Antibody-induced Multicellular Behaviors of Salmonella Typhimurium

Author

Samantha K. Lindberg, SUNY AlbanyFollow

ORCID

https://orcid.org/0000-0002-1377-609X

Date of Award

Winter 2025

Language

English

Embargo Period

1-11-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

School of Public Health

Program

Biomedical Sciences

First Advisor

Nicholas Mantis

Committee Members

Joseph Wade, Kathleen McDonough, Pallavi Ghosh, Craig Altier

Keywords

antibody, immunoglobulin A, Salmonella enterica, agglutination, motility, fimbriae

Subject Categories

Bacteriology | Immunology of Infectious Disease

Abstract

Salmonella enterica is a globally distributed bacterial pathogen that is commonly acquired through consumption of contaminated food and water. The enterica subspecies includes over 1500 unique subvariants, or serovars, that are classified by antigen presentation. Due to its global environmental prevalence, broad host range, and increasing pathogenicity, serovar Typhimurium is of particular concern for human and animal health, as well as food safety. Once in the gastrointestinal tract, Salmonella Typhimurium (STm) exploits host immune mechanisms, implements an arsenal of virulence factors, and outcompetes the microbiota to invade the intestinal epithelium. While STm infections are generally self-limiting and relatively mild, emerging multidrug-resistant and hyper-invasive isolates of STm cause more severe and potentially systemic infections, as well as limit the efficacy of current treatment options.

Secretory IgA (SIgA) antibodies represent the first line of defense at mucosal surfaces to protect against STm and thus represent a potential alternative to antibiotics. By virtue of its dimeric structure and high avidity, SIgA mediates a key effector function known as immune exclusion, a process in which invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. While immune exclusion is essential for protection in vivo, the underlying mechanism of IgA-mediated agglutination, and the extent of active bacterial contribution in this process, remains unknown.

The anti-lipopolysaccharide (LPS) monoclonal IgA Sal4 was generated from a mouse hybridoma in the early 1990s and demonstrates the capability to protect mice against STm infection through multiple routes of administration. In addition, previous studies have shown that Sal4 triggers motility arrest, inhibition of type 3 secretion, exopolysaccharide production, and agglutination of STm in vitro. Sal4 targets the O5 antigen component of LPS expressed on the surface of STm, and thus the impacts of Sal4 are dependent on expression of this epitope, which is directly and exclusively generated by the transacetylase encoded by the gene oafA. The results from these in vitro characterization studies of Sal4 collectively indicate that STm senses and adapts its behavior in response to Sal4 binding. From this, I hypothesized that Sal4 IgA acts as an environmental stress signal that drives STm to transition from a planktonic to sessile state, which leads to motility arrest, agglutination, and ultimately, biofilm formation.

In Chapter 3, I developed a method known as the snow globe assay to visualize and quantify Sal4-mediated agglutination of STm. Bacterial aggregation was shown to occur in a time-, dose-, and O5-antigen expression-dependent manner in the snow globe assay, thus validating results from previous studies. Non-motile strains of STm did not agglutinate in homogenous culture with Sal4 treatment, however, aggregation was induced with a nucleating factor, such as agitation or the presence of motile cells, that could generate collisions and consequently, the formation of aggregates. Alternatively, selected cyclic-di-GMP metabolizing enzymes previously implicated in motility arrest and EPS production did not appear to play a significant role in the process of Sal4-mediated agglutination in the snow globe assay. This indicates that the cellular collisions caused by motility drive agglutination of STm in the presence of Sal4 IgA and establishes the snow globe assay as an approachable and effective technique for measuring antibody-induced aggregation of STm in vitro.

In Chapter 4, I confirmed that a mutant strain of STm lacking oafA expression can outcompete WT over the course of four treatments with Sal4 IgA, even when outnumbered at a ratio of 1:6000. With this modified version of the snow globe assay, I performed a genome-wide CRISPR interference (CRISPRi) screen to identify genes that actively promote bacterial agglutination in response to repeated exposure to Sal4 IgA. A previously generated strain of STm 14028s containing a library of 36,651 plasmid-based spacers was treated twice with Sal4 IgA and next-generation sequencing was performed to determine the frequency of each spacer within the subpopulation present at the ALI. From this, I developed a custom analysis process to (1) exclude spacers below the cutoff for minimum read frequency, (2) calculate fold enrichment of spacers detected at the ALI between treatment groups, (3) align spacers to their target gene, and (4) identify genes that were targeted by more than one unique spacer. Ten percent of the de-enriched spacer list contained spacers that targeted genes linked to flagellar biosynthesis and complex assembly. Alternatively, oafA and fimW each had three unique spacers appear on the enriched list, indicating that silenced expression of these genes leads to enrichment of STm at the ALI. The gene product of fimW is a negative regulator of Type I Fimbriae (T1F) expression, and this function was confirmed with a quantitative mannose-sensitive yeast agglutination assay. The non-agglutinating phenotype of a fimW mutant was successfully complemented in trans, and further characterization revealed that this strain does not escape Sal4-mediated motility arrest or inhibition of epithelial cell invasion. However, the fimW mutant demonstrated enhanced biofilm formation relative to WT STm upon Sal4 treatment, indicating that overexpression of T1F can prime STm for the planktonic-to-sessile transition induced by Sal4.

This study presents the snow globe assay as a standardized, approachable, and adaptable technique to quantify antibody-mediated bacterial agglutination. Furthermore, the results from this dissertation provide compelling evidence that the anti-LPS monoclonal IgA Sal4 acts as an environmental stressor of STm. I propose a model where Sal4 binding induces a stress response in STm that involves motility arrest, agglutination, and biofilm formation occurring sequentially. Future studies are necessary to elucidate the molecular steps that facilitate Sal4-induced biofilm formation of STm and the underlying mechanisms of Sal4-mediated protection against STm.

License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Lindberg, Samantha K., "Antibody-induced Multicellular Behaviors of Salmonella Typhimurium" (2025). Electronic Theses & Dissertations (2024 - present). 101.
https://scholarsarchive.library.albany.edu/etd/101