The human stomach was considered to be microbiologically sterile before the successful culturing of Helicobacter pylori from gastric biopsy tissue (Marshall and Warren, 1984). It was shown that gastritis and stomach ulcers in humans are caused by the Gram-negative, urease producing bacterium (Marshall and Warren, 1984). Later, it also became clear that this bacterium is a major risk factor in the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (Kusters et al., 2006). In developing countries, 70–90% of the population is infected with H. pylori; while in developed countries, the prevalence of H. pylori is 25–50% (Solnick et al., 2003; Obiageli and Ivan, 2016).

Besides H. pylori, the human stomach can also contain transient oral, esophageal or intestinal bacteria and is highly dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Dicksved et al., 2007). These microorganisms may either be permanent members of the gastric microbiota but not picked up due to limitation of conventional microbiological culturing methods or may be in transit in the stomach (e.g., together with food intake). However, a change of the physiological conditions of the stomach, as occurs during acid-reducing drug therapy, corpus atrophy or gastric cancer, provides an opportunity for foreign microbes to enter and colonize the stomach (Dicksved et al., 2007).

Streptococci are members of the normal intestinal flora of healthy individuals which exert antagonistic activities against many intestinal pathogens (Heczko et al., 2006). A strong correlation was found between the presence of Streptococcus salivarius and H. pylori where 83% of the S. salivarius positive biopsies also harbored H. pylori. S. salivarius is known to have urease activity which creates a less acidic environment and could further enhance the survival and incidence of H. pylori (Ryan et al., 2008). Hence, streptococci may potentially survive and develop in an acidic gastric environment as an indigenous microbiota of the gastric mucosa, which may in turn inhibit the colonization by H. pylori (Adolfsson et al., 2004; Johnson-Henry et al., 2004; Uziel et al., 2004). In our previous studies, we have shown that Streptococcus mitis can be isolated from human gastric tissue biopsies (Khosravi et al., 2014a) and co-culturing S. mitis and H. pylori released metabolites that induced H. pylori to transform into viable but non-culturable (VBNC) coccoidal form in vitro (Khosravi et al., 2014b). On the other hand, culturability of S. mitis in the co-culture was enhanced. In this current paper, we completed our analysis by analyzing the changing protein profiles of H. pylori and S. mitis to understand the different response of the two bacterial species in a multi-species micro-environment. While it is not surprising that H. pylori changes to coccoid in a multi-species micro-environment, the enhancement of S. mitis survival capability deserve further investigation to determine any potential pathogenic role of this bacterium in the human gastric environment in the presence of H. pylori.

A total of 1514 H. pylori and 1414 S. mitis proteins - identified based on −logP ≥20, ≥1 unique peptide(s) (FDR <1%) and were detected in ≥2 of the triplicates - in both mono- and co-cultures (day 1, 2, and 4) are presented as Venn diagrams (Figure 1). Complete list of proteins identified can be found in the Supplementary Materials with confidence of identification and peptide data.

H. pylori proteins that were found to be significantly different between mono-cultured and co-cultured H. pylori were mapped to 12 and six biological pathways respectively (Table 1). Proteins involve in RNA degradation, nucleotide excision repair, mismatch repair, and lipopolysaccharide (LPS) biosynthesis were relative more abundant in co-cultured H. pylori. On the other hand, proteins involve in citrate cycle, glycolysis/ gluconeogenesis, aminoacyl-tRNA biosynthesis, translation, metabolism, and cell signaling were less abundant in co-cultured H. pylori. This is consistent with the observation that in the presence of S. mitis, H. pylori was transformed to coccoid (Khosravi et al., 2014b). H. pylori coccoid has been demonstrated to have low metabolic enzymes (FBA, EDD, AcnB, FumC, OorA, and ICD as shown in Table 2B) but proteins involved in DNA biosynthesis remained high (Loke et al., 2016). Despite that H. pylori coccoid cannot be cultured in vitro, it has been reported that the coccoid had a stronger inhibitory effect on proliferation and weaker apoptotic effect than its spiral counterpart (Li et al., 2013), which suggest that the coccoid may be an important factor in gastric cancer progression. However, contradictory to the earlier report (Loke et al., 2016), proteins involve in epithelial cell signaling during H. pylori infection were reduced and those involved in LPS biosynthesis (Table 1) were increased in H. pylori coccoid induced by co-culturing with S. mitis. These differences may highlight differences between H. pylori coccoids induced by various means (prolonged culturing vs. co-culturing with S. mitis) and age of coccoids (3 months old vs. 4 days old). Furthermore, the role of H. pylori coccoids in a multi-species environment and its impact on gastric pathogenesis has not been fully assessed.

Among proteins identified, 27 proteins satisfied the criteria to be selected for label-free quantification analysis using the Peaks software. In contrast to 23 proteins that were significantly different in expression level between mono- and co-cultured H. pylori (Table 2B), only 4 proteins were found to be significantly different between mono- and co-cultured S. mitis (Table 2A). This suggests that multi-species environment may have a greater impact on H. pylori than S. mitis.

Among the differentially expressed proteins, phosphoglycerate kinase (PGK), which is required for ATP generation in both the glycolytic pathway of aerobes and the fermentation process of anaerobes (Yoshida and Tani, 1983), was only expressed by co-cultured S. mitis (Table 2A and Figure 2A). In addition, PGK is one of the predominant surface-associated proteins of streptococci, such as S. oralis (Wilkins et al., 2003) and group B streptococci (Hughes et al., 2002). Interestingly, sera directed against PGK was shown to protect neonatal animals from S. agalactiae infection suggesting that this protein may be essential for multiplication or adhesion of streptococci in vivo (Hughes et al., 2002). The expression of S. mitis PGK in the presence of H. pylori might have contributed to the enhanced survival of S. mitis, which was demonstrated earlier by our group (Khosravi et al., 2014b).

Consistent with the reduction in abundance of enzymes involve in citrate cycle detected in the co-cultured H. pylori (Table 1), the expression of citrate cycle enzymes (AcnB, FumC, OorA, and ICD) were also found to be lower in co-cultured H. pylori (Table 2B), The citrate cycle is most sensitive to reactive oxygen species (ROS; Janero and Hreniuk, 1996). Thus, reduced level of expression of citrate cycle enzyme may indicate reduced oxidative stress response of H. pylori in the presence of S. mitis. This viewpoint is further supported by reduced expression of glutathione metabolism enzymes (ICD and PepA), thioredoxin (TrxA), flavodoxin (FldA), and thiol peroxidases (TPX and TsaA) in co-cultured H. pylori.

The expression of thioredoxin (TrxA), a small redox-regulating protein that is involved in maintaining the thiol/ disulfide balance in both prokaryotes and eukaryotes (Holmgren, 1985), was significantly reduced in 4 days old co-cultured H. pylori (Figure 2B). This protein is essential for protecting bacteria, such as Bacillus subtilis (Scharf et al., 1998; Uziel et al., 2004), Bacteroides fragilis (Tally et al., 1975; Rolfe et al., 1997) and Salmonella species (Bjur et al., 2006), against oxidative stress for survival and replication. Interestingly, TrxA is also highly expressed in many cancers, including lung (Kim et al., 2003), cervix (Hedley et al., 2004), pancreatic (Han et al., 2002), colorectal (Raffel et al., 2003), hepatocellular carcinomas (Choi et al., 2002), gastric carcinomas (Grogan et al., 2000) and breast cancer (Cha et al., 2009). TrxA has been postulated to contribute toward cancer progression by playing crucial roles in maintaining cellular redox homeostasis and cell survival (Trachootham et al., 2008). Up-regulation of TrxA and related proteins has been postulated to present a dynamic redox change to drive proliferation and malignant progression of tumors (Karlenius and Tonissen, 2010). In the presence of S. mitis, the expression of H. pylori TrxA was reduced suggesting that S. mitis may potentially reduce the risk of H. pylori-associated gastric cancer development and/ or progression in the human stomach.

Alkylhydroperoxide reductase of H. pylori, which protects the bacterium from a hyperoxidative environment by reduction of toxic organic hydroperoxides, has been shown to function as a molecular chaperone for prevention of protein misfolding under oxidative stress (Chuang et al., 2006). This study highlights the importance of translation (elongation factors) and protein folding (chaperones) to H. pylori in response to oxidative stress. Thus, low level of expression of chaperones, such as TrxA, JHP_0216 and DnaK (aka 70 kDa chaperone; Table 2B), implies relatively low oxidative stress level in co-culture H. pylori. It has been shown that both bacterial factors and host inflammatory response causes oxidative stress on the gastric epithelium during H. pylori infection that may lead to apoptosis and tissue damage (Ding et al., 2007). Thus, low oxidative stress confers by H. pylori in a multi-species environment can be expected to be less pathogenic.

In conclusion, using S. mitis and H. pylori as model organism, data from this in vitro study suggest that in a multi-species setting, S. mitis may be able to benefit from cross-talking with H. pylori to enhancing its survival in the adverse gastric environment. Simultaneously, S. mitis may protect H. pylori from excessive oxidative stress. This in vitro co-culture model emphasizes the possibility that inter-species interaction may protect the host against bacterial-associated pathogenesis and carcinogenesis. However, this study is preliminary and the human gastric environment is highly complex and dynamic. Therefore, more evidences are required in order to fully understand the implication of H. pylori-gastric microbiota crosstalk and its impact on the development of gastroduodenal diseases in human.

Conceived and designed the experiments: YK, MFL, KLG, JV. Performed the experiments: YK. Analyzed the data: YK, MFL.

University of Malaya-Ministry of Higher Education (UM-MOHE) High Impact Research (HIR) grant (reference UM.C/625/1/HIR/MOHE/CHAN-02; account no. A000002-50001, “Molecular Genetics”).