CD9 peptides against Pseudomonas aeruginosa in human keratinocytes
* Corresponding author E-mail address: hassanain@uitm.edu.my (H. Al-Talib)
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Received: ,
Accepted: ,
Abstract
Multidrug-resistant Pseudomonas aeruginosa strains are becoming a public health problem worldwide, causing numerous nosocomial infections. Adhesion of bacteria to host cells is a crucial step in infection. Hence, interruption of this stage can reduce bacterial infection. Tetraspanin CD9 was chosen for this study as it has been implicated in the pathogenesis of bacterial infections in a previous study. The aim of this study is to investigate the adhesion inhibition of tetraspanin CD9 peptides against P. aeruginosa in human keratinocytes. HaCaT cells were infected with P. aeruginosa, prior to treatment with CD9 peptides. The CD9 peptides cytotoxicity testing was determined by MTT assay. Bacterial adhesion was also determined quantitatively by counting viable bacterial cells and qualitatively by Giemsa staining and transmission electron microscope (TEM). Inflammatory markers (IL-8 and IL-6) expression was measured by ELISA assay. CD9 peptides did not affect the viability and inflammatory markers release of HaCaT cells. This study successfully demonstrated that CD9 peptides reduced P. aeruginosa adherence. Colonies produced by P. aeruginosa isolates treated with CD9 peptides were significantly reduced. Giemsa staining and TEM showed that treated samples had lower bacterial density and were located farther from the cells. These data highlight tetraspanin CD9 peptides as a potential therapeutic approach against P. aeruginosa due to their ability to inhibit bacterial adhesion without killing the bacteria, whereby, at the same time, it does not adversely affect the host cells.
Keywords
Anti-adherence
Anti-adhesion
CD9 peptides
HaCaT cells
Human keratinocytes
Multidrug-resistance
P. aeruginosa
Tetraspanin
1. Introduction
Pseudomonas aeruginosa is the most common Gram-negative bacterium causing nosocomial infections, especially in cystic fibrosis, burn, and wound patients (Botelho et al., 2019). Reports have shown that P. aeruginosa is resistant to β-lactams, carbapenems, aminoglycosides, and fluoroquinolone antibiotics, which limits the effective choice of treatment options against P. aeruginosa infections (Pachori et al., 2019). According to Bassetti et al. (2018), the mortality rate among patients with carbapenem-resistant Pseudomonas infections has increased to 71% due to limited effective treatment. With increasing concerns about the rising rates of multidrug resistance (MDR) and P. aeruginosa nosocomial infections, several new alternative agents are urgently needed to reduce the extent of resistance and infections. The development of new antibiotics is limited, costly, and time-consuming, and sooner or later, P. aeruginosa will develop resistance to them (Dutescu and Hillier, 2021). Therefore, new treatment options are urgently needed worldwide.
Recently, the development of anti-adherence and antimicrobial peptides (AMPs) has been critical for targeting MDR isolates, with a focus on low toxicity and low risk of bacterial resistance (Beaudoin et al., 2018; Lau and Dunn, 2018; Pfalzgraff et al., 2018). Defensins, cathelicidin LL-37 indolicidin, and tritrpticin are good examples of AMPs that inhibit P. aeruginosa (Batoni et al., 2016; Zhang et al., 2021). The utilisation of peptides, as opposed to antibiotics, presents several advantages, including a lower risk of bacterial resistance and a more targeted or selective mode of action with a lower risk of cytotoxicity (Park et al., 2022; Wei and Zhang, 2022). A tetraspanin-based treatment has been patented for the treating cancer, allergic diseases, and anaphylaxis (Marni et al., 2022; Robert et al., 2020).
Earlier studies have shown promise in combating various gram-negative and positive bacteria as well as viruses, although they are still far from application in healthcare settings, such as against Staphylococcus aureus, Salmonella typhimurium, Neisseria meningitidis, and Escherichia coli on the respective human cells (Green et al., 2011; Hassuna et al., 2017; Murad et al., 2022; Ventress et al., 2016). However, to date, there are limited studies investigating the role of tetraspanin CD9 on P. aeruginosa (Alrahimi, 2017; Jadi et al., 2023). Tetraspanin CD9 was selected for this study because it has been implicated in the pathogenesis of bacterial infections in a previous study (Murad et al., 2022). Our team has previously reported the direct antimicrobial effects of CD9 peptides, which include anti-biofilm activities against P. aeruginosa (Murad et al., 2023). Therefore, this study aims to further demonstrate the role of tetraspanin CD9 peptides as an alternative strategy to attenuate bacterial adherence to human keratinocytes without the risk of developing resistance.
2. Material and Methods
2.1 Bacterial strains
P. aeruginosa reference strain (Schroeter) Migula (ATCC 27853) and a clinical sample isolated from MDR- P. aeruginosa isolate (resistant to ciprofloxacin, ceftazidime, and gentamicin) were used in this study. The profile of bacterial resistance was also provided from the databases of the Centre for Pathology Diagnostic & Research Laboratories, Hospital Al-Sultan Abdullah, Malaysia. For analysis, bacterial colonies were inoculated in Luria Bertani broth (LB, Oxoid, UK), and the turbidity was adjusted to 0.5 McFarland’s standard or 1.0 ×108 CFU/mL using a McFarland densitometer (Biomerieux, USA).
2.2 Tetraspanin CD9 peptides
Tetraspanin CD9 peptides were derived from the primary sequence of the large extracellular (EC2) domain of CD9, represented by a 15 amino-acids sequence (EPQRETLKAIHYALN) with tetramethyl rhodamine (TMR) tagging, as used in the pioneering work of Ventress et al. (2016). CD9 peptides were purchased from GenScript (USA) with 98% purity in lyophilised form.
2.3 Cell line
The human keratinocytes, HaCaT cell line, was purchased from iCell (Shanghai, China) and routinely maintained in a 25 cm2 tissue culture flask. The cells were grown to confluence at 37°C in a humidified 5% CO2 incubator in complete growth media containing Dulbecco’s Modified Eagle Medium (DMEM, Nacalai Tesque, Japan) supplemented with 10% foetal bovine serum (FBS, Tico Europe, Netherlands) and 1% penicillin-streptomycin (Nacalai Tesque, Japan).
2.4 Cytotoxicity testing of CD9 peptides on HaCaT cells
The HaCaT cells were grown in a 96-well plate at 1.5×105 cells per well (100 µL/well) for 24 hrs and treated with CD9 peptides (0.5 µM) and 0.1% dimethyl sulfoxide (DMSO) alone at 37°C in the presence of 5% CO2 for another 24 hrs. The untreated cells were supplemented with DMEM and 10% FBS only. Cell viability assay was performed following the protocol of the MTT kit by Nacalai Tesque, Japan. Absorbance values were measured at 570nm (Tecan, Switzerland).
2.5 Bacterial adherence assay
The HaCaT cells were grown in a 24-well plate at 2.0×105 cells per well and incubated at 37°C in 5% CO2 for up to 24 hrs until 90% confluency. The cells were washed with phosphate buffer saline (PBS, 1st BASE, Singapore). For the treated wells, cells were infected with 500 µL of bacterial suspension in DMEM supplemented with 10% FBS (adjusted to 0.5 Mc Farland), followed by the addition of 500 µL of CD9 peptides (0.5 µM). Meanwhile, the untreated samples were added with DMEM supplemented with 10% FBS only. Cells were incubated at 37°C for 3 hrs in 5% CO2. The cells were washed three times with PBS to remove the non-adherent bacteria.
The cells were detached using trypsin (Nacalai Tesque, Japan), and 900 µL of LB broth was added to each well. Ten-fold serial dilutions (1:10 to 1:100,000) of P. aeruginosa suspension in fresh LB broth were performed, and 100 µL of three dilutions (1:1000; 1:10,000; and 1:100,000) were plated on Mueller Hinton agar (Oxoid, UK). The plates were incubated overnight at 37°C, and colonies on the plates were counted. Bacterial adhesion was expressed by colony forming unit per millilitre (CFU/mL) on agar plates based on the following formula: (number of colonies × dilution factor)/volume (mL).
2.6 Morphology of bacterial adherence
The adhesion of bacteria to the HaCaT cells was carried out according to the method mentioned in section 2.5. For Giemsa staining, the infected cells were washed with 1× PBS and were fixed with 100% methanol (Merck, USA) for 5 mins. The fixed cells were stained with 3% Giemsa (J.T. Baker, USA) for 30 mins, washed three times with 1× PBS, and observed at 1000× magnification oil immersion (Olympus BX53, Japan). For transmission electron microscope (TEM) analysis, samples were cut ultra-thin and stained with uranyl and lead acetate, and were performed at the Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Selangor, Malaysia. TEM observation (JEOL JEM-2100F Field Emission TEM, Japan), operated at 200kV, was performed by the professional staff at the Institute of Biosciences, Universiti Putra Malaysia.
2.7 ELISA assay of IL-8 and IL-6
The ELISA kit (Elabscience, USA) was used according to the manufacturer’s protocol to measure the expression of IL-6 and IL-8 in the supernatant harvested from the infection of HaCaT cells (Elabscience, 2022a, 2022b).
2.8 Statistical analysis
Statistical Package for the Social Sciences (SPSS, Chicago, US) version 28.0 and Fiji ImageJ (US) were used for the statistical analysis and image observation. The results were expressed as the mean of three replicates ± standard deviation of three readings in three independent experiments.
3. Results
3.1 CD9 peptides on human keratinocytes viability
The cell viability results of HaCaT have been shown in Fig. 1. No changes are observed in the cell viability and morphology (Giemsa stain and TEM images) of the treated cells compared to the untreated group (control). The HaCaT cells treated with CD9 peptides (0.5 μM) dissolved in DMSO showed no significant effect after 24 hrs of treatment compared to the control cells, with a P-value of 0.48 (P > 0.05). The analysis shows that HaCaT cells tolerated the 24-hour treatment of 0.1% DMSO alone. The analysis within three groups showed non-significant changes in the cell viability with a P-value of 0.282 (P > 0.05). Overall, CD9 peptides dissolved in DMSO did not cause toxicity to the cells or affect the efficacy of the CD9 peptides.
- Cell viability of HaCaT cells after 24 hrs of treatment with CD9 peptides and 0.1% DMSO alone. Absorbance values were measured at 570 nm.
3.2 CD9 peptides on the adherence of p. aeruginosa to human keratinocytes
The adhesion rate of P. aeruginosa to HaCaT cells was determined by counting the viable cells of bacteria adhering to the cell surface, expressed as the mean CFU/mL of adherent bacteria. The percentage of adherence inhibition was also determined. Fig. 2 shows the transformed data for bacterial adherence rate (log10 CFU/mL). In HaCaT cells infected with P. aeruginosa reference strain (ATCC 27853), the treated isolates show an average adherence rate of 3.41×107 CFU/mL, a significant decrease compared to the untreated cells with 5.64×1010 CFU/mL. Meanwhile, in HaCaT cells infected with MDR-P. aeruginosa treated with CD9 peptides, the rate of bacterial adherence is 1.72×107 compared to the untreated cells at 5.46×1010 CFU/mL. These results show a statistically significant difference at a P < 0.01 in colonies formed by both P. aeruginosa isolates treated with CD9 peptides compared to untreated samples. In addition, the adherence rate is significantly reduced by up to 90% in both isolates treated with CD9 peptides compared to untreated cells.
- Colony-forming unit (CFU) of the adherent P. aeruginosa isolates treated with CD9 peptides and untreated. All analysed data were transformed into a log10 of CFU/mL. *P < 0.01.
3.3 CD9 peptides on the morphology of p. aeruginosa and HaCaT Cells
Giemsa staining and TEM analysis were performed to visualise and identify the morphology of HaCaT cells infected with P. aeruginosa isolates, treated and untreated with CD9 peptides. TEM was performed to reveal the internal structure of HaCaT cells and the bacteria by randomly oriented cells in dissected ultrathin sections. Observations were made to evaluate the location and density of P. aeruginosa in under-treated and untreated conditions.
Giemsa staining results in Fig. 3 reveal that the cytoplasm of HaCaT cells is stained bluish-purple, while the nuclei are dark blue. P. aeruginosa is seen as dark purple small rods surrounding the HaCaT cells. The stained cells treated with CD9 peptides show clear cell pores and nuclei, which is not the case for the untreated cells. Fig. 4 shows the TEM images of the bacteria with normal bacilli shapes, while some in circular shapes. This could be due to the transverse and longitudinal sections through the rod-shaped P. aeruginosa during the preparation. Interestingly, P. aeruginosa replication (binary fission) is observed outside the HaCaT cells by TEM.
- (a-f) Comparison of treated and untreated CD9 peptides on the morphology of HaCaT cells and P. aeruginosa viewed using Giemsa staining at 1000× magnification and oil immersion. (a) HaCaT cells alone. (b) Morphology of stained P. aeruginosa. (c) HaCaT cells infected with ATCC 27853 P. aeruginosa. (d) HaCaT cells infected with ATCC 27853 P. aeruginosa and treated with CD9 peptides. (e) HaCaT cells infected with MDR- P.aeruginosa. (f) HaCaT cells infected with MDR- P. aeruginosa and treated with CD9 peptides. The green arrows show the clear outline of HaCaT cell nuclues, red arrows show the presence of stained bacteria and yellow arrows show the clear outline of HaCaT cell membrane.
- Transmission electron microscopy (TEM) photomicrograph of P. aeruginosa and HaCaT Cells. (a) HaCaT cells alone and the green arrow shows the clear outline HaCaT cells under TEM. (b) Morphology of stained P. aeruginosa. (c) HaCaT cells infected with ATCC 27853 P. aeruginosa. (d) HaCaT cells infected with ATCC 27853 P. aeruginosa and treated with CD9 peptides. (e) HaCaT cells infected with MDR- P. aeruginosa. (f) HaCaT cells infected with MDR- P. aeruginosa and treated with CD9 peptides. The images were viewed at 200 kV 2000× (a, c, d, e and f) and 3000× (b) magnification. All red arrows show the presence of P. aeruginosa and yellow arrows show the HaCaT cell membrane.
Qualitative analysis shows a significant reduction in bacterial density in the cells treated with CD9 peptides compared to untreated cells Fig. 2. The results show a decrease in the adherence of P. aeruginosa to the HaCaT cells treated with CD9 peptides compared to the untreated cells. In addition, P. aeruginosa is distanced away from the cells in the treated condition in comparison to the untreated (Fig. 4). However, Giemsa staining analysis and TEM images indicate no significant changes in the size of infected HaCaT cells compared to control cells. Analysis between treated and untreated cells also revealed no significant difference in the size of HaCaT cells. Overall, these results are consistent with the MTT assay findings and indicate that CD9 peptides are not toxic to the cells.
3.4 CD9 peptides on inflammatory markers expression
Pro-inflammatory cytokines such as IL-8 and IL-6 play an important role in the immune system to mitigate bacterial infection. Fig. 5 shows the measured amounts of IL-8 and IL-6 by HaCaT cells after infection with P. aeruginosa treated and untreated with CD9 peptides. Control cells treated with CD9 peptides show a non-significant increase in IL-8 protein secretion compared to untreated cells (control). These results are also consistent with the IL-6 response after the addition of CD9 peptides compared to the cells alone. The analysis shows that the treatment of control cells with CD9 peptides did not significantly alter the levels of IL-8 and IL-6 (P > 0.05). Moreover, as expected, the levels of IL-8 and IL-6 are significantly increased after infection with P. aeruginosa compared to the control (P < 0.05). The analysis signifies that the inflammatory markers are not significantly altered in the treated standard P. aeruginosa strain compared to the untreated conditions for the release of IL-8 and IL-6 (P > 0.05). In general, cells treated with CD9 peptides showed no significant differences in the levels of IL-8 and IL-6 compared to untreated cells.
- Expression of (a) IL-8 and (b) IL-6 by HaCaT cells infected with P. aeruginosa isolates after a 3-hour treatment with CD9 peptides and those not treated, measured from the supernatants by ELISA. The data presented are not significant, as *P > 0.05.
4. Discussion
This study successfully provides additional insights into a potential antipseudomonal agent, as the CD9 peptides block the adhesion of P. aeruginosa strains, particularly MDR strain. The most important factor in an antimicrobial drug candidate is its safety, causing no or very low cell toxicity. The CD9 peptides, although dissolved in 0.1% DMSO, did not show significant adverse effects on the viability of HaCaT cells. Alrahimi (2017) also reported that 30 mins of treatment with 500 nM (0.5 μM) CD9 peptides did not cause a significant loss in viable cells. The current results are also consistent with the study by Ventress and colleagues (2016), which showed that CD9 peptides 200 nM (0.2 μM) had no negative effects on skin cell metabolism. Furthermore, the qualitative results by Giemsa staining and TEM support these findings and suggest that CD9 peptides do not cause cellular metabolic activity or alter cell morphology (Figs. 3 and 4). The findings indicate that CD9 peptides do not cause toxicity when applied directly to the infected skin because the skin is composed of keratinised stratified squamous. Previous research showed that CD9 peptides exhibited little direct inhibitory effect on the growth of P. aeruginosa, indicating that CD9 peptides did not possess bacterial killing activity (Murad et al., 2023).
In addition, a wide range of synthetic peptides have been approved by the Food and Drug Administration (FDA), generally characterised by high specificity, affinity, and good efficacy, besides passing the toxicity tests (Al Musaimi et al., 2023; Lau and Dunn, 2018). Unlike antibiotics, CD9 peptides produce little to no harmful metabolites and carry a lower risk of bacterial resistance. Interestingly, P. aeruginosa opportunistically attach to injured epithelial cells, usually in patients with burns and wounds, more strongly than to normal cell surface; hence, anti-adhesion treatment may weaken the interaction of bacteria with the host cell receptors. This study successfully demonstrated that at the concentration of 0.5 μM, CD9 peptides significantly decreased the bacterial counts and adherence of both P. aeruginosa isolates to HaCaT cells by up to 90% compared to the untreated cells. These findings were deduced by Giemsa staining and TEM, supported by the reduction of colonies formed on the agar plate. Images of Giemsa staining and TEM also confirmed the results, where the treated cells had low bacterial density, and some of the bacteria were located further away from the cells than in untreated cells. The results clearly demonstrate the difference in bacterial distance from the cells in treated and untreated conditions.
These results are similar to other studies that reported the reduction of adherence of Gram-negative (N. meningitidis, S. typhimurium, and E. coli) and Gram-positive (S. aureus and Streptococcus pneumoniae) bacteria to the studied cell lines after treatment with tetraspanins monoclonal antibodies and/or recombinant peptides (Murad et al., 2022). Furthermore, the Giemsa staining shown in Figs. 4(d) and (f) revealed that the CD9-treated cells had a clearer outline of the HaCaT membrane, and more visible pores and nuclei than untreated cells (Figs. 4(c) and (e). These findings may reflect the non-cytotoxic effects and protective mechanism of CD9 peptides that act as a barrier to the host cells from the destructive effects of P. aeruginosa infection. This situation may result in less virulence of P. aeruginosa to the HaCaT cells upon treatment with CD9 peptides, resulting in less damage and maintaining the cell structures.
Nevertheless, a considerable number of P. aeruginosa colonies were still observed on the plates. This condition was also noted by Ventress et al. (2016), who found that despite treatment with CD9 peptides, some bacteria were found in the epidermis of the tissue-engineered skin model, but only a small amount penetrated the deeper layers. It suggests that tetraspanin CD9 peptides have an indirect inhibitory effect on the attachment of bacteria to the host cells, disrupting the tetraspanin-enriched microdomain. This condition may alter the microdomain while disrupting the interaction of tetraspanins with bacterial receptors on the surface of host cells. Karam et al. (2020) reported that CD9 acts as a co-receptor for the diphtheria toxin of Corynebacterium diphtheriae, which enhanced the interaction of the diphtheria toxin with its receptor and leads to infection.
These results reflect tetraspanin’s indirect and non-specific receptors for bacterial adherence, perhaps due to their properties not only as receptors but as mediators of the adhesion platform for bacterial pathogenesis (Murad et al., 2022; Van Spriel and Figdor, 2009). The CD9 peptides can exploit the properties of a receptor analogue, which competitively prevents the host CD9 receptor from interacting with the bacterial adhesins (Robert et al., 2020). Thus, the CD9 peptides reduced the “true”-bacterium-host interaction. Furthermore, a study in 2011 showed that antibodies and/or recombinant tetraspanin CD63 peptides against N. meningitidis strains lacking the gene for type iv pili only slightly reduced the bacterial adherence, compared to the wild type strains, suggesting that tetraspanins are not the direct receptor of bacterial adherence (Green et al., 2011).
The tetraspanin CD9 is supposedly involved in cell signalling and host immune response. The results showed that P. aeruginosa infection remarkably increased the release of interleukins, consistent with the previous study reporting no effects on cytokine levels in tissue-engineered skin treated with CD9 peptides (Ventress et al., 2016). This is in line with the theory reported previously, i.e., pyocyanin production by P. aeruginosa caused the release of TNF-α, which induced the expression of IL-8, IL-1, and IL-6 in the infected host cells (Chai et al., 2014). The minimal changes in the release of IL-8 and IL-6 may be due to the ability of CD9 peptides to restore the physiological balance between pro- and anti-inflammatory markers during infection. This could be the case for the slight increase of IL-6 and IL-8 in the standard P. aeruginosa strain, possibly due to CD9 peptides cross-linking with other immune mediators, activating the dendritic cells, and leading to cytokine production (Brosseau et al., 2018). While there were no significant changes in the interleukin release, it is possible that the CD9 peptides could provide some level of protection to the cells from further inflammation. This suggests that CD9 peptides may have a potential beneficial effect on the immune response.
Another important point in this study is that the anti-adhesion properties of CD9 peptides may be due to the electrostatic forces introduced by the CD9 peptides, which may induce negatively charged components and slowly neutralise the surface of P. aeruginosa. In addition, synthetic peptides may exhibit excellent antimicrobial activity, are relatively inexpensive, and have low immunogenicity, rendering them favourable candidates (Pfalzgraff et al., 2018; Wang et al., 2022). This makes the CD9 peptides more promising than antibiotics, which develop resistance relatively quickly.
The limitations of this study include the limited synthetic tetraspanin peptide used, which means that the results of this study are not representative of the other tetraspanin members. Therefore, it is recommended to include a different amino acid sequence of EC2-CD9 peptides, as well as other peptides derived from other tetraspanin members. In addition, future studies should investigate the expression of the P. aeruginosa quorum sensing system through molecular investigation, as it is an important virulence factor for bacterial biofilm formation and attachment to host cells, to better understand the anti-adherence mechanism of CD9 peptides.
5. Conclusion
We have shown that CD9 peptides can reduce the adherence of P. aeruginosa to the human keratinocyte cell line without negatively affecting the cell viability and the immune response. The broad specificity and low toxicity of tetraspanin-derived peptides render them good candidates for broad-spectrum antimicrobial treatment due to their mechanism of directly targeting the components of the host cells rather than the bacteria. Despite the limitations, this is a pilot study for the potential large-scale future work, formulation, and recommendation as a more targeted treatment strategy for infections caused by P. aeruginosa.
This may help lead to a better quality of treatment for P. aeruginosa without resistance development and reduce the economic burden on the healthcare system in combating MDR isolates.
CRediT authorship contribution statement
Khairiyah Murad: Conceptualization, Literature Search, Experimental Studies, Data Acquisition, Data Analysis, Statistical Analysis, Manuscript Preparation and Manuscript Editing and Review. Sharaniza Ab Rahim: Supervision and Manuscript Editing and Review. Hassanain Al-Talib: Conceptualization, Funding acquisition, Supervision and Manuscript Editing and Review. All authors approved the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Acknowledgements
We thank Prof Dr. Zolkapli Eshak from the Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Selangor, Malaysia for assistance in preparing the samples TEM and Mr Rafiuz Zaman Haroun from the Institute of Biosciences, Universiti Putra Malaysia in TEM viewing. Many thanks to the Institute of Medical Molecular Biotechnology (IMMB), Universiti Teknologi MARA (UiTM) for providing the facilities.
Funding
600-RMC/GIP 5/3 (088/2021) and 600-IRMI 5/3/LESTARI (012/2019) from Universiti Teknologi MARA, Malaysia.
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