The role of flagella in Salmonella serovar Typhimurium virulence has been extensively studied in vitro. The in vivo function of flagella is less well understood, and it was unclear whether the flagella play a role in murine serovar Typhimurium colitis. We found that flagella and, more specifically, chemotaxis are required for efficient induction of murine Salmonella-induced colitis. In contrast, the systemic infection which occurred parallel to the enterocolitis did not depend significantly on flagellum function.
The role of motility during systemic stages of serovar Typhimurium infection in typhoid mouse models has been somewhat controversial. Carsiotis et al. found that a nonflagellated strain was attenuated compared to a wild-type strain in oral, intraperitoneal, and intravenous infections (5
). However, nonmotile and nonchemotactic mutants that both possessed intact flagella were not attenuated. Similarly, Weinstein et al. showed that a Salmonella
wild-type strain survived longer than a nonflagellated mutant survived in macrophage infection assays and exhibited faster net growth in spleens of infected mice (53
). During systemic infection Salmonella
cells are thought to reside and replicate mainly inside phagocytic cells (21
). Interestingly, expression analyses have demonstrated that flagellar genes are downregulated inside J774-A.1 macrophages, which may indicate that flagella are not required during the intracellular stages of the Salmonella
life cycle (13
). It has been reported by other workers that fla+ mot
mutants, in contrast to fla mot
mutants, are slightly attenuated in mice upon oral infection but not when the mice are infected by the intraperitoneal route (34
). In another study an flhD
mutant, but not an fliC fljB
flagellum mutant, was described as having a significantly lower 50% lethal dose in the mouse typhoid model for Salmonella
serovar Typhimurium and exhibited faster net growth in mouse macrophages. This effect was attributed to uncharacterized regulatory functions of flhD
The initiation of a systemic infection (mLN, liver, spleen) was not significantly attenuated in fliGHI
mutants. This contrasts with the significant role of flagella in the early phase of murine colitis, but it is consistent with previous studies performed with the murine typhoid model (40
). This obvious discrepancy might be explained by peculiar features of the Peyer's patches of the distal ileum, which are thought to function as the port of entry to systemic sites (6
). The structure of the follicle-associated epithelium, which contains specialized M cells, is different in many ways from the structure of the surrounding villous epithelium. For example, it lacks the overlying mucus layer (36
). Hence, flagella might not be required for the interaction with M cells, and this could explain the similar organ counts for flagellum mutants and wild-type Salmonella
serovar Typhimurium in our experiments.
Little is known about the role of flagella in intestinal salmonellosis. Robertson et al. investigated S. enterica
serovar Enteritidis-induced salmonellosis in the rat. This serovar is monophasic, and deletion of the only flagellar subunit gene (fliC
) attenuated intestinal inflammation. The levels of several inflammatory parameters measured in the small intestine contents of infected rats were reduced in the early stages of infection (1 and 2 days p.i.) with an fliC
flagellum mutant compared to the levels in rats infected with the wild type (37
). In bovine ligated ileal loops the enteropathogenic response to Salmonella
serovar Typhimurium flhD
or fliC fljB
mutant strains was decreased (40
). Here, the flhD
mutant provoked a statistically reduced secretory response and PMN influx. These inflammation parameters were also attenuated upon infection with the fliC fljB
double mutant, although the differences were not statistically significant. In line with these observations in the bovine model, we found that an fliGHI
mutant is impaired in terms of eliciting colitis in streptomycin-pretreated mice. This effect is very pronounced at earlier time points (10 or 24 h p.i.), while the mutant catches up later, causing severe colitis to almost the same level as wild-type serovar Typhimurium at 48 h p.i. This suggests that flagella are required for establishing an infection in streptomycin-pretreated mice.
In spite of many similarities to the bovine model, there are several aspects which need to be kept in mind when data from the murine colitis model are compared with data obtained with bovine ligated ileal loops. Both models suffer from the absence of or a severe reduction in the level of a complete adult-type commensal flora. Both models are characterized by mucosal edema, PMN influx, destruction of the epithelial cell layer, and fast tissue regeneration. However, in the bovine system there is massive luminal fluid secretion which is not observed in the murine model. The reason for this is currently unknown.
In several studies the workers have addressed the role of flagella in invasion of cultured epithelial cells. Various mutants that lacked flagella or had impaired motility or chemotaxis were less invasive or had a defect in attachment (10
). Jones et al. found that an fla
mutant and a smooth-swimming chemotaxis mutant were not attenuated for invasion of murine ligated ileal loops, while a mot
mutant and a tumbly che
mutant were less invasive than the wild-type strain (26
). Altogether, these results demonstrate that functional flagella are necessary to efficiently invade epithelial cells, a process which also requires the SPI-1 TTSS. However, it is still a matter of dispute whether flagella merely allow the bacteria to approach the host cell in order to position the SPI-1 TTSS optimally for injection or whether flagella might have additional functions. The similar levels of attenuation of fliGHI
(no flagella) and cheY
(flagellated but non chemotactic) mutants observed in our experiments suggest that chemotactic movement is the major virulence function of flagella in the streptomycin-pretreated mouse model.
Could flagella have additional functions in this model? Flagellin has been shown to elicit innate immune responses. This phenomenon has been studied extensively in tissue culture. Flagellin (FliC) binds TLR5, activates NF-κB, and induces proinflammatory responses (11
). Similar observations have been made in vivo. After intraperitoneal injection into mice, Salmonella
flagellin leads to systemic release of interleukin-6, a key mediator of inflammation (19
). This indicates that the manner in which murine tissues respond to flagellin is similar to the manner in which human cells respond to flagellin. So far, no TLR5 knockout mice have been described. However, knockout mice lacking a central element of TLR signaling cascades (MyD88) do not respond to flagellin (19
). These observations suggest that flagellin could contribute to the inflammatory response in the streptomycin-pretreated mouse model.
Interestingly, bacteria of the normal gut flora are known to produce and release a wide variety of TLR ligands (pathogen-associated molecular patterns [PAMPs], including flagellin) that can induce innate immune responses (43
). However, these ligands normally do not lead to intestinal inflammation. This apparent discrepancy has been resolved by evidence which suggests that TLRs are localized on the basolateral side of the intestinal epithelium (16
). Hence, bacterial flagellin released in the intestinal lumen cannot readily come in contact with TLR5.
Do serovar Typhimurium flagellin-TLR5 interactions contribute to colitis in streptomycin-pretreated mice? This question is quite difficult to answer as flagella in this case would serve two different functions, innate immune signaling and chemotaxis. In vitro, purified Salmonella
flagellin can induce inflammatory responses when it is added to tissue culture cells (11
). Therefore, interactions between flagellin released from bacteria and TLR5 might be involved in murine serovar Typhimurium colitis. The SPI-1 TTSS is a key virulence factor in this model (3
). Serovar Typhimurium mutants with a disrupted SPI-1 TTSS do not cause significant inflammation in streptomycin-pretreated mice even though they efficiently colonize the murine large intestine and have a fully functional flagellar system (3
). Thus, the mere presence of serovar Typhimurium flagella (or other PAMPs released by serovar Typhimurium) in the intestinal lumen is not enough to induce colitis. However, in the wild-type infection, SPI-1-induced processes (i.e., disruption of the epithelial barrier) might facilitate access of flagellin to TLR5 receptors. In this case, the flagellin-TLR5 interactions might contribute to the SPI-1-dependent inflammation observed with wild-type serovar Typhimurium. We did not observe a significant difference between the (delayed) inflammatory responses elicited by fliGHI
(no flagellin secretion) and cheY
(flagellated or secreted flagellin but nonchemotactic) mutants. Therefore, the flagellin-TLR5 interaction does not seem play a major role in murine serovar Typhimurium colitis. However, the effects caused by the flagellin-TLR5 interaction might simply be masked by other inflammatory stimuli attributable to other serovar Typhimurium virulence factors or PAMPs.
The competition experiments indicated that chemotaxis is required for efficient colonization of the murine intestine. This is in line with previous observations made with the chicken model (2
). Overall, these observations suggest that Salmonella
spp. actively move through the mucus layer in a chemotactic manner towards the epithelium in order to inject effector proteins via the SPI-1 TTSS. In experiments with Vibrio cholerae
, Freter et al. demonstrated that motile bacteria penetrated the intestinal mucus layer in mice more efficiently than either nonmotile or nonchemotactic mutants penetrated the intestinal mucus layer (15
). Interestingly, these authors found that nonchemotactic mutants of V. cholerae
still invaded the mucus at a low rate similar to the rate of inert particles. A similar mechanism could explain the delayed onset of disease symptoms with the nonmotile mutant in our experiments. We suppose that fliGHI
mutants might contact the epithelium less efficiently than the wild-type strain and therefore translocate SPI-1 effector proteins at a lower frequency. Additional work is required to address this possible correlation between motility and the efficiency of SPI-1 effector protein translocation.
In summary, the data presented here clearly show that motility and chemotaxis are required for the efficient induction of colitis in streptomycin-pretreated mice. Further studies are needed to identify the other Salmonella virulence factors involved and to elucidate the inflammatory cascades leading to colitis in streptomycin-treated mice.