Probiotics are defined as active microorganisms that are beneficial to the host, they can confer several benefits such as improving growth, digestion, and immunity (Parker, 1974; Zhang et al., 2018). Probiotics are widely used as alternatives to antibiotics. For example, Lactobacillus (e.g., L. casei, L. gasseri, L. helveticus, L. lactis, L. rhamnosus, L. reuteri, and L. acidophilus) and Lactiplantibacillus (L. plantarum and L. pentosus) have health-promoting effects (Kankainen et al., 2009; Zhang et al., 2018; Partrick et al., 2021; Wang et al., 2021), and are commonly accepted probiotics strains in the market.

Adhesion to the gut is important to the colonization of probiotics, which is conducive to the proliferation of probiotics but harmful to the pathogens in the intestine (Reid and Burton, 2002; Chen et al., 2007; Yadav et al., 2017). The molecules such as surface proteins, cell wall polysaccharides, peptidoglycans, bacteriocins, secreted proteins, and organic acids derived from probiotics Lactobacilli can mediate bacterial adhesion (Teame et al., 2020). The surface protein is one of the major adhesion molecules involved in probiotic mechanisms among different taxonomic groups of probiotics (Sanders et al., 2018). For example, L. casei surface layer protein could reduce the adhesion of pathogens to collagen and HT-29 cells, and thus decrease the pathogens-induced apoptosis of HT-29 cells (Meng et al., 2021). L. acidophilus surface layer protein also presented antibacterial effects against Staphylococcus aureus in combination with nisin (Wang et al., 2020).

Results from our previous studies suggested that the molecules located on the surface of L. plantarum HC-2 might participate in the competitive exclusion of Vibrio parahaemolyticus (Sha et al., 2016a), and the surface layer proteins of L. plantarum HC-2 were subsequently found to play an important role in its adhesion and colonization in the intestine of shrimp to competitively exclude the pathogens and promote beneficial bacteria growth (Du et al., 2019). However, whether other genes or proteins of L. plantarum HC-2 are involved in the adhesion and exclusion of pathogens remains unclear. Probiotics and pathogens compete for common cell receptors for binding to the intestine mucosa (Van den Abbeele et al., 2016; Singh et al., 2018). Thus, understanding the surface proteins of probiotics involved in host-probiotic interaction would provide new strategies for disrupting host-pathogen interaction.

Moreover, L. plantarum HC-2 could reduce the negative effects of aflatoxin by positively affecting the growth and resistance of P. vannamei (Fang et al., 2020). Thus, some other modes of action may exist for L. plantarum HC-2 to find a niche in the shrimp intestine. Different bacterial species may have unique biological activities, which may vary from one strain to another strain. So, the unique property is fundamental to understand and apply a specific probiotic strain (Minj et al., 2021). The characteristics of the genome and proteome could reflect the adaptability and function of the bacteria to specific niches, so that the probiotic properties of L. plantarum HC-2 can be predicted by analyzing its whole genome and proteome (O'sullivan et al., 2009; De Angelis et al., 2016).

This study mainly aimed to investigate which and how the genes and proteins of L. plantarum HC-2 affect the adhesion and exclusion of pathogens. We hypothesized that (i) the homologous genes shared between L. plantarum HC-2 and V. parahaemolyticus would affect the adhesion of L. plantarum HC-2 and competitive exclusion against pathogens in the mucus of shrimp by regulating the quantity of membrane proteins; (ii) the genome of L. plantarum HC-2 harbored key genes contributing to its adaptability to shrimp intestine niche. So, we first tested whether the shared genes between L. plantarum HC-2 and V. parahaemolyticus participated in adhesion of L. plantarum HC-2 to mucus, and examined the proteins involved in the adhesion of L. plantarum HC-2 to mucus and the response to V. parahaemolyticus exposure. Secondly, we analyzed the other probiotic characteristics of L. plantarum HC-2 based on its genome. We found that ftsH gene could affect the adhesion of L. plantarum HC-2 by regulating several membrane proteins, some of which play important roles in the competitive exclusion of pathogens. Arsenal genes in L. plantarum HC-2 genome are predicted to be involved in carbohydrate metabolism and bacteria-host interactions. These findings advance our mechanistic understanding of the selective adhesion of probiotics and competitive exclusion of pathogens in the intestine of shrimp, and have an important practical significance for the screening of probiotics, the optimization of feed additive formulations and the defense against diseases.

Probiotics are widely used as a kind of alternatives to antibiotics (Parker, 1974; Zhang et al., 2018). Adhesion of probiotics to the gut mucus is the first step to exert different effects against the activity of pathogens (e.g., competition, exclusion, displacement, and repression) (Reid and Burton, 2002; Chen et al., 2007; Yadav et al., 2017). This study clarified the genes and proteins of L. plantarum HC-2 involved in adhesion to shrimp intestine and competitive exclusion against pathogenic V. parahaemolyticus. The results generally supported our core hypothesis that the shared ftsH gene could influence the adhesion of L. plantarum HC-2 to the shrimp mucus by regulating membrane proteins. Also, the genome of L. plantarum HC-2 harbors many genes relating to carbohydrate utilization and probiotic-host interactions.

Probiotic bacteria adhering to the gut may compete the colonized niches with pathogens, and thus prevent pathogen infestation (Servin, 2004; Fukuda, 2017). The major driving forces for enhancing bacterial adhesion are protein–protein and protein-carbohydrate interactions. The bacteria mainly share molecular mechanisms of initial adhesion to the host, and the presence of common adhesion proteins on the surface of bacteria could affect the adhesion-related molecules (Westerlund and Korhonen, 1993; Pizarro-Cerdá and Cossart, 2006; Kline et al., 2009; Iniguez-Palomares et al., 2011; Singh et al., 2018). L. plantarum HC-2 and V. parahaemolyticus can be adhered to the intestine of Litopenaeus vannamei (Sha et al., 2016a), thus the adhesion-related proteins encoded by homologous genes of these strains may compete for adhesion niches in the intestine as they may play a common functional role (Edgar and Batzoglou, 2006). In this study, homologous sequences obtained among L. plantarum HC-2 and all V. parahaemolyticus genomes were finally assigned to 11 genes which can attribute to the essential cellular metabolic processes. Several of them show multiple functions in different subcellular localization, like proteins related to transcription and translation which not only attribute to the essential cellular metabolic processes in cells but also act as adhesins when emerged on the cell surface of bacteria, such as genes numbered HC2_GM000845 (rplF), HC2_GM000995 (prfC), and HC2_GM000828 (fusA). Moreover, the gene numbered HC2_GM000464 was annotated as ftsH and was found that there was only one copy in this genome. The ftsH gene was firstly found in Escherichia coli and then proved in almost all bacteria to perform cellular roles for FtsH (Schumann, 1999). FtsH is a ubiquitous membrane-integrated protein, an ATP-and Zn²⁺-dependent metalloprotease, which belongs to ATPase associated with different cellular activities (AAA) protein family and in responsible for regulating the protein stability of specific and critical regulators to control cellular processes. Given the critical role of FtsH in cellular processes, the ftsH gene was selected as the target gene for mining adhesion-related biomarkers among 11 genes. In this study, the double-crossover mutagenesis was performed and obtained nearly 5,000 mutants one ftsH mutant. The number of mutants showed desired genotype only 0.02% due to the fact that the second homologous crossover is random. Thus, it is difficult to obtain the desired genotype at the absence of selection pressure, and a suitable method should be developed to get target mutagenesis of L. plantarum.

FtsH protease is very important for controlling the membrane protein, heat shock response, lipopolysaccharide biosynthesis, viral infection, and mediating colicin activity in E. coli (Schumann, 1999), and it could also regulate the cytoplasmic substrates in Caulobacter crescentus and Bacillus subtilis (Langklotz et al., 2012). However, the FtsH is dispensable for normal growth of bacteria except that survival upon stress conditions like heat shock or osmotic stress (Beier et al., 1997; Deuerling et al., 1997; Fischer et al., 2002; Lithgow et al., 2004; Fiocco et al., 2009; Langklotz et al., 2012). In L. plantarum, the FtsH mutant reduced the capacity to form biofilms and changed the physico-chemical surface properties, which possibly linked to the alterations of surface charge and/or architecture (Bove et al., 2012). Given that in present study, the bacteria were washed three times with phosphate buffer solution before being used to test their adhesion to mucus, and there was basically no difference in charge on the surface of cells. However, we found that the FtsH mutant increased the adhesion ability to the mucus (Garrett et al., 2008). This is consistent with previous studies, which identified that the down-expression of FtsH could promote biofilm formation in L. plantarum and E. coli (Langklotz et al., 2012; Gu et al., 2021). Therefore, the absence of ftsH in L. plantarum HC-2 might mainly alter the membrane architecture depending on the regulatory protease activity of FtsH (Garrett et al., 2008; Langklotz et al., 2012; Gu et al., 2021).

The obtained mutant strains increased the adhesion ability to mucus of shrimp, and comparative proteomic analysis was further performed to create a molecular map of the adhesion regulation processes. 1355 proteins were present in both groups, while 13 and 20 proteins were uniquely identified in wild type and mutant strains, respectively (Figure 1B). There was basically no change in the number of total proteins expressed, but the number of uniquely expressed proteins exceeded 1% in each group. A total of 80 differential expressed proteins were found, including 54 up-regulated proteins. Many increased functional activities in the mutant strains reveal that the improvement of L. plantarum HC-2 adhesion requires the mobilization of its more basic biological and cellular processes. In addition, the proteomic results showed that the expression of FtsH was still existed in the mutant strains with 10.56% down-regulation (data not shown) and showed no significant difference from the wild type strain, suggesting that the mutants were partially repaired. This speculation could be supported by the elevated abundance of proteins for reducing damage such as proteins involved in enzyme metabolism and DNA repair pathways (Figure 2). L. acidophilus also responded to oxidative stress in the similarly way (Calderini et al., 2017). However, further studies are needed to clarify how the missing ftsH gene is repaired. A 10.56% decrease in FtsH expression already altered the adhesion ability of this group, so the results in this study can be used for subsequent analysis to test our hypotheses.

Surface proteins play an important role in the adhesion of bacteria to mucus in two ways. The first one is that surface proteins are bound to the membrane by hydrophobic transmembrane domain(s) and lipoprotein, and the second is that surface proteins are attached to the cell surface by noncovalent or recognizing some cell-wall-binding domains (e.g., LPXTG, CWBD1, and GW proteins) (Deepika and Charalampopoulos, 2010). We found that the differentially expressed proteins were predicted to be located in the cytoplasm and membrane rather than the cell wall, suggesting that FtsH mainly affects the adhesion of L. plantarum by regulating the expression of the membrane proteins.

Cell division inhibitor MinD (protein ID: G0M1M7) participates in the division of cell, and the overexpression of MinD could result in filamentation of cell by enhancing MinC-mediated inhibition of cell division (Nguyen et al., 2013). However, the minC gene was identified as antimicrobial activities other than being involved in cell division in L. acidophilus (Nguyen, 2012; Nguyen et al., 2013). Thus, the members of cell division inhibitors may play different roles in different strains. In this study, the increased MinD in membrane may result in filamentation of cell which may participate in the adhesion as an anchoring site for other adhesion molecules. The protein I9L3P8 encoded by choS was reported to have a compatible solutes glycine betaine/carnitine/choline transport system (Rossi et al., 2016). Moreover, choline was found to be a direct precursor of phosphatidylcholine, which was a major lipid membrane component in the Rhizobiaceae family (Dupont et al., 2004). The choS was also involved in tolerance to stress from the external environment (Rossi et al., 2016), so the higher expression of gene encoding for I9L3P8 may be a response to protect itself against pathogens and enhance adhesion to the host. The A0A2S9W1Y1 is annotated as ATP synthase subunit a, which is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation (Ishmukhametov et al., 2017). These three proteins were significantly up-regulated in L. plantarum HC-2 when it cocultured with V. parahaemolyticus E1 for 6 h and 12 h while down-regulated at other time points. Biological membranes are a mix of membrane proteins and their lipid partners. One potential consequence of membrane crowding is that this conformational change could cause a change in free energy that is associated with the area available to the rest of the proteins, resulting in depletion forces (Phillips et al., 2009). These changes play a critical role in sensory and signaling pathways. Therefore, during this experiment, it was observed that the expression of genes can at times be up or down. The similar gene-expression patterns were also found in Pseudomonas.syringae pv. actinidiae during the first five days after infection of kiwifruit (McAtee et al., 2018), in which the expression of several genes from the plasmid-borne putative aromatic pathway showed a fluctuating pattern over the infection time course. In addition, compared with V. parahaemolyticus E1 after incubation alone, aphA in V. parahaemolyticus E1 was also observed to be up-regulated at 2 h and 6 h but down-regulated at 4 h and 8 h when V. parahaemolyticus E1 was cocultured with the same number of its own cells at 28°C (Sha et al., 2016a). The probiotics and pathogens share similar nutritional substrates, especially the amino acids. Thus, the gene encoding amino acid permeases (A0A241RSP9) was induced at earlier time. As above mentioned, four proteins (G0M1M7, I9L3P8, A0A2S9W1Y1, A0A241RSP9) may be involved in the competitive exclusion against pathogens in the mucus of shrimp by regulating their quantity of proteins on the surface of L. plantarum HC-2.

The application of strains is benefit from their adaption to the host intestinal environment and interaction with the host. As a kind of facultatively heterofermentative LAB, L. plantarum HC-2 genome possesses genes for both the phosphoketolase and Embden-Meyerhof pathways (EMP). Thus, it mainly ferments carbohydrate via EMP by using glucose. However, in the absence of six-carbon sugars, it can also ferment five-carbon carbohydrates such as arabinose or ribose via the phosphoketolase pathway (Bustos et al., 2005; Abriouel et al., 2017). As animals have limited evolved abilities to digest complex polysaccharides (Cantarel et al., 2012), the enzymes produced by intestinal microorganisms are needed to metabolize carbohydrate (e.g., xylan, β-glucan, cellulose, and chitosan). The genes encoding carbohydrate-modifying enzymes could be used to predict the possible adaption and enrichment of bacteria in the intestine (Abriouel et al., 2017). In present study, many enzymes involved in degrading of xylan and β-glucan are found in L. plantarum HC-2 genome. As the accessory enzymes for the degradation of xylan, acetyl xylan esterase (four genes, Table 3), glycerol-3-phosphate cytidylyltransferase, eukaryotic-like serine/threonine-protein kinase, xylan endo-1,3-beta-xylosidase activity and xylanase were predicted in L. plantarum HC-2 genome sequence, which can remove the terminal acetate residues from xylan polymers and act in the xylan catabolic pathway (Kim et al., 2020). Furthermore, 12 genes coding for enzymes involved in the degradation of beta-glucan were also predicted in L. plantarum HC-2 (Supplementary Table S3). Xylan occurs in nearly all plants and some marine algae, and could act as immune modulators to enhance animal immunity (Yang et al., 2019). However, as a kind of soluble non-starch polysaccharides (SNPs), the presence of xylan in diet could also interfere with feed utilization and negatively affect animal performance (Sinha et al., 2011; Yao et al., 2019). Beta-glucan often used as a feed additive in aquaculture, which can enhance fish and shrimp innate immune responses (Chang et al., 2003; Yamamoto et al., 2018), but it is also a kind of SNPs. Therefore, the presence of these enzymes is predicted to contribute to the degradation of xylan and beta-glucan in the intestine. Interestingly, the L. plantarum HC-2 genome has 22 genes responsible for the cellulose synthesis, including cellulose synthase (Supplementary Table S3). The cellulose synthase (17 genes) could accumulate cellulose on the cell wall surface as an extracellular matrix for adhesion of cell and formation of biofilm for protecting the bacteria (Abriouel et al., 2017). Over 7% (229 of 3,259 genes) of the identified genes in L. plantarum HC-2 are involved in carbohydrate metabolism, which is similar to the genome of L. pentosus MP-10 (Abriouel et al., 2017). The abundance of carbohydrate metabolism genes is important for L. plantarum HC-2 to adapt the intestinal environment, which may enhance its possibilities of survival and persistence and competitiveness to pathogens within the intestine of host.

Probiotic lactobacilli can express adhesion-related proteins to mimic the colonization process of pathogens, resulting in several probiotic effects. The identification and characterization of these proteins, often strain-specific, involved in interaction with the host are necessary to evaluate a priori the probiotic potential of Lactobacillus sp. Candidates (Abriouel et al., 2017). In this study, the possible interaction of L. plantarum HC-2 with the host was bioinformatically predicted from the genome sequence. Several adhesion-related proteins were predicted, such as cell surface proteins, LPXTG cell wall anchor domain-containing proteins and elongation factors. However, the role of these proteins in L. plantarum HC-2 has not yet been determined, requiring further mutation or proteomic studies for this purpose.

In summary, this study identified and clarified the underlying mechanisms of genes and proteins of L. plantarum HC-2 involved in adhesion to intestine and competitive exclusion against pathogenic V. parahaemolyticus. The findings advance our mechanistic understanding of the selective adhesion of probiotics and competitive exclusion of pathogens. Also, the repertoire of genes identified in the L. plantarum HC-2 genome have an important implications for its applications in other animals as a potential probiotic.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, SRR21010423. http://www.proteomexchange.org/, PXD036035. https://www.ncbi.nlm.nih.gov/, PRJNA868485.

The animal study was reviewed and approved by Science and Technology Ethics Committee of Dezhou University (Dezhou University, Dezhou, China).

YS: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, resources, supervision, visualization, writing—original draft, and writing—review and editing. QY: formal analysis, writing—original draft, and visualization. JL: methodology and funding acquisition. JY: conceptualization and writing—review and editing. SX: writing—review and editing. ZH: writing—review and editing. JR: conceptualization and writing—review and editing. JQ: conceptualization and writing—review and editing. SZ: writing—review and editing. GW: data curation and methodology. WD: data curation and methodology. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (31802309), the Natural Science Foundation of Shandong Province (CN) (ZR2017BC027), Research Foundation for Advanced Talents of Dezhou University (2019xgrc35).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.