# 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
### Secretion of Outer Membrane Vesicles  
When considering the extracellular structures of _H. pylori_, which play an important role in biofilm formation, membrane vesicles should be mentioned. Membrane vesicles are spherical structures with nanometric dimensions that transport a variety of chemicals, delivering them to the target site in an effective concentration and conditioning protection against environmental factors, such as the action of nucleases and peptidases [92; 93; 94]. Membrane vesicles are produced in all phases of microbial growth and secreted by both Gram-positive and Gram-negative bacteria, including _H. pylori_[95; 96]. The research group of [PERSON] et al. [97] found that significant amounts of eDNA can be found in _H. pylori_ biofilm samples. However, the existence of denoting differences in intracellular and extracellular DNA profiles was determined with the help of random amplified polymorphic DNA (RAPD) analysis, indicating that cell lysis is not the main source of this component in biofilm. Further studies by this team determined that the vast majority of eDNA in _H. pylori_ biofilm is related to OMVs [25]. It was also found that OMVs secreted by biofilm cells were secreted more intensively (approximately 24,800 OMVs/mL), had four times more eDNA, and possessed a more negative charge (\(-\)30 mV) than that produced by planktonic cells (18,000 OMVs/mL and \(-\)25 mV) [25; 98]. On this basis, the participation of OMVs in genetic variation and scaffolding of biofilm has been suggested [25; 97; 98]. The assistance of these extracellular structures in the formation of _H. pylori_ biofilm may play an important role in the early stages of this structure formation, which seems to be confirmed by the observations of [PERSON] et al. [24] and [PERSON] et al. [99]. This could be related to the generation of negative surface charge by OMVs, which is preferred for \(H\). _pylori_ adhesion [24]. Although this type of mechanism has not yet been confirmed experimentally, the SEM analysis of \(H\). _pylori_ cells adhering preferentially to OMVs clusters seem to support the above hypothesis (Figure 5). The involvement of OMVs in the formation of \(H\). _pylori_ biofilm was also independently investigated by a Japanese team led by [PERSON] et al. [99; 100; 101]. During the analysis of eight strains of \(H\). _pylori_, it was noticed that one of them--\(H\). _pylori_ TK1402--was characterized by several times more intense biofilm production [99]. During microscopic observations, it was noticed that the main component of the matrix were OMVs. Further analysis of this group showed that OMVs from the \(H\). _pylori_ TK1402 strain possessed an outer membrane protein with a specific amino acid sequence [100; 101]. This protein was Omp21 (also referred to as HopB or AlpB) and the variable region at positions 121-146 was crucial for its adhesive function [101]. In the comparison of \(H\). _pylori_ TK1402 with other strains, it was found that the former expressed _alpB_ faster (24-h culture) than the others (48 h). Additionally, in \(H\). _pylori_\(\Delta\)_alpB_ mutants a 10-fold reduction in biofilm formation capacity was demonstrated. The above data show that the proteins included in OMVs can play an important, eDNA-independent, structural function in \(H\). _pylori_ biofilm. In proteomic studies of \(H\). _pylori_ OMVs, it was determined that the growth phase of this bacterium influences the size, composition, and preferential cargo of these extracellular structures [102; 103]. Among 260 proteins found in OMVs, 171 were specific for these structures (not found in bacterial cells) [102]. Proteome of OMVs was represented by outer membrane proteins, efflux transporter/pump proteins, cytotoxic proteins (e.g., VacA and CagA), as well as numerous flagella components and enzymes with various activities, including catalase, urease, carbonic anhydrase, peptidoglycan modification enzymes, and beta-lactamases. It should be pointed out that many of these enzymes play an important role in the environmental adaptation of \(H\).