A Review on In vitro Cell Culture Model for Bacterial Adhesion and Invasion: From Simple Monoculture to Co-Culture Human Intestinal Epithelium Model

Aim: This paper reviews the different in vitro models of human intestinal epithelium that have been utilized for studying the adhesion and invasion properties. Problem Statement: The cell adhesion and invasion are the key mechanisms of bacterial pathogenicity that determines their possible routes of transmission. Numerous investigations Mini-review Article Hasbullah et al.; JPRI, 33(43B): 97-106, 2021; Article no.JPRI.72713 98 related to the adhesion and invasion ability of bacterial isolates have been reported on monoculture human intestinal cells. However, the use of monoculture cells has several major disadvantages, such as the inability to reproduce the complex structure that defines the intestine and the inability to accurately predict the mechanism of bacterial adhesion and invasion. Approach: Co-culture models of human intestine have been developed as an alternative to improve the monoculture epithelial cell for adhesion and invasion studies, which provide more flexibility and overcome some of the limitations Conclusion: With the use of diverse in vitro approach, it could provide thorough information on different ability of bacterial adhesion and invasion and it could help to clarify the intricacy of hostpathogen interactions that underpin bacterial pathogenesis.


INTRODUCTION
The intestinal adhesion and invasion of the epithelium are important stages in initiating the bacterial pathogenesis, as the virulent effects of intestinal pathogens rely on their ability to colonize and invade the intestinal mucosa [1][2][3]. Hence, the entry of bacteria into epithelial cells is essential for its pathogenicity, intracellular replication, spread to other tissues, and cause intestinal disease [4][5][6][7][8]. The ingested bacteria undergo the infection process in both intestinal phagocytic and non-phagocytic cells, which include bacterial adherence, colonisation, invasion, and propagation [9-10].
Due to the scarcity of accurate and specific experimental models, the molecular complexity of host-pathogen interactions in many infectious diseases, especially in humans, remains poorly understood [11]. As a result, a systematic cell culture method for examining the adhesion and invasion capabilities of bacteria is critical for understanding this host-pathogen interaction in vitro. Various intestinal epithelium models have been used to investigate the potential for bacterial pathogenicity via adhesion and invasion. The mucosa surface of the intestinal epithelium is a complex environment consisting of several cell types which are enterocytes, goblet cells, Paneth cells and endocrine cells. However, absorptive and goblet cells are the two main constituents of the intestine [12][13]. The Caco-2 cell lines are derived from intestinal absorptive, whereas HT29 and HT29-MTX are known as goblet cells. Caco-2 cells are originated from colon carcinoma that can be differentiated into enterocytes-like cells [14][15][16]. This monoculture is often used as in vitro model for studying cell adhesion and invasion [5,9,17]. However, the use of Caco-2 and other monocultures of epithelial cells has their own disadvantage. As no mammalian system consists of a single cell-type, these monocultures may not accurately depict intestinal physiology in vivo, and they do not closely simulate the composition of the normal epithelial layer which contains a variety of cell types [18][19][20].
Hence, in order to attain better physiological conditions, co-cultivation of two cell lines will therefore provide a model consisting of two different cell types that predominately represented in normal human intestinal epithelium, namely enterocytes and goblet mucus-secreting cells. Thus, this review aims to discuss the establishment of an in vitro co-culture cell model completely resembles the small intestinal epithelial layer, based on intestinal enterocytes (Caco-2) and mucus-secreting goblet cells (HT29 and HT29-MTX), to evaluate the adhesion and invasion capabilities of pathogenic and non-pathogenic bacteria. OF  HUMAN  INTESTINAL  CELLS  FOR  ADHERENCE  AND  INVASION  STUDIES The gut mucosa, which is lined with epithelial cells, is thought to be the most outer defence barrier, preventing microbes and endotoxins from reaching systemic organs and tissues [21]. Thus, studies for adherence and invasion properties of microorganisms have been carried out using single cells, also known as monocultures of human epithelial cell lines such as Caco-2 (nonmucus secreting), HT29 (low mucus secreting) and HT29-MTX (high mucus secreting) ( Table 1).

MONOCULTURE
Caco-2 cells are derived from human colon carcinoma and are employed as a model for mature human enterocytes since they can express protein features of both colonocytes and small intestinal enterocytes immediately after confluence [27,33,43]. This cell line, however, is classified as non-mucus producing cell. Caco-2 cell lines have been extensively utilized as a model to examine in vitro adhesion and invasion ability of bacteria such as Salmonella enterica, Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli), Campylobacter jejuni (C. jejuni) and probiotics.  [41] demonstrated the potential probiotic effect of Carnobacterium strains to attenuate the pathogenesis L. monocytogenes. The number of probiotic strains that adhered to HT29-MTX was substantially larger than the number of strains that adhered to HT29. Their report was attentively followed the adherent trend of C. jejuni, which showed higher numbers adherent to HT29-MTX cells than the HT29 cells [40]. These results are consistent with those of Alemka et al.
[39], who found 10-fold greater levels of C. jejuni infection in HT29-MTX than in HT29 cells. On the other hand, intestinal mucus, had no effect on E. coli colonization in HT29 and HT29-MTX, according to Kerneis et al. [37]. The level of bacterial adherence to the mucus secreting intestinal cells HT29-MTX also appeared to be higher than attachment to enterocyte-like Caco-2 cells [36].
From literature, there are few studies that comparing the pattern of bacterial adhesion and invasion using all three types of intestinal monoculture that have been discussed in this review. Gagnon et al. [27] studied the suitability of the mucus-secreting HT29-MTX cell model to test adhesion and invasion of Salmonella strains and compared with data obtained with the more commonly used Caco-2 and HT29 models. They found that Salmonella adhesion and invasion were more effective in HT29-MTX than in nonand low-mucus producing Caco-2 or HT29 cells, respectively. They also suggested that Salmonella might potentially permeate the protective mucus layer and subvert the mucus to facilitate invasion. Similarly, in 2019, Li et al. [30] investigated the function of MUC1 (highly expressed mucins in stomach and intestinal tract) during the invasion of Salmonella to Caco-2, HT29 and HT29-MTX cell lines. According to the results, Caco-2 and HT29 had lower level of invasion than HT29-MTX cells. They also discovered that, as compared to HT29-MTX, Caco-2 and HT29 cells express comparatively low levels of MUC1. Therefore, these findings prove that mucus aids Salmonella adherence and invasion of. The presence of mucus by HT29-MTX cells, in particular, is thought to play crucial function as protective component of the normal intestinal epithelium, enhancing pathogen adherence and invasion.
Since adhesion and invasion are the most important stages in bacterial pathogenesis, an anti-adhesion and anti-invasion therapies are needed as therapeutic strategies or antibiotic therapies to prevent bacterial adhesion/invasion to the host or detachment from the tissues at the early stages of infection [44][45][46]. For example, designing a synthetic peptide that mimic the structure of pilus protein will inhibit pilus assembly (pilicides) [47]. This approach is a key strategy for preventing adhesion of E. coli and Salmonella that used the pili for initiating their virulence factor [48]. Furthermore, the adhesion of pathogenic bacteria could also be disrupted some dietary supplements that acts as receptors analogs [44,46). Human milk which rich in oligosaccharides and Bovien Muc1 derived from cow milk are proven to inhibit adherent of E. coli and Salmonella to the Caco-2 cell lines [49][50][51]. Aside from that, several compounds with antiinvasion properties were discovered to be able to inhibit adhesion and invasion in monoculture Caco-2 cells. Citrus extracts effectively reduced Salmonella and L. monocytogenes adhesion and invasion to Caco-2, according to Barbosa et al. [23]. Barzelighi  In addition, probiotic bacterial strains can be considered as one of the anti-adhesion therapies as they compete with the pathogens for vital growth nutrients [52]. In 2019, Śliżewska and colleagues [28] investigated the competition between probiotics and pathogenic bacteria. They discovered that probiotics reduced the adherence of pathogenic Salmonella Typhimurium (S. Typhimurium), Salmonella Enteritidis (S. Enteritidis), and L. monocytogenes to Caco-2 cells by up to 60%. The findings are similar to those of Tuo et al. [34], who discovered that probiotic Lactobacillus strains prevent E. coli from binding to Caco-2 cells. The anti-invasive ability of probiotics strains was also examined on Salmonella enterica, L. monocytogenes, E. coli and C. jejuni. The strains were found to have reduced invasive capacity of S. Typhimurium [29], S. Enteritidis, L. monocytogenes, E. coli and C. jejuni [31] with varying degrees depending on the bacterial species. The ability of probiotics as both anti-adhesion and anti-invasion were also proved to be effective against S. Typhimurium and S. Enteritidis towards Caco-2 cells [26].

CO-CULTURE HUMAN INTESTINAL MODEL FOR ADHERENCE AND INVASION STUDIES
It is undoubted that one single cell line or monoculture does not adequately represent the human intestine. Consistent with the hypothesis that good adherence and invasion especially in the presence of mucus, the goblet cell has been successfully used to co-cultured with Caco-2 cells as an alternate technique to imitate in vivo human intestinal physiology. This consideration has led to a method for bridging the gap between simple in vitro models and in vivo biological process [12,53].
The co-cultures previously proposed in literature for evaluating the mechanism of bacterial adhesion and invasion were obtained by using mucus secreting HT29 subclones which is HT29-MTX to generate a mixed population of enterocytes and mucus secreting cells (Caco-2/HT29-MTX) resembling as closely as possible the intestinal epithelium ( Table  2).

Table 2. Caco-2/HT29-MTX co-culture model for bacterial adhesion and invasion
Description of study References Identification of potentially effective synbiotics for probiotics adherence towards intestinal mucosa Adhesion ability of probiotic, commensal and pathogenic bacterial strains Bacterial adhesion is influenced by surface characteristics, which are influenced by the structure and composition of the cell wall [1,12]. Krausova et al. [1] aimed to identify potentially effective synbiotics by analyzing the adherence of bacterial strains to a Caco-2/HT29-MTX coculture cell line model that mimicking the intestinal epithelium. They reported high adherence for all strains tested after integrating HT29-MTX cells for mucin synthesis in this model. Several intestinal bacteria have been found to be able to permeate the mucus glycan and use it as a carbon source and attachment site [57][58].
However, their results were in conflict with those obtained by Laparra and Sanz [33], who used similar model and found very low adherence by lactobacilli and bifidobacteria. They differentiated the adhesion pattern of probiotic, commensal and potentially pathogenic bacteria (E. coli and L. monocytogenes) on both Caco-2 monoculture and co-culture of Caco-2/HT29-MTX. Their findings demonstrated that bacterial adhesion values on Caco-2 monoculture were higher than those in Caco-2/HT29-MTX co-culture, implying that a mucin layer formed by HT29-MTX cells could cover putative recognition components in Caco-2 plasma membrane, rendering them inaccessible to bacteria. Above findings are supported by the fact that the presence of mucus in the model system is significant for estimating intestinal permeability as the mucus acts as a barrier against the absorption of certain compounds [54,59]. The lack of mucus in Caco-2, on the other hand, permits easy access to the cells, leading to an overestimation of their permeability [13]. Consequently, the presence of intestinal mucus may significantly affect adherence, as observed in studies with cell lines that secrete or do not secrete mucus [1].
With the goal of providing an epithelial monolayer covered with mucus that better mimicked the situation in vivo, Dostal et al. employed the Caco-2/HT29-MTX co-culture model to explore the interactions of the enteropathogen S. Typhimurium through adhesion and invasion towards intestinal cell under different iron concentrations. Under high iron conditions, Salmonella adhesion was increased by 8-fold compared to normal iron conditions while invasion was not significantly lowered. On the other hand, in low iron conditions, the invasion of Salmonella was significantly increased as compared to normal iron condition. Meanwhile, in 2020, Limage et al. [54] used Caco-2/HT29-MTX co-culture as in vitro model of gastrointestinal tract to determine how the mucus layer was affected by the presence of Gram-positive, commensal Lactobacillus rhamnosus (L. rhamnosus) and Gram-negative, opportunistic E. coli. They discovered that, when these bacteria were present and adhered to cells, the secretion of neutral and acidic mucins was altered, and the thickness of the mucus layer was enhanced. In contrast to Caco-2/HT29-MTX, monoculture of Caco-2 cells do not produce mucus and only displayed background levels of staining. However, co-culture of Caco-2/HT29-MT showed more intense staining following adhesion of L. rhamnosus and E. coli compared to that of unexposed cells.
Similarly, believing that co-cultures of Caco-2/HT29-MTX are better representation of the complex mucosa, Volštátová et al. reported their research in 2016 [55] and 2016 [60] regarding the adherence of probiotic Lactobacillus strain. All strains tested in their study (Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei) were all shown to adhere effectively to the co-culture cell lines. Kim et al. [56] also studied the adhering ability of probiotics to the identical Caco-2/ HT29-MTX model used herein since the ability of bacterial strains to adhere to intestinal epithelium cells was regarded as an important selection criterion for probiotics. They discovered that prebiotic fructooligosaccharides reduced the ability of probiotic E. coli (Nissle 1917) to adhere to cocultures Caco-2/ HT29-MTX.
It is of interest to note that until now, no research on the use of co-culture Caco-2 and HT29 cells (low mucus production) to study bacterial adhesion and invasion has been published. Mostly, the co-culture model of Caco-2/HT29 were used to study intestinal permeability of the nanogels and several peptide drugs [61][62]. Therefore, the co-culture of Caco2/HT29-MTX covered with mucus could be considered as the most suitable in vitro model of human intestinal epithelium for understanding the bacterial adhesion and invasion, thus the results obtained by using this co-culture model will simulate the human intestine as close as possible.

CONCLUSION
Caco-2 cell line has been undoubtedly the most used and accepted in vitro intestinal cell model to study the bacterial adhesion and invasion. However, the mechanism of adhesion and invasion are largely depending on the presence of mucus, an important protective component of the normal intestinal epithelium. Therefore, the HT29-MTX cell model may be more suitable for studying bacterial adhesion and invasion in vitro compared to Caco-2 and HT-29 cell models that secrete no or little mucus respectively. The coculture of Caco-2 cells with the mucus-producing HT29-MTX cell line has been referred as a more predictable experimental cell model than monoculture alone due to the production of mucus that is the most important feature similar to the human intestinal mucosa. Data from this review suggest that the development of coculture Caco-2/HT29-MTX cell model, with a better physiologically relevant characteristics of mucus layer formation, herein create an excellent in vitro system for characterizing cells-pathogens interactions via adhesion and invasion study.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.