# Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of _Helicobacter pylori_
## 4 Phenotypic Variability as a Modulator of _H. pylori_ Biofilm Structure
### Metabolic Activity and Matrix Production  
Metabolomic studies on a selected collection of _H. pylori_ strains, defined as weak and strong biofilm producers, showed that the former were characterized by a more intense and wider range of produced metabolites than strong biofilm producers [57]. These data are consistent with the observations of [PERSON] et al. [21; 22], who showed a significant reduction in the expression of genes related to metabolism in biofilm _H. pylori_ forms. Nevertheless, it should be remembered that biofilm forms are still metabolically active, and this activity may influence the composition of biofilm matrix [57].  
Using confocal laser scanning microscopy (CLSM) and selective staining of biofilm components, it was found that the _H. pylori_ biofilm matrix consists of proteins, extracellular DNA (eDNA), and sugars [21; 24]. However, it was pointed out that proteins seem to be the most important units of the biofilm, because the treatment with proteinase K at any stage of biofilm development caused its significant degradation. The eDNA was also important (confirmed by the degradative activity of DNase), but only in the early stages of the biofilm formation.  
In addition to the above illustrative results showing the components of _H. pylori_ biofilm, others focused on the detailed chemical characteristics of this structure [20; 56; 90]. In the first study, it was determined that this polymer consists of various sugars, including fucose, glucose, galactose, N-acetylglucosamine, and N-acetylineuraminic acid, which largely constitute components of LPS and peptidoglycan [20]. Fatty acids were also represented by the compounds derived from _H. pylori_ LPS, i.e., tetradecanoic acid (C\({}_{14}\)), hexadecanoic acid (C\({}_{16}\)), and octadecanoic acid (C\({}_{18}\)). Covering amino acids, these were predominated by: asparagine/aspartic acid, glutamine/glutamic acid, glycine, alanine, and leucine. Another study focused on the protein and sugar components of _H. pylori_ biofilm [56]. The main sugar in mature biofilms was mannose (approximately 80%), the dominance of which was associated with numerous proteomannans (1,3- and 1,4-mannose bonds). Among proteins, attention was also paid to the importance of neutrophil-activating protein A (NapA) in biofilm formation (\(\Delta napA\) mutants showed reduced ability to create this structure) [56]. This may be linked with the strongly positive charge of this protein [91], explaining the preference of this bacterium for adhesion to negatively charged surfaces [24]. The last study determined surface-related _H. pylori_ sugars, and found that these components mainly include LPS components, mannose-rich glycosides, and \(\alpha\)-glucans [90]. A correlation was also found between the culture conditions (and thus the type of biofilm) and the sugar composition of the _H. pylori_ cell surface. In strains grown on solid media, an increase in the number of amyloids (\(\alpha\)-(1,4)-D-glucan amyloids) and a decrease in the number of LPS-related sugars was observed, while in liquid cultures the relationship was reversed.  
All the above results indicate that the chemical components (their composition and quantity) may have a direct impact on the structure and stabilization of _H. pylori_ biofilm. Moreover, many of these compounds are derived from structural organelles of bacterial cells. Thus, in order to better understand the complexity of the biofilm matrix, it is also necessary to take a closer look at the role and function of individual organelles in the physiology of _H. pylori_.