Background
Gastrointestinal (GI) phase of
Listeria monocytogenes infection is complex and successful extra-intestinal dissemination of the pathogen from the GI tract to liver, spleen, gall bladder, central nervous system and the placenta (in case of pregnant women) is essential for systemic disease, listeriosis. During the early stage of infection,
L. monocytogenes interacts with host intestinal cells. Thus, understanding this interphase between host and bacteria may aid in developing preventive or therapeutic strategies against listeriosis. Several virulence factors are responsible for initial interaction with the host. Among those, Internalin family of proteins including InlA and InlB play important roles during infection [
1,
2]. InlA interacts with E-cadherin and InlB with c-Met as host receptors during
Listeria infection. Additionally, adhesion- and invasion-associated proteins including autolysin amidase (Ami), virulence invasion protein (Vip), fibronectin binding protein, LapB, InlJ, CtapB and several others are involved [
3]. During intestinal phase, listerial survival under various intestinal environments such as acids, biles, antimicrobial peptides, mucus and resident microflora and their metabolites is critical and a majority of the stress response genes are regulated by Sigma B and/or PrfA [
4,
5].
Previously, our group showed that
Listeria adhesion protein (Lap), a 104-kDa alcohol acetaldehyde dehydrogenase (
lmo1634), is responsible for bacterial adhesion and transepithelial translocation through epithelial barrier [
6‐
8]. Lap uses the host heat shock protein 60 (Hsp60) as a receptor [
9], and the Lap-mediated
Listeria adhesion is severely impaired in epithelial cells when
hsp60 gene is partially silenced by shRNA [
7]. Cytosolic Lap is secreted to extracellular milieu with the help of the auxiliary secretion system, SecA2 [
6,
10]. Although both pathogenic and nonpathogenic
Listeria species express Lap, only in pathogenic
Listeria, secreted Lap is re-associated on the bacterial cell surface through an unknown mechanism, promoting
Listeria interaction with the host cell [
8]. Thus, we wanted to investigate the relationship between amounts of secreted Lap and the ability of the strain to adhere and translocate through epithelial cells among clinical isolates of
L. monocytogenes. We examined the amounts of secreted Lap from different clinical isolates and determined cellular localization of Lap in the intracellular (cytosolic), cell wall and supernatant fractions among different isolates. Protein expression data were verified by
lap-specific transcript (mRNA) analysis. Finally, the adhesion, invasion and paracellular translocation properties of these isolates were determined in an
in vitro cell culture model.
Discussion
L. monocytogenes is an opportunistic foodborne pathogen that infects primarily immunocompromised individuals such as the elderly, neonates, organ transplant patients or cancer patients receiving chemotherapy and pregnant women. Intestinal phase of listeriosis is complex and remarkably,
Listeria has developed unique survival skill through expression of several virulence genes that ensure its survival in the presence of acids, enzymes, bile salts, mucus, antimicrobial peptides, resident microflora and their metabolites in the gut [
4,
5]. Crossing of intestinal epithelial barrier is a critical event for initiating systemic listeriosis. M cells [
18] in Peyer’s patches and in solitary intestinal lymphoid tissue (SILT) and mucus secreting goblet cells [
17], in part, facilitate,
L. monocytogenes translocation. In addition, several virulence proteins play important role during bacterial dissemination through epithelial barrier [
4]. Among these, InlA following interaction with E-cadherin promotes bacterial invasion and translocation through intracellular route. E-cadherin is a major protein in adherence junction (AJ) and is localized at the basolateral side of the cell junction; thus it is normally unavailable for interaction with InlA for luminal bacteria [
17]. During epithelial necrotic cell extrusion process at the villus tip, InlA is able to interact with E-cadherin facilitating
L. monocytogenes invasion [
19]. Recently, we have shown that Lap, after interaction with epithelial Hsp60, increases tight junction permeability and promotes bacterial translocation through paracellular route [
7]. During this paracellular translocation process, we hypothesize that
L. monocytogenes will also have increased interaction with E-cadherin promoting listerial crossing of epithelial barrier by either InlA-dependent or InlA-independent pathway such as seen for the Δ
inlA-mutant [
7]. A Lap-positive but Δ
inlA-mutant strain efficiently translocates through epithelial barrier in the trans-well model (Figure
5) reinforcing the notion that bacteria are indeed capable of translocating using the paracellular route [
7] to cause systemic infection. It is speculated that deletion of
inlA gene may favor
L. monocytogenes to use the paracellular route to cross epithelial barrier as an alternative strategy for successful systemic spread.
In a previous experiment, surface Lap expressing
Lactobacillus paracasei was able to cross epithelial barrier through paracellular route at a significantly higher percentage than the WT strain [
20]. Furthermore, the Lap-expressing recombinant probiotic strain was also able to reduce
L. monocytogenes translocation by 46% in a trans-well model emphasizing again the significance of Lap in bacterial paracellular translocation [
20].
Lap is alcohol acetaldehyde dehydrogenase, a house keeping enzyme [
8]. It is produced by both pathogenic and nonpathogenic
Listeria and only in pathogenic species (
L. monocytogenes and
L. ivanovii); the secreted Lap re-associates on the surface using an unknown mechanism and promotes Lap-mediated adhesion and paracellular translocation. Surface re-association mechanism possibly is defective in nonpathogenic bacteria [
8]. Since Lap plays a critical role during intestinal phase of infection and secreted Lap is an important determinant for Lap-mediated pathogenesis [
6,
8], it is relevant to determine if variation in Lap secretion among the clinical isolates would affect bacterial paracellular translocation. We tested 47 clinical isolates and 9 food isolates from our culture collection and the total Lap from whole cell preparations (except supernatant proteins) appears to be very similar for all the clinical strains tested. In general, food isolates have reduced level of total Lap compared to clinical isolates; however, importance of such findings is not pursued any further in these isolates.
In clinical isolates, Lap in the supernatant fraction was highly variable with some isolates secreting larger quantities than the others. Further cell fractionation analysis revealed that the high Lap-secreting isolates had very low levels of cytosolic Lap, while low secreting isolates accumulated higher amounts of cytosolic Lap (Figure
2). Cell wall-associated Lap remained mostly uniform for both groups and its involvement in Lap-mediated pathogenesis is not fully understood. This experiment indicates that total amount of Lap to be constant for both groups and only observable differences were in distribution of Lap among the cellular compartments. To verify if protein expression is in agreement with transcript (mRNA) level in cells, qRT-PCR was performed and data were in agreement with protein data indicating possible variation in protein secretion may be a strain-dependent phenomenon where protein translocation by SecA2 system is involved (see below). Interestingly, high Lap-secreting strains (observed only in H4 and H13) had significantly lower levels of
inlA transcript while low Lap secreting strains had significantly higher amounts of
inlA transcripts (Figure
3). This suggests a possible balance in virulence factor expression in pathogens may happen, which is quite intriguing and warrant further investigation.
We have shown earlier that Lap secretion to the extracellular milieu, but not to the cell wall, is facilitated by the SecA2 system [
6,
10]. We determined if reduced Lap secretion in certain isolates may be associated with impairment in SecA2 function in these isolates. To verify, we analyzed the secretion of N-acetyl muramidase (NamA), an autolytic enzyme, whose secretion is dependent on SecA2 [
21]. Western blot analysis revealed that there was no difference in secreted (supernatant) and the cell wall associated NamA (Figure
2) in both high and low Lap secreting isolates indicating that SecA2 system is functional. However, protein (substrate)-dependent SecA2 transport mechanism cannot be ruled out in bacterial strains [
22]. Involvement of SecA2 in Lap secretion was further verified by performing paracellular translocation experiment. SecA2 deletion mutant from two different strains (F4244 and 10403S) showed reduced paracellular translocation, providing a circumstantial evidence that impaired secretion of Lap possibly affecting bacterial paracellular translocation. It is important to note that SecA2 has been involved in translocation of many other virulence factors [
21] and some of which may also contribute to bacterial translocation through epithelial barrier that cannot be ignored.
Lap being a house keeping enzyme [
8], its sequence is highly conserved. Thus, variation in
lap gene sequence in high and low Lap secreting isolates is not expected to be involved in strain dependent protein secretion. We compared the Lap sequence of our F4244 (WT) strain with published whole genome sequence from nine
L. monocytogenes isolates in the NCBI database, and the sequences were found to be highly conserved among isolates (99.4-99.8% similarity) (data not shown) providing a circumstantial evidence that sequence variation may not be a contributory factor for variation in Lap secretion in isolates.
Since the secreted Lap is critical in pathogenesis [
8], we examined if variable Lap secretion from clinical isolates from patients and some of whom suffered from fatal listeriosis, would exhibit differential pathogenic attributes such as adhesion, invasion and paracellular translocation. We observed that high Lap-secreting isolates exhibited significantly increased adhesion to, invasion into and paracellular translocation through Caco-2 cells compared to low Lap-secreting isolates. Even though the low Lap-secreting isolates translocated at a reduced level they still are able to cross the epithelial barrier, albeit at lower number, to cause systemic disease but severity of infection may be lower than the high Lap secreting isolates. Since we do not have detailed clinical history of patients from whom each of the isolates was isolated, it is difficult to validate our observation. However, in our previous mouse experiment, we have demonstrated that WT strain secreting increased amounts of Lap under anaerobic condition exhibited higher extra-intestinal dissemination to liver and spleen compared to the strain that was grown under aerobic condition secreting a reduced level of Lap [
6]. Furthermore, high Lap-secreting isolates induced enhanced epithelial cell junction permeability as evidenced by increased dextran permeability through the cell monolayers than the low secreting strains (Figure
5). These findings strongly suggest that variation in Lap secretion by
L. monocytogenes isolates could serve as a predictor of pathogenic potential and clinical outcome.
Competing interest
We all declare that there are no competing interests.
Authors’ contribution
AKB and HK designed the experiments and HK performed them. Both HK and AKB analyzed the data and wrote the manuscript. Both authors approved the manuscript for publication.