Background
Salmonella is a significant foodborne bacterium associated with enteric disease outbreaks in humans due to the consumption of contaminated food.
Salmonella serovars, like
Salmonella enterica serovar Enteritidis (SE), are the leading cause of death among the major foodborne pathogens [
1]. SE phage type (PT) 8 is one of the most common PTs associated with egg-associated outbreaks in the United States while SE PT4 is the most common in Europe [
2,
3]. Therefore, the identification and evaluation of
Salmonella virulence factors could help develop new ways to control salmonellosis in the farm to fork food processing cycle.
A hallmark of
Salmonella virulence is its ability to invade host intestinal epithelial cells [
4]. This is a multi-step process mediated by a type 3 secretion system (T3SS) encoded within
Salmonella pathogenicity island-1 (SPI-1) [
5,
6]. The first step in the invasion process is the adhesion of
Salmonella to the host intestinal epithelial cells. Several pathogenic factors have been implicated in adhesion to host cells. The best characterized are the fimbrial adhesins which include type 1, plasmid-encoded, long polar, and thin aggregative fimbriae [
7‐
10]. A further study has suggested that the T3SS itself can mediate host cell adhesion by showing that SipB, SipC, and SipD are required for the intimate association of
Salmonella with mammalian cells [
11]. Inhibition of
Salmonella adhesion at the initial stages of infection is potentially the most effective strategy for controlling salmonellosis in production animals which could result in reduced contamination of our food supply [
12].
In this study, we identified stdA as an adhesion mutant of SE by transposon mutagenesis. The stdA deletion mutant (∆stdA) displayed a normal growth profile when compared to the wild-type (WT) SE PT8 and complemented strains. A motility assay showed a significant decrease in motility for ∆stdA. Adhesion and invasion assays showed ∆stdA was deficient in cell culture models of Salmonella adhesion and invasion. Furthermore, ∆stdA was deficient in a poultry model of Salmonella adhesion and invasion with the systemic infection deficiency most likely due to the decreased adhesion. Taken together, these data indicated a major role for StdA in the adhesion ability of SE host cells.
Discussion
In this study, an adhesion mutant of SE was created and characterized. Transposon mutagenesis identified StdA as a potential adhesion mutant of SE. A ∆stdA strain of SE was created using the lambda Red recombination system, and was deficient in adhesion in both cell culture and chicken models of infection. Additionally, this adhesion defect lead to a deficiency in invasion of T84 intestinal epithelial cells, and decreased overall systemic infection ability in a poultry model as evidenced by reduced bacterial counts in the livers and spleens of chickens inoculated with ∆stdA. These data indicated that StdA plays a significant role in the adhesion ability of SE to the intestinal mucosa of poultry.
StdA is a 19-kDa fimbrial protein that is part of the
std operon which was originally identified during sequence analysis of the
Salmonella enterica serovar Typhi CT18 strain [
13]. It was later found to be in other serovars of
Salmonella including
Salmonella enterica serovar Typhimurium (STM) [
14‐
17]. In STM, the Std fimbriae play a role in
Salmonella adhesion to specific sections of the intestinal mucosa as evidenced by
std operon deletion mutants having reduced intestinal persistence in mice [
18,
19]. This correlates to the data observed in our study, where deletion of
stdA significantly alerted the adhesion ability of SE in the intestinal mucosa of poultry.
The synthesis of Std fimbriae is tightly regulated, but the mechanisms involved in
std expression are unclear. In the study by Balbontin
et al., gene expression profiling of a
dam mutant of STM demonstrated that transcription of the
std operon is repressed by Dam methylation [
20]. In another study, Jakomin
et al. showed that uncontrolled expression of Std fimbriae contributes to the attenuated virulence observed in
dam mutants of STM [
21]. They also described a regulatory role for SeqA as a repressor of the
std operon and HdfR as an activator of
std expression whose activity may be antagonized by SeqA [
21]. Further regulatory evidence was displayed in the study by Chessa
et al. which identified RosE as a novel transcriptional regulator of Std fimbrial expression in STM [
18]. Further investigation into the regulation of
stdA, and how it affects
Salmonella adhesion to the intestinal mucosa of poultry will be conducted in our laboratory.
An interesting observation in our study is that ∆
stdA displayed a significant decrease in motility. Motility is hypothesized to be a pathogenic mechanism because it promotes contact with the surface of epithelial cells by allowing the bacterium to penetrate the thick mucus layer covering the intestinal mucosa [
22,
23]. Some studies suggest a role for flagella in bacterial adhesion to host tissue [
24,
25]. The study by Erdem
et al. suggests a role for FliC in
E. coli adhesion to bovine intestinal tissue while the study by Olsen
et al. suggests a role for FliC in
Salmonella binding to intestinal epithelial cells [
24,
25]. Further studies will be needed to determine how StdA affects
Salmonella motility and if this motility reduction contributes to the adhesion and invasion defect seen in ∆
stdA.
Additional studies will also be needed in order to gauge the level of attenuation of the ∆stdA SE strain in chickens. Depending on the outcome of these studies, further studies could be conducted to determine if ∆stdA is a good candidate for use in a live-attenuated poultry vaccine.
Conclusions
Transposon mutagenesis identified StdA as a potential adhesion mutant of SE. A ∆stdA strain of SE was created using the lambda Red recombination system, and was deficient in adhesion both in vitro and in vivo. Additionally, this lack of adhesion lead to a deficiency in invasion of T84 intestinal epithelial cells, and decreased overall systemic infection ability in a poultry model as evidenced by reduced bacterial counts in the livers and spleens of chickens inoculated with ∆stdA. Overall, our data suggest StdA plays a role in the adhesion ability of Salmonella to the intestinal mucosa of chickens, and could be an important factor in the early stages of Salmonella infection in poultry.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DS carried out the in vivo and in vitro experimental work, performed the statistical analysis, and drafted the manuscript. NE performed the transposon screening and identified the adhesion mutants. DM created the stdA mutant strain. AF designed and coordinated the study, and edited the manuscript. All authors read and approved the final manuscript.