# The Influence of Lipopolysaccharide O‐Antigen Chain Length on Biofilm Formation Capacity and Outer Membrane Proteome Shape of Salmonella Enteritidis

## TL;DR

This study shows how the length of a part of Salmonella's cell wall affects its ability to form biofilms and changes the proteins in its outer membrane.

## Contribution

The study reveals how O-antigen chain length in Salmonella Enteritidis influences biofilm formation and outer membrane proteome composition.

## Key findings

- Shorter O-antigen is linked to reduced biofilm biomass, depending on the culture medium.
- Shortened O-antigen alters bacterial aggregation, hydrodynamic size, zeta potential, and hydrophobicity.
- Changes in O-antigen affect proteome composition, including upregulation of LPS biosynthesis proteins.

## Abstract

Biofilm formation is a phenomenon of great medical importance, also affecting food production. In the present work, we investigated the effect of the O‐antigen length of lipopolysaccharide (LPS) of
Salmonella
Enteritidis on biofilm production and the physicochemical properties of Salmonella cells, using bacterial deletion mutants. We also analysed the influence of LPS O‐antigen shortening on the composition of the outer membrane (OM) proteome of
S.
Enteritidis. We have shown that the shortening of the LPS O‐antigen part is associated with decreased biofilm biomass formation in some mutants and that it also depends on the composition of the culture medium. Physicochemical properties of bacterial cells changed with the shortening of the O‐antigen, promoting bacterial aggregation and influencing their hydrodynamic size, zeta potential, or hydrophobicity. We have also shown that shorter O‐antigen alters the bacterial proteome in comparison to regular size O‐antigen: flagellar FliC protein was down‐regulated in most mutants, while the HptG as well as 50S ribosomal protein L7/L12 protein were up‐regulated, suggesting increased protein synthesis activity. In some mutants, proteins involved in LPS biosynthesis were also upregulated: lipopolysaccharide core heptose(II)‐phosphate phosphatase, acyl carrier protein, and undecaprenyl‐phosphate alpha‐N‐acetylglucosaminyl 1‐phosphate transferase, implying that the increased LPS biosynthesis is aimed at the replacement of the lacking LPS modal fractions in the
S.
Enteritidis mutants.

Shortening of the LPS O‐antigen part is related to decreased biofilm biomass formation, which also depends on the composition of the culture medium. Physicochemical properties of bacterial cells changed with the shortening of the O‐antigen, promoting bacterial aggregation, hydrodynamic size, zeta potential, and hydrophobicity. Additionally, shorter O‐antigen alters the bacterial proteome in comparison to regular‐sized O‐antigen.