The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.

The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.
Lambert MA, Smith SG. The PagN protein of Salmonella enterica serovar
Typhimurium is an adhesin and invasin. BMC Microbiol. 2008 Sep 8;8:142.

Salmonella is a gram negative bacteria and is a member of the family Enterobacteriaceae. The nomenclature of Salmonella is complex. The earlier nomenclature was based on clinical considerations such as S. typhi-murium (mouse typhoid fever). When serological analysis was adopted, a Salmonella species was identified as a “group of related fermentation phage type” and each Salmonella serovar (serological variants) was classified as a separate species. Later, molecular studies led to the hypothesis that all Salmonella serovars form a single DNA hybridization group i.e., a single species and this species was composed of seven sub-species. The species name that was adopted was Salmonella enterica. Each of the sub-species contains various serovars. S. enterica serovar Typhimurium is one such species (belongs to sub-species 1 and serovar is Typhimurium).
It infects a wide range of animal hosts and caused self limiting gastroenteritis. The first step in pathogenesis is attachment of S. enterica serovar Typhimurium to the intestinal epithelial cells with the help of the fimbrae. The outer membrane proteins Rck and OmpD also mediate attachment. After attachment the subsequent process of uptake of Salmonella into mammalian cells is complex process. This process is coordinated by a series of proteins that are encoded within the SP-1 and SP-5 pathogenecity islands. The SP-1 locus encodes a type 3 secretion system (T3SS) that delivers effective protein into the host cells. The net result of ejecting of these effector proteins is entry of the organism into non-phagocytic cells.
Transcription of the virulence genes of Salmonella is regulated by the PhoPQ system. This system consists of PhoQ membrane bound sensor kinase and cytoplasmic DNA binding protein PhoP. When a specific external stimuli is present, this kinase phosphorylates the PhoP, which increases its binding affinity to the specific DNA targets. As a result, some genes are activated (Pags, PhoP activated genes) while others are suppressed (Prgs, PhoP repressed genes).
PagN is a PhoP activated genes. Its function is unknown, although it shows 54% homology with Hek and Tia adhesins/invasins of the pathogenic E. coli. In this exhaustive work done by Dr. MA Lambert and Stephen GJ Smith, Trinity College, Dublin, the authors show that when PagN was expressed in E. coli, it caused agglutination of erythrocytes and adhesion and invasion of mammalian cells. The authors also observed that S. Typhimurium mutants defective in PagN displayed reduced adhesion to and invasion of mammalian cells. Furthermore, it was observed that multi-copy expression of PagN can compensate for the loss of the SP-1 encoded T3SS.
The major results and methodologies used in the study are described as follows:
PagN is a heat sensitive hemagglutinin but not a autoagglutinin: The Hek protein of E. coli promotes hemagglutination. To determine whether PagN also promotes agglutination of erythrocytes, the authors performed a microtitre hemagglutination assay. For this assay, E. coli K12 strainXL-1 blue containing the PagN expression vector, pML1 or the empty vector control, pTrc99a, were incubated with human RBCs. It was observed that the presence of PagN expression vector caused the recombinant E. coli to agglutinate erythrocytes. As Hek is a heat resistant agglutinin, the authors next investigated whether PagN is also heat resistant. Unlike Hek, PagN showed a decrease in HA titres after heating thus showing that PagN is heat sensitive. The authors also observed that unlike Hek, PagN is not an autoagglutinin by a specteophotometric assay. The authors next wanted to know if PagN could support hemagglutination when expressed in multi-copy S. Typhimurium. Thus, plasmid pPagN2.3 was introduced into S. Typhimurium LT-2 and cultured in a medium that caused maximal induction of the PhoP dependent PagN promoter. To confirm the expression of PagN, the outer membrane extracts were analyzed by SDS-PAGE. To their surprise, the investigators observed that S. Typhimurium containing pPagN2.3 did not cause hemagglutination. Laboratory strains of E. coli such as K12 and B completely lack the O antigen of LPS. In contrast, the O antigen of S. Typhimurium is very large. Thus, the authors hypothesized that the large O antigen of S. Typhimurium may serve to mask the PagN and thus may limit access to its receptor. To investigate this, the authors used a S. Typhimurium strain CH133, a gal mutant, which lacks the O antigen component of LPS (rough strain). The authors transformed this strain with the plasmid pPagN2.3 and observed the agglutination of RBCs. These results suggested that LPS is capable of masking PagN and preventing functional receptor binding during hemagglutination.

PagN is an adhesin and invasin: To determine whether PagN could mediate adhesion to and invasion of eukaryotic epithelial cells, like its homologues Tia and Hek E. coli DH5 alpha harboring either control plasmid (pTrc99a) or PagN expression vector, pML-1, were induced with IPTG and incubated with eukaryotic cell lines CHO-K1. Approximately 60% of PagN-expressing bacteria were observed to invade the CHO-K1 cells. As invasion of epithelial cells by bacteria is often accomplished by inducing cytoskeletal rearrangements by manipulating polymerization of actin. Cytochalasin D is one of the cytochalasin that inhibits elongation of actin filaments at the fast growing end and thus it affects polymerization of actin. Thus, confluent CHO-K1 monolayers were pre-incubated with cytochalasin D for 30 minutes before infection with bacteria. When these cells were infected with bacteria, a 30 fold reduction in invasion was observed.

PagN contributes to Salmonella adhesion to and invasion of epithelial cells: A pagN mutant strain ML6 was constructed. It was observed that both wild type SL1344 and pagN mutant strain showed the same doubling time, thus removing PagN does not affect growth of S. Typhimurium. Also, the LPS profiles of both strains were also similar. The contribution of PagN to the invasion of CHO-K1 cell lines by S. Typhimurium was determined by using a standard invasion assay. These data suggested that wild type strains caused invasion to a greater extent than mutant strains. Earlier the authors observed hemagglutination by Salmonella expressing PagN in only rough strains (lacking O antigen of LPS), but adherence and invasion was observed in case of smooth strain SL1344. Thus, they checked for enhanced adherence and invasion of eukaryotic cells by a rough strain. When they compared adherence and invasion of epithelial cells by rough S. Typhimurium strain and rough S. Typhimurium strain lacking PagN, a reduction in cell association was observed as observed in case of smooth strain, however, rough mutant strain displayed nearly five fold decrease in cell association which was greater than the decrease observed in smooth strain. The magnitude of the decrease in cell association observed in case of rough strains lacking pagN was similar to that observed in case of infection of cell lines with S. Typhimurium lacking SP-1 encoded T3SS by mutation in invA. Furthermore, loss of both pagN and invA can be complemented by adding the PagN multicopy plasmid, pPagN2.3.