# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 3 Transcriptomic and Proteomic Analysis of Biofilm _H. pylori_ Forms  
The development of biofilm structure and the transition from planktonic to biofilm phase are influenced by changes in the expression and translation of _H. pylori_ genes. These changes were determined experimentally using 'omics' techniques (transcriptomics, proteomics, and metabolomics), and despite some differences between the results obtained by independent research teams, it is still possible to identify the most important features of the biofilm forms of this bacterium. In these studies, an increased expression has been demonstrated for: global pleiotropic regulators [21; 22; 49; 50], adhesins (especially outer membrane proteins from the Hop and Hom family) [21; 22; 51; 52], lipopolysaccharidesaccharide (LPS) [21; 22; 51], efflux pumps [22; 37; 38; 39; 53; 54; 55], flagella proteins [21; 43; 51], components of T4 SS systems [21; 22; 43; 51], enzymes regulating pH (e.g., urease or arginase) [43; 56] and responsible for obtaining alternative energy sources (e.g., hydrogenase) [22; 43], proteins related to the cell wall rearrangement [22; 43], and proteins of the toxin-antitoxin system [47] (Figure 1). Covering the components for which a decrease in the expression in biofilm _H. pylori_ forms has been shown, these were factors involved in metabolism [21; 22; 57], translation [21; 22], and quorum sensing related to the autoinducer-2 (AI-2) activity [22; 46; 48; 55; 58; 59] (Figure 1). The influence of the transition of _H. pylori_ into the biofilm form on the amount of antioxidant proteins is difficult to determine, because [PERSON] et al. [56] and [PERSON] et al. [43] showed their increase, while [PERSON] et al. [22] showed an opposite situation. It is possible that this difference results from the type of analyzed biofilm. [PERSON] et al. [56] and [PERSON] et al. [43] studied biofilm cells that were exposed to oxidative stress (air-liquid biofilms or these formed as microaggregates in the medium), while the one studied by [PERSON] et al. [22] was a sedentary biofilm produced at the bottom of wells, where the oxygen concentration is significantly lower. Among the proteins mentioned above, it is worth paying attention to the production of global regulators of _H. pylori_ physiology, because they will influence the expression of other genes under  
Figure 1: Diagram showing transcriptomic/proteomic changes in _H. pylori_ cells during the transition from planktonic to biofilm phase. The scale bar shows 1 \(\upmu\)m.  
their control. In general, negative regulators bind to the promoter, reducing the attachment of RNA polymerase and lowering the expression of specific genes. In the case of positive regulators, binding is observed in the promoter's upstream region, which determines the recruitment of polymerase and the increase in gene expression [60; 61]. [PERSON] et al. [43] showed that aconitase production increases in the biofilm phase. Aconitase acts as a pleiotropic regulator supervising antioxidant functions, motility and production of flagella, as well as the activity of some enzymes (urease and hydrogenase) [62]. It was established that the high activity of _H. pylori_ hydrogenase is a feature of strains with a high carcinogenic potential [63]. Through hydrogenase, this bacterium can use molecular hydrogen (H\({}_{2}\)) to switch to the chemolithoautotrophic mode and use the energy from this process to bind CO\({}_{2}\)[64]. Hydrogenase is also crucial for the functioning of the T4 SS systems, both Cag-T4 SS (involved in cytotoxicity and oncogenicity) and ComB-T4 SS (involved in DNA transformation) [63]. It is worth noting that the ComB system is inactive at acidic pH and is stimulated in the environment with pH \(>\) 6.5, indicating that the transformation process takes place in close proximity to the gastric mucosa [65] and explaining the increase in urease production during biofilm formation (when microbial transformation is strongly intensified) [56]. The increased expression of _H.