A Peptidoglycan Amidase Activator Impacts Salmonella enterica Serovar Typhimurium Gut Infection

Nao Nakamura; Yusuke Hoshino; Takuro Shiga; Takeshi Haneda; Nobuhiko Okada; Tsuyoshi Miki

doi:10.1128/IAI.00187-20

. 2020 May 20;88(6):e00187-20. doi: 10.1128/IAI.00187-20

A Peptidoglycan Amidase Activator Impacts Salmonella enterica Serovar Typhimurium Gut Infection

Nao Nakamura ^a, Yusuke Hoshino ^a, Takuro Shiga ^a, Takeshi Haneda ^a, Nobuhiko Okada ^a, Tsuyoshi Miki ^a,^✉

Editor: Manuela Raffatellu^b

PMCID: PMC7240094 PMID: 32284369

KEYWORDS: EnvC, Salmonella, cell division, gut infection, peptidoglycan amidase activator

ABSTRACT

Salmonella enterica serovar Typhimurium is an important foodborne pathogen that causes diarrhea. S. Typhimurium elicits inflammatory responses and colonizes the gut lumen by outcompeting the microbiota. Although evidence is accumulating with regard to the underlying mechanism, the infectious stage has not been adequately defined. Peptidoglycan amidases are widely distributed among bacteria and play a prominent role in peptidoglycan maintenance by hydrolyzing peptidoglycans. Amidase activation is required for the regulation of at least one of two cognate activators, NlpD or EnvC (also called YibP). Recent studies established that the peptidoglycan amidase AmiC-mediated cell division specifically confers a fitness advantage on S. Typhimurium in the inflamed gut. However, it remains unknown which cognate activators are involved in the amidase activation and how the activators influence Salmonella sp. pathogenesis. Here, we characterize the role of two activators, NlpD and EnvC, in S. Typhimurium cell division and gut infection. EnvC was found to contribute to cell division of S. Typhimurium cells through the activation of AmiA and AmiC. The envC mutant exhibited impairments in gut infection, including a gut colonization defect and reduced ability to elicit inflammatory responses. Importantly, the colonization defect of the envC mutant was unrelated to the microbiota but was conferred by attenuated motility and chemotaxis of S. Typhimurium cells, which were not observed in the amiA amiC mutant. Furthermore, the envC mutant was impaired in its induction of mucosal inflammation and sustained gut colonization. Collectively, our findings provide a novel insight into the peptidoglycan amidase/cognate activator circuits and their dependent pathogenesis.

INTRODUCTION

Peptidoglycan (PGN, also called murein) is a peculiar and essential component of most bacterial cell walls (1, 2). PGN is a polysaccharide polymer that consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by a β-1,4-glycosidic bond. The two MurNAc molecules in the units are cross-linked by short peptides, giving PGN its dynamic structure. The interpeptide cross-links are critical for determining cell size and shape, as well as for cell growth and division (3). Accordingly, particularly in bacterial pathogens, the defined PGN is required for colonization in the host by protecting the bacterial cell from harmful osmotic pressure and antimicrobials (4).

PGN hydrolases contribute to the formation of the defined PGN structure by regulating bacterial cell wall growth. Since adequate bacterial cell wall growth is required for cell division, its defect results in the formation of chains of bacterial cells by incomplete cell separation of daughter cells. Thus, PGN hydrolases are essential for septal splitting and cell separation. Many bacteria have a large number of PGN hydrolases, such as PGN amidases, endopeptidases, carboxypeptidases, N-acetylmuramidases, N-acetylglucosaminidases, lysozymes, or lytic transglycosylases, that play redundant roles (3, 5, 6). Among these, the PGN amidases play the largest role in cell division by splitting the cell wall shared by dividing daughter cells. It has been shown that in Escherichia coli, several redundant PGN amidases containing AmiA, AmiB, and AmiC participate in PGN hydrolysis by cleaving the amide bond between MurNAc and l-alanine (7, 8). The activators NlpD and EnvC for the PGN amidases also participate in cell wall growth and cell division (9). NlpD and EnvC contain a lysostaphin-like metalloprotease (LytM) domain of the peptidase_M23 family. In E. coli, NlpD activates AmiA and AmiC, whereas EnvC regulates AmiB (9). In contrast, in Vibrio cholerae, both NlpD and EnvC contribute to the activation of AmiB, which is the only amidase for this bacterium (10). Thus, the activation of PGN amidases requires distinct cognate activators in the individual bacteria.

The Salmonella enterica serovar Typhimurium is an enteropathogenic bacterium causing gastroenteritis in humans and mice and typhoid-like systemic infection in mice (11, 12). The healthy individuals are normally less susceptible to the infection with S. Typhimurium since the very dense commensal microbes, called the microbiota, protect from the infection (13, 14). In contrast, antibiotics disrupt the balanced microbiota and thereby dramatically enhance the susceptibility to infection by various enteropathogens, including Salmonella spp. (13, 15). The streptomycin mouse model for Salmonella-induced colitis allows us to study the gastroenteritis caused by gut infection with S. Typhimurium (16, 17). In contrast, in the streptomycin-untreated mice inoculated with S. Typhimurium via the oral route, the mucosal inflammation is not induced. In both models, the pathogen subsequently traverses the intestinal mucosa and disseminates to systemic sites, which resembles the pattern of typhoid fever dissemination in humans. We have previously shown that the S. Typhimurium PGN amidases AmiA and AmiC are involved in the cell division process, and further, that the AmiC-mediated cell division contributes to S. Typhimurium colonization in the inflamed gut in a streptomycin mouse model (18). However, it remains unknown which cognate activator(s), NlpD, EnvC (also called YibP), or both, participate in the AmiC activation and its dependent Salmonella pathogenesis. Studies on PGN amidases and their cognate activators have been extensively performed in E. coli, indicating that they contribute to the cell division process (9, 19, 20). In contrast, little information has been gathered on their role in S. Typhimurium. PGN hydrolysis is involved not only in a large number of bacterial physiological functions but also aspects of bacterial pathogenesis, such as immune evasion and colonization (3, 4). Therefore, in the current work, we investigated and identified the roles of NlpD and EnvC in the cell division process and pathogenesis of S. Typhimurium. Our study provides novel insights into the PGN amidase/cognate activator circuit and its contribution to Salmonella pathogenesis.

RESULTS

The S. Typhimurium activators NlpD and EnvC for PGN amidases are very similar to those in E. coli.

Bioinformatic analysis showed that three PGN amidases of S. Typhimurium, namely, AmiA, AmiB, and AmiC, possess the signal sequences for a twin-arginine transport system (AmiA and AmiC) or Sec-dependent secretion system (AmiB), suggesting that they are periplasmic or extracellular proteins (see Fig. S1A in the supplemental material). Moreover, the Amidase_3 domain is present at the C-terminal region. PGN amidases of S. Typhimurium are very similar to those of E. coli (Fig. S1A). In particular, the Amidase_3 domains of all amidases have high amino acid sequence identities with the respective homologues of E. coli. Similarly, activators for PGN amidases including NlpD and EnvC are also very similar between S. Typhimurium and E. coli (Fig. S1B). NlpD and EnvC of S. Typhimurium have the Sec-dependent signal sequence. Moreover, the Peptidase_M23 domain is found at the C-terminal region in both activators, which show very high sequence identities of amino acids with the homologues of E. coli. The metal-binding sites of the peptidase_M23 domain are completely identical between S. Typhimurium NlpD and E. coli NlpD or between S. Typhimurium EnvC and E. coli EnvC (Fig. S1C). These results suggest that the S. Typhimurium cognate-specific activators NlpD and EnvC for PGN amidases may be functional and prompt us to hypothesize that the PGN amidase/cognate activator circuit in S. Typhimurium might be identical to that in E. coli.

S. Typhimurium EnvC is the main activator of PGN amidases, contributing to cell division.

In E. coli, mutants for PGN amidases or their cognate activators form chains of bacterial cells by a failure of cell division (7, 20). Thus, to evaluate the activity of the S. Typhimurium NlpD and EnvC, we first tested the involvement of the amidases and activators in the process of bacterial cell division. Test strains were grown in LB medium or high-osmotic LB medium (LB supplemented with 0.5 M NaCl) in which the AmiC-dependent cell division is induced (18). In the wild-type strain or amiB mutant grown in LB medium, almost all of the cells were single or paired, and few chains were observed (wild-type chains, 0.45%; amiB mutant chains, 0.81%; Fig. 1A and B). In contrast, proportions of single cells of the amiA mutant or amiC mutant grown in LB medium were significantly reduced compared to those of the wild-type strain, and paired cells and chains were significantly increased (amiA mutant paired cells, 23.0%; amiA mutant chains, 6.93%; amiC mutant paired cells, 43.7%; amiC mutant chains, 12.4%; Fig. 1A and B). A slight increase in the proportions of chains of the nlpD mutant was observed in comparison with those of the wild-type strain, but the difference was not significant (nlpD mutant chains, 2.55%; Fig. 1A and B). Notably, the proportions of single or paired cells of the envC mutant were significantly reduced, whereas the proportions of chains of the envC mutant were substantially increased (envC mutant chains, 47.6%; Fig. 1A and B). Similarly, on growth in high-osmotic LB, the proportions of chains of the amiA mutant, amiC mutant, and envC mutant were significantly increased compared to those of the wild-type strain (amiA mutant chains, 4.57%; amiC mutant chains, 37.6%; envC mutant chains, 38.8%; Fig. 1C and D).

In the comparison between growth in LB medium and growth in high-osmotic LB medium, the proportions of chains of the amiC mutant in high-osmotic LB were significantly increased compared with those in LB medium (LB, 12.4%; LB–0.5 M NaCl, 37.6%; Fig. 1E). This is in line with previous results that AmiC-dependent cell division was induced in a culture with high-osmotic medium (18). In an nlpD mutant grown in high-osmotic LB medium, the proportion of chains showed a 3.5-fold increase, but the change was not significant (Fig. 1E). In contrast, the proportions of chains of the amiA mutant and envC mutant were not increased.

Previous results (Fig. 1A to E) suggested that EnvC is required for cell division through the activation of AmiC and AmiA in S. Typhimurium. To confirm the link between EnvC and AmiA/AmiC, we compared the cell division of the amiA amiC, envC, and amiA amiC envC mutants. The proportions of chains of the envC mutant were similar to those of the amiA amiC mutant when grown in high-osmotic LB medium (amiA amiC mutant chains, 29.2%; envC mutant chains, 26.0%; Fig. 1F). In contrast, almost all of the amiA amiC envC mutant cells form chains (amiA amiC envC mutant chains, 98.7%; Fig. 1F). These results raised the possibility that NlpD might activate AmiA and/or AmiC in the envC mutant, contributing to cell division. Thus, we next investigated the chain proportions of the nlpD envC mutant. As expected, similar to the amiA amiC envC mutant, nearly all cells of the nlpD envC mutant displayed chains (nlpD envC mutant chains, 94.9%; Fig. 1F).

To ask whether the PGN amidases have redundant roles, we next constructed the amidase-null mutant strain (amiA amiB amiC mutant) and transformed it with the complementation plasmid expressing the AmiA, AmiB, or AmiC protein, respectively. Test strains were grown in high-osmotic LB medium, and the cell morphology was analyzed. All of the amiA amiB amiC mutant cells and the pMW118-transformed strain cells displayed chains (Fig. S2A). In contrast, transformed strain cells with the plasmids expressing AmiA, AmiB, or AmiC were complemented, in which the proportions of chains were reduced (pamiA chains, 7.7%; pamiB chains, 21.4%; pamiC chains, 22.2%; Fig. S2A). These results indicate the redundant role of the Ami amidases in S. Typhimurium.

Finally, we revealed the PGN amidase/activator circuit in S. Typhimurium. Additional mutations with the nlpD or envC gene were introduced in the amiA amiB amiC mutant; subsequently, the resulting mutant strains were transformed by the complementation plasmids expressing AmiA, AmiB, or AmiC, respectively. The proportions of chains of the amiA amiB amiC nlpD mutant harboring the plasmids expressing AmiA, AmiB, or AmiC were reduced to the levels of wild-type strain (pamiA chains, 11.5%; pamiB chains, 9.4%; pamiC chains, 1.9%; Fig. S2B). In contrast, in the amiA amiB amiC envC mutant, the only plasmid expressing AmiC complemented the failure of the daughter cell separation (pamiA chains, 95.5%; pamiB chains, 93.8%; pamiC chains, 9.9%; Fig. S2C).

Collectively, these results suggest that AmiC, and to a lesser extent AmiA, are required for cell division, whereas AmiB is likely to have no role in this cellular process when incubated in LB medium and high-osmotic LB medium. Importantly, the EnvC-mediated AmiA and AmiC activation plays a central role in cell division process. In contrast, PGN amidases and their cognate activators have a redundant role in cell division. Finally, these data reveal the PGN amidase/activator circuit in S. Typhimurium; EnvC activates all of amidases, including AmiA, AmiB, and AmiC, whereas NlpD involves the activation of AmiC but not AmiA or AmiB.

To ask whether chain formation by a cell division defect in the envC mutant could affect bacterial cell growth, we examined growth curves in liquid LB medium and colony formation on LB agar plates of the wild-type strain and the envC mutant. We observed that the growth rate and colony formation were similar between the two strains (Fig. S3), suggesting that a cell division defect caused by the PGN amidase/activator loss of function does not affect bacterial cell growth.

S. Typhimurium EnvC contributes to gut colonization.

We next examined whether NlpD and EnvC would be involved in the gut colonization in a streptomycin mouse model for Salmonella colitis. Streptomycin-pretreated C57BL/6 mice were infected with a 1:1 mixture of the wild-type strain and the amiA amiC, nlpD, or envC mutant by gavage (5 × 10⁷ CFU in total). We analyzed the bacterial loads in the feces at days 1 and 4 postinfection (p.i.) and enumerated the pathogen loads in the cecum lumen, the mesenteric lymph node (mLN), and the spleens at day 4 p.i. (Fig. 2A to C). In the comparison of the wild-type strain versus the amiA amiC mutant, fecal bacterial loads of the amiA amiC mutant were reduced in comparison to those of the wild-type strain at day 4 p.i. but not day 1 p.i. (competitive index [CI] at day 4 p.i., 0.009; Fig. 2A). Similarly, the amiA amiC mutant featured a colonization defect at the cecum lumen but not at the mLN or spleen. These data are similar to our earlier data obtained with an attenuated S. Typhimurium strain (18).

FIG 2 — The *envC* mutant is impaired in the gut lumen on coinfection with wild-type strain. (A to C) Streptomycin-pretreated C57BL/6 mice (n = 5 per group) were infected by gavage via the oral route with a 1:1 mixture (total, 5 × 10⁷ CFU) of the S. Typhimurium wild-type strain (wt) and the *amiA amiC* mutant (*amiA amiC*) (A), the wt and *nlpD* mutant (*nlpD*) (B), or the wt and *envC* mutant (*envC*) (C). CI values of S. Typhimurium loads in feces at days 1 and 4 p.i. and in the cecum lumen, mLN, and spleen. dpi, day postinfection. Bars show the median values. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Mann-Whitney U test.

In the comparison of the wild-type strain versus the nlpD mutant, no colonization defects were observed in the feces or in organs other than the spleen (Fig. 2B). In contrast, the envC mutant already exhibited a pronounced colonization defect in feces by day 1 p.i. (competitive index [CI] at day 1 p.i., 0.03; CI at day 4 p.i., 0.014; Fig. 2C). Similarly, the bacterial loads of the envC mutant at day 4 p.i. in the cecum lumen, mLN, and spleen were reduced compared to those of the wild-type strain. These results demonstrate that EnvC, but not NlpD, is involved in the gut colonization of S. Typhimurium. Remarkably, the faster colonization defect in the envC mutant was not observed in the amiA amiC mutant, and therefore, this defect may not be attributable to a failure in activation of AmiA and AmiC. To address this, we next performed the similar mouse infection experiment with the amiA amiC mutant and the amiA amiC envC mutant. The amiA amiC envC mutant in feces at day 1 p.i. was drastically outcompeted by the amiA amiC mutant (CI at day 1 p.i., 0.000001; Fig. S4). The results indicate that abrogation of AmiA, AmiC, and EnvC has a synergy effect on the colonization defect. Furthermore, these results lend support to the hypothesis that the faster colonization defect in the envC mutant is independent of deficient activities of AmiA and AmiC. Otherwise, chain formation by the severe defects of cell separation might confer stronger attenuation of the gut colonization on the amiA amiC envC mutant. Accordingly, we analyzed the envC mutant in further experiments.

Reduced colonization of the envC mutant is partly attributable to the hypersensitivity to bile acids.

Resistance of S. Typhimurium to luminal bile acids plays a critical role in murine gut colonization (21, 22). In addition, chains of S. Typhimurium lacking the amiA and amiC genes are more highly sensitive to deoxycholate, a component of bile acids, and colonization defects of the virulence-attenuated S. Typhimurium amiA amiC mutant are conferred by this impaired resistance to luminal bile acids in the gut (18). Thus, we next asked whether bacterial killing by bile acids is associated with a colonization defect of the envC mutant. The MICs of deoxycholate were reduced in the amiA amiC mutant in comparison with the wild-type strain (Table 1). Notably, the MICs for the envC mutant were further reduced compared to those of the amiA amiC mutant. This impaired resistance of the envC mutant was restored by the introduction of a plasmid carrying the envC gene. These data suggest that the amiA amiC, and envC mutant strains are impaired in their resistance to bile acids.

TABLE 1.

MICs of deoxycholate toward S. Typhimurium^a

Strain	Genotype	MIC of deoxycholate (%)
SL1344	Wild type	4
T407	amiA amiC	1
T516	envC	0.25
T255	envC/pMW118	0.1
T256	envC penvC	4

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Each experiment was repeated three times independently.

We next examined whether higher sensitivity to luminal bile acids is a cause of the colonization defect of the envC mutant. To this end, a bile sequestrant colestimide resin was used to lower the luminal levels of bile acids. Mice were fed with rodent chow supplemented with colestimide resin, which sequesters luminal bile acids and thereby increases the excretion of the resin-bound bile acids in the colon. Beginning at 3 days prior to the streptomycin treatment, we fed mice with chow containing colestimide resin or normal chow and then infected both groups with a 1:1 mixture of the wild-type strain and amiA amiC mutant or a 1:1 mixture of the wild-type strain and envC mutant via the oral route. Colonization defects of the S. Typhimurium mutants were observed in mice of the group fed with normal chow (Fig. 3A and B). In contrast, in the mice fed chow containing colestimide resin, the colonization defect of the amiA amiC mutant was completely restored (Fig. 3A). In the mixed infection with the wild-type strain and the envC mutant, colestimide resin partly restored the colonization defect of the envC mutant, but a competitive disadvantage was still observed (Fig. 3B). Elevated levels of fecal bile acids in the mice fed chow containing colestimide resin were observed in comparison with those in the mice fed normal chow, suggesting that the colestimide resin reduces the luminal levels of bile acids (Fig. 3C). Collectively, these results indicate that the luminal levels of bile acids play at least a partial role in the EnvC-dependent gut colonization. Notably, the competitive disadvantage of the envC mutant is largely conferred by mechanisms other than the bacterial killing by bile acids.

FIG 3 — Involvement of bile acid-mediated killing in the gut colonization of the S. Typhimurium *amiA amiC* or *envC* mutant. (A and B) C57BL/6 mice (n = 8 or 9 per group) on rodent chow with colestimide resin or normal chow were administered with streptomycin and infected with the S. Typhimurium wild-type strain (wt) and *amiA amiC* mutant (*amiA amiC*) (A) or the wt strain and *envC* mutant (*envC*) (B). CI of S. Typhimurium loads in feces at days 1 and 4 p.i. dpi, day postinfection. Bars show the median values. ns, not significant; *, P < 0.05; ***, P < 0.001, Mann-Whitney U test. (C) Fecal concentrations of total bile acids. The horizontal bars indicate the median values. *, P < 0.05, unpaired Student's t test.

The envC mutant displays the impairment in gut colonization in the streptomycin-untreated mice, in which the complex microbiota is present with no inflammation.

In the streptomycin model, antibiotic pretreatment allows S. Typhimurium to colonize the gut lumen by reducing microbiota abundance by >10-fold and altering the composition of the microbiota (13). Thus, we next asked whether the colonization defect of the envC mutant at day 1 p.i. is associated with the microbiota. To test this, we prepared two groups of mice, where one group was treated with streptomycin and the other was left untreated. We then infected the mice of both groups with a 1:1 mixture of the wild-type strain and envC mutant via the oral route (5 × 10⁷ CFU in total). In feces, the envC mutant had a colonization defect at day 1 p.i. at similar levels in both groups of mice (Fig. 4A). Gut inflammation was verified by measuring the fecal levels of lipocalin-2 (Lcn-2), an inflammatory marker (Fig. 4B). The results of the Lcn-2 enzyme-linked immunosorbent assay (ELISA) showed that S. Typhimurium induced inflammation in the streptomycin-pretreated mice, whereas the mice not treated with streptomycin had no inflammation. These results show that perturbation of the microbiota and/or changes in inflammation levels do not enhance or abrogate the colonization defect of the envC mutant.

FIG 4 — The *envC* mutant is impaired in gut lumen containing complex microbiota. Shown is a CI experiment of the wild-type (wt) strain versus the *envC* mutant (*envC*) in naive C57BL/6 mice or streptomycin-pretreated mice (n =7 or 8; total, 5 × 10⁷ CFU by gavage; analysis at day 1 p.i.). (A) CI of S. Typhimurium loads in feces at day 1 p.i. (B) Fecal lipocalin-2 was monitored by ELISA. dpi, day postinfection. Sm, streptomycin. Bars show the median values. ns, not significant; *, P < 0.05; ***, P < 0.001; Mann-Whitney U test.

A colonization defect of the envC mutant is conferred by reduced chemotactic movement.

To decipher the molecular mechanism by which the envC mutant is outcompeted in the mixed infection with the wild-type strain, we investigated bacterial phenotypes contributing to the gut colonization of S. Typhimurium. Motility and chemotaxis have been shown to be contributing factors enabling S. Typhimurium to colonize the gut lumen (23). Thus, we first tested the ability of the envC mutant to move on semiagar (0.3% agar). As controls, the fliGHI mutant and the cheY mutant were tested. fliGHI belong to class II genes of the flagellum regulon. Especially, an S. Typhimurium strain lacking a fliG gene is known to be a nonflagellated bacterium (24). In contrast, CheY is involved in chemotaxis (25), and the cheY mutant assembles flagella but cannot move in response to chemotactic stimuli (26). Both the fliGHI and the cheY mutant failed to move on swimming motility agar (Fig. 5A and B). Compared to that of the wild-type individuals, the swimming motility of the envC mutant was significantly attenuated, albeit not diminished (Fig. 5A and B). This attenuated motility in the envC mutant was restored by the introduction of an envC-encoded plasmid. We further examined the swarming motility of the envC mutant. The motility and chemotaxis mutants (the fliGHI and the cheY mutant) did not display the chemotactic movement on swarming agar (Fig. 5C). Similarly, swarming of the envC mutant was impaired, and this defect was also rescued in the complementation strain (Fig. 5C). These results suggest that the abrogation of EnvC leads to reduced swimming motility and swarming.

FIG 5 — Causal link between a colonization defect of the *envC* mutant and impairment in motility and chemotaxis. (A) Swimming motility test by using LB with 0.3% agar. (B) The halo of the experiment in panel A was measured. The assay was repeated 3 times independently. Data are shown as the means ± standard deviations. ns, not significant; **, P < 0.01; ***, P < 0.001, unpaired Student's t test. (C) Swarming test by using LB with 0.5% agar. (D) The amount of FliC in the sheared fraction (sheared) and whole-cell fraction (cell lysate) from the wild-type (wt) strain, *fliC* mutant (*fliC*), *envC* mutant (*envC*), and the complementation strain (*envC*/penvC) was analyzed by Western blotting with anti-*Salmonella* type H-i serum. (E) Relative band intensity from the experiment in panel D. Data represent the relative value of the wild-type strain as 1 and are shown as the means ± standard deviations of the results from seven independent experiments. NA, not applied; ns, not significant, one-sample t test (theoretical mean, 1). (F) CI experiment of *envC fliGHI* versus *fliGHI* or *envC* mutant in streptomycin-pretreated mice (n = 7 per group; total, 5 × 10⁷ CFU by gavage; analysis at day 1 p.i.). (G) CI experiment of the *envC cheY* mutant versus the *cheY* or *envC* mutant in streptomycin-pretreated mice (n = 8 per group; total, 5 × 10⁷ CFU by gavage; analysis at day 1 p.i.). dpi, day postinfection. Bars show the median values. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Mann-Whitney U test.

Next, we compared the abilities to produce FliC flagellin and transport it to the bacterial surface. In the wild-type strain, FliC was produced and transported to the bacterial surface, whereas the fliC mutant did not produce and transport flagellin (Fig. 5D and E). Notably, the levels of transported FliC proteins in the envC mutant and the complementation strain were equivalent to those in the wild-type strain. Similarly, FliC production was similar among the wild-type strain, envC mutant, and complementation strain (Fig. 5D and E).

Finally, we tested whether the reduced motility and swarming were involved in the poor colonization of the envC mutant. To this end, we performed competitive infection experiments by using S. Typhimurium mutants harboring additional mutations on the fliGHI or cheY genes, which are associated with swimming motility and swarming, respectively. Two groups of the streptomycin-pretreated C57BL/6 mice were gavaged with a 1:1 mixture of the envC fliGHI mutant and fliGHI or envC mutant by the oral route (5 × 10⁷ CFU in total). S. Typhimurium loads in the feces at day 1 p.i. were determined, and colonization levels were compared by calculating the CI. In the CI experiment, the envC fliGHI mutant displayed no colonization defect compared to the fliGHI or envC mutant (Fig. 5F). Surprisingly, the envC fliGHI mutant rather colonized more efficiently than did the individual mutant. Similarly, CI experiments to compare the colonization levels of the envC cheY, cheY, or envC mutant were performed. The envC cheY mutant exhibited no colonization defect, in contrast with the cheY or envC mutant (Fig. 5G). Taken together, these results show that the reduced ability of the envC mutant to colonize the gut was attributable to impaired swimming motility and chemotaxis.

The S. Typhimurium envC mutant exhibits attenuated triggering of enterocolitis.

The data presented above demonstrate that the colonization defect of the envC mutant may be linked to reduced motility and chemotaxis. Nonmotile and nonchemotactic mutants of S. Typhimurium have a reduced ability to induce gut inflammation and colonize the gut lumen (23, 27). Thus, in order to examine whether the reduced motility and chemotaxis in the envC mutant is associated with the induction of gut inflammation, we performed further infection experiments with the streptomycin mouse model for Salmonella enterocolitis. Streptomycin-pretreated mice were infected with the wild-type strain, envC mutant, or complementation (envC/penvC) strain by gavage, and gut colonization levels were monitored at days 1 and 4 p.i. In addition, to evaluate the induction of gut inflammation, fecal levels of lipocalin-2 (Lcn-2) were determined at days 1 and 4 p.i. At day 1 p.i., the three tested strains displayed similarly high levels of gut colonization (Fig. 6A). Subsequently, the wild-type strain continued to colonize the gut at high levels. In contrast, at day 4 p.i., the colonization levels of the envC mutant were decreased (Fig. 6A). This defect was restored in the complementation strain. Lcn-2 ELISA showed that a low level of gut inflammation tended to be induced at day 1 p.i. in the mice infected with the wild-type strain and the complementation strain, whereas at day 4 p.i., mice infected with the wild-type strain showed robust inflammation, and those infected with the envC mutant did not develop the gut inflammation (Fig. 6B). These results are in line with those from previous reports (23, 27) showing that nonmotile and nonchemotactic mutants of S. Typhimurium colonize the gut lumen at day 1 p.i. at the same levels as the wild type, whereas the levels of gut inflammation are reduced in mice infected with these mutants. Collectively, these results lend further support to the hypothesis that the gut colonization defect of the envC mutant is attributable to attenuated motility and chemotaxis.

FIG 6 — The *envC* mutant is impaired to induce gut inflammation and sustained colonization. Streptomycin-pretreated C57BL/6 mice (n = 10 or 11 per group) were infected for 4 days with 5 × 10⁷ CFU intragastrically of the wild-type (wt), *envC* mutant (*envC*), or the complementation (*envC*/penvC) strains. (A) Bacterial loads of S. Typhimurium in feces at days 1 and 4 p.i. (B) Fecal lipocalin-2 at days 1 and 4 p.i. dpi, day postinfection. Bars show the median values. ns, not significant; *, P < 0.05; **, P < 0.01, Mann-Whitney U test.

DISCUSSION

Our data demonstrate that S. Typhimurium EnvC is required for cell division and constitutes a virulence factor contributing to gut infection, which is involved in the resistance to bile acids and in flagellin-based motility and chemotaxis (Fig. 7). The PGN amidase/cognate activator circuit in S. Typhimurium is quite different from that in E. coli. Indeed, we found that S. Typhimurium EnvC potentiated the activity of AmiC, which is not observed in E. coli. This is surprising because these corresponding homologues are very similar between S. Typhimurium and E. coli, and key domains such as amidase or peptidase essential for their enzymatic activities are present in the respective proteins. In addition to E. coli, the AmiC/NlpD circuit has been reported in Neisseria gonorrhoeae (28, 29) and the phytopathogen Xanthomonas campestris (30). To the best of our knowledge, the current study provides the first demonstration that EnvC activates the AmiC amidase.

FIG 7 — Role of the EnvC in *Salmonella* gut infection. *Salmonella* EnvC activates the peptidoglycan amidases AmiA and AmiC. This is required for the successful cell division, which also confers the resistance of S. Typhimurium to bile acids. The resistance to bile acids contributes to sustained colonization in the gut lumen, especially the inflamed gut. Alternatively, EnvC might interact with unknown factor(s) involved in the flagellin-based motility and chemotaxis. In addition, the factor might contribute to the resistance to bile acids. Flagellin-based chemotactic movement is a virulence factor, contributing to *Salmonella* gut infection (23, 27, 31, 32). Therefore, the EnvC-mediated gut infection is conferred by the resistance to bile acids and activities of the chemotactic movement.

It should be noted that AmiA/AmiC and EnvC appeared to make distinct contributions to the gut colonization. Indeed, the colonization of the envC mutant was already impaired at day 1 p.i. in the CI experiment, which was not observed in the amiA amiC mutant. We have previously shown that the AmiA/AmiC-related gut colonization depends on host inflammatory responses, since a colonization defect of the amiA amiC mutant is induced in the inflamed gut but not the normal gut (18). Our data showed that little gut inflammation is likely to be induced at day 1 p.i. with a CI experiment. Moreover, the envC mutant failed to colonize even in the streptomycin-untreated mice, for whom gut inflammation is not induced even after S. Typhimurium infection; we therefore conclude that the colonization defect of the envC mutant does not depend on inflammatory responses. In addition, our data showed that reduced resistance to bile acids is exclusively responsible for conferring the colonization defect of the amiA amiC mutant, whereas it plays only a limited role in conferring the colonization defect of the envC mutant. Finally, our results showed that the colonization defect of the envC mutant was attributable to reduced motility and, more specifically, reduced chemotaxis, as it was not observed in the amiA amiC mutant (18). Flagellin-based chemotactic movement is necessary to reach the gut epithelial cells, leading to the induction of gut inflammation (23, 27, 31). In addition, since mucin is a rich energy source for Salmonella growth in the gut lumen, chemotaxis is useful for the acquisition of nutrients, resulting in further expansion of luminal Salmonella spp. (27, 32). Collectively, these facts raise the possibility that in addition to AmiA and AmiC, EnvC could interact with unknown factor(s) associated with chemotactic movement. In contrast, the tatC mutant of S. Typhimurium, which fails to export the AmiA and AmiC into periplasmic space through the twin-arginine translocation system, has been shown to display reduced motility (33). It has been concluded that the reduced phenotype is associated with less flagellin on the bacterial surface (33). Notably, our data show that the amount of transported flagella on the bacterial surface in the envC mutant was identical to that of the wild-type strain, leading us to conclude that the envC mutant is competent for the flagellar export. Therefore, we currently do not know how abrogation of EnvC attenuates the motility and chemotaxis in S. Typhimurium. More detailed analyses will be needed to reveal the molecular mechanism by which a loss of function of EnvC confers reduced motility and chemotaxis.

The gut colonization defect at day 1 p.i. of the envC mutant implied the possibility that this mutant fails to invade the gut ecosystem. An earlier work showed that S. Typhimurium invasion into the gut ecosystem, including the complex microbiota, relies on the presence of H₂ (34). The H₂ production largely depends on the gut microbiota. Thus, the streptomycin pretreatment in the Salmonella colitis mouse model abolishes this phenotype (34). Our data showed that the streptomycin pretreatment and microbiota are not involved in the colonization defect of the envC mutant, suggesting that the attenuated colonization of the envC mutant is not attributable to a failure in the use of H₂.

In earlier work, S. Typhimurium EnvC has been shown to be required for the resistance to certain antimicrobial peptides such as the human neutrophil peptide and the cathelicidin-derived antimicrobial peptide LL-37, which is partly associated with cell surface properties (35). Furthermore, the envC mutant has been shown to be impaired in full virulence of S. Typhimurium in the mouse model for typhoid fever (35). In contrast, our data showed that EnvC is involved in the resistance to bile acids, an antimicrobial factor in the gut lumen. We further revealed that EnvC contributes to gut infection with S. Typhimurium by using the streptomycin mouse model for Salmonella colitis. This partly depends upon the resistance to bile acids and, importantly, is largely conferred by chemotactic movement of S. Typhimurium. Collectively, S. Typhimurium EnvC plays critical roles in infectious stages of both gut infection and systemic infection by distinct and/or overlapped modes.

It has been shown that a mutation of the envC gene causes an alteration in the composition of the outer membrane and periplasmic proteins in Haemophilus influenzae and E. coli, leading to hypersensitivity to antimicrobial peptides (35, 36). Since this could be applied to the S. Typhimurium envC mutant, analyses for extracellular proteins will be useful to decipher the EnvC function.

In conclusion, our data establish that S. Typhimurium EnvC is required for the cell division process due to its activation of AmiA and AmiC. Moreover, EnvC constitutes a virulence factor, contributing to the gut colonization and induction of mucosal inflammatory responses. The fact that S. Typhimurium is strikingly different from E. coli in requiring the activator for PGN amidase activation may suggest that the repertoire of the PGN amidase/activator circuit considerably influences bacterial virulence.

MATERIALS AND METHODS

Bioinformatic analysis.

Bioinformatic analyses were done using the following online applications: PfamScan (https://www.ebi.ac.uk/Tools/pfa/pfamscan/), SignalP (http://www.cbs.dtu.dk/services/SignalP/), ClustalW (https://www.genome.jp/tourols-bin/clustalw), and BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are listed in Table 2. S. Typhimurium strain SL1344 is wild-type and a mouse virulent strain. S. Typhimurium strains harboring chromosomal in-frame deletions were created using a lambda red homologous recombination system (37) or phage P22-mediated transduction. The primers used for construction of the mutant strains are the following: for the amiB mutant, SL amiB-red-FW (5′-TTACTGGCGCGTTTAGCCGATTAGCTATAAAGGTGGCGGGGTGTAGGCTGGAGCTGCTTC-3′) and SL amiB-red-RV (5′-CAAGCTGCGGCGGCAGAACCTGAATCGGCATGAAATCTCCCATATGAATATCCTCCTTAG-3′); for the nlpD mutant, nlpD-red-FW (5′-ACGTCACTGGTTGTTAACCAAATTTTTTCCTGGGGGATAAGTGTAGGCTGGAGCTGCTTC-3′) and nlpD-red-RV (5′-CCTTGACGGAACTAGCAAGTCAAAGCCTGGTTCCGCCGCTCATATGAATATCCTCCTTAG-3′); for the yibP mutant, yibP-red-FW (5′-GACTGGTAAGCCGCTGTTCATCGTGGAATAATCCCTCCCCGTGTAGGCTGGAGCTGCTTC-3′) and yibP-red-RV (5′-TGGCCAGCGTGAGAATGGAGCGACGAAACTGAGGCAAAACCATATGAATATCCTCCTTAG-3′); and for the fliGHI mutant, fliG-red-FW (5′-GCTGGTCATTCGCCAGTGGATGAGTAACGATCATGAGTAAGTGTAGGCTGGAGCTGCTTC-3′) and fliI-red-RV (5′-GAGCGCCATGTTGTGCCATGATCGTCGCCCTCCTGCTTTACATATGAATATCCTCCTTAG-3′).

TABLE 2.

Strains and plasmids used in this study

Strain or plasmid	Genotype or description	Reference or source
S. enterica serovar Typhimurium strains
SL1344	Wild-type S. Typhimurium, hisG	42
T405	SL1344 ΔamiA::kan	This study
T686	SL1344 ΔamiB::cat	This study
T406	SL1344 ΔamiC::kan	This study
T407	SL1344 ΔamiA::kan ΔamiC	18
T422	SL1344 ΔnlpD::kan	This study
T516	SL1344 ΔenvC::cat	This study
T255	T516 harboring pMW118	This study
T256	T516 harboring penvC, expressing EnvC	This study
T293	SL1344 ΔamiA ΔamiC	This study
T294	SL1344 ΔamiA ΔamiC ΔenvC::cat	This study
T517	SL1344 ΔnlpD::kan ΔenvC::cat	This study
T297	SL1344 ΔamiA ΔamiB::cat ΔamiC	This study
T819	T297 harboring pMW118	This study
T820	T297 harboring pamiA, expressing AmiA	This study
T821	T297 harboring pamiB, expressing AmiB	This study
T822	T297 harboring pamiC, expressing AmiC	This study
T299	SL1344 ΔamiA ΔamiB ΔamiC ΔnlpD::kan	This study
T807	T299 harboring pMW118	This study
T808	T299 harboring pamiA, expressing AmiA	This study
T809	T299 harboring pamiB, expressing AmiB	This study
T810	T299 harboring pamiC, expressing AmiC	This study
T823	SL1344 ΔamiA ΔamiB ΔamiC ΔenvC	This study
T824	T823 harboring pMW118	This study
T825	T823 harboring pamiA, expressing AmiA	This study
T826	T823 harboring pamiB, expressing AmiB	This study
T827	T823 harboring pamiC, expressing AmiC	This study
T523	SL1344 ΔfliGHI::cat	This study
T273	SL1344 ΔfliGHI	This study
T253	SL1344 ΔenvC	This study
T271	SL1344 ΔenvC ΔfliGHI::cat	This study
T323	SL1344 ΔcheY::kan	This study
T296	SL1344 ΔenvC ΔcheY::cat	This study
TM1172	SL1344 ΔfliC::kan	Lab stock
Plasmids
pMW118	Low-copy-number expression vector	Nippon Gene
pamiA	pMW118 containing amiA, expressing AmiA	18
pamiB	pMW118 containing amiB, expressing AmiB	This study
pamiC	pMW118 containing amiC, expressing AmiC	18
penvC	pMW118 containing envC, expressing EnvC	This study

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Bacterial morphology analysis.

S. Typhimurium strains were grown overnight in LB at 37°C, diluted 1:100 in fresh LB broth or LB containing 0.5 M NaCl, and grown for 2.5 h. The resulting bacteria were placed on a 1.5% agarose pad, sealed under a glass coverslip, and imaged at ×400 using the Zeiss Axiovert A1 microscope.

Mouse infection experiments.

C57BL/6 mice were bred at the institute of experiments of animals at School of Pharmacy, Kitasato University or purchased from Japan SLC. Mouse infection experiments were performed in 6- to 12-week-old mice, as described previously (38). Briefly, mice were pretreated with 25 mg streptomycin 24 h prior to infection. For infection, bacteria were grown for 12 h in 0.3 M NaCl-supplemented LB medium diluted 1:20 and subcultured for 4 h in the same medium. On coinfection experiments, the cultures were mixed in a 1:1 ratio. For oral infections, mice were infected with 5 × 10⁷ CFU bacteria by gavage. Mice were sacrificed on day 1 p.i. or day 4 p.i. by cervical dislocation. Collected fecal pellets, cecum content, and organs such as mLN and spleen were homogenized in phosphate-buffered saline (PBS) (if necessary, with 0.5% Tergitol). Differential plating on LB agar supplemented with the appropriate antibiotics (50 μg/ml streptomycin, 50 μg/ml kanamycin, 10 μg/ml chloramphenicol, or 100 μg/ml ampicillin). The competitive index (CI) was calculated by dividing the population size of the mutant strain by the population size of the corresponding background strain. To reduce luminal levels of bile acids, mice were fed a normal rodent chow containing 1.5% colestimide resin (Mitsubishi Tanabe Pharma). To verify gut inflammation, concentrations of fecal lipocalin-2 were determined as follows: fecal pellets collected at the indicated time points were homogenized and diluted in PBS. The resulting dilutions were then analyzed using the mouse lipocalin-2 ELISA DuoSet (R&D), according to the manufacturer’s instructions.

Plasmid construction.

To construct a complementary plasmid carrying the amiB or envC gene, DNA fragments containing the amiB or envC gene were amplified by PCR using the following primer sets: SL amiB-SacI-FW (5′-AAAGAGCTCCGCAACAAGGTAAAGGCG-3′) and SL-amiB-SphI-RV (5′-AAAGCATGCCGGCAGAACCTGAATCGGC-3′), or yibP-KpnI-FW (5′-TTTGGTACCGTGCCGCTGATTTATGTCGG-3′) and yibP-SalI-RV (5′-CCCGTCGACGAACCTGGTTTTCCGTGTGC-3′), and S. Typhimurium strain SL1344 chromosomal DNA as the template; these were digested with SacI and SphI or KpnI and SalI and then ligated between the same sites of pMW118, yielding pamiB or penvC.

Determination of MICs.

MICs were determined as previously described (18, 39). Briefly, S. Typhimurium strains were diluted to 1 × 10⁶ CFU per ml with different concentrations of deoxycholate (Nacalai tesque) in sterile LB broth and incubated at 37°C. After 22 h of incubation, the A₆₀₀s were measured using a microplate reader (Bio-Rad). A positive control contains no deoxycholate, whereas in the negative control, S. Typhimurium cells were not present. MICs were determined as the lowest concentrations of deoxycholate that were shown to prevent bacterial growth by more than 50% in comparison with the growth of the positive control.

Quantification of total bile acids in feces.

Fecal levels of bile acids were measured as previously described (40). Ethanol-extracted total bile acids in feces were analyzed using the Total bile acids-test Wako (FujiFilm Wako Pure Chemical), according to the manufacturer’s instructions.

Swimming motility and swarming assay.

S. Typhimurium strains grown overnight in LB at 37°C, subcultured in fresh LB broth, and further grown for 2 h. A 5-μl aliquot at an optical density at 600 nm (OD₆₀₀) of 1.0 was placed on a 0.3% agar LB plate (for swimming) or 0.5% agar LB plate supplemented with 0.5% glucose (for swarming) and left for 5 min. The plates were incubated at 37°C for 5 h (swimming) or 8 h (swarming).

Analysis of flagellin production and transport.

The bacterial pellet grown in LB medium was resuspended in 1 ml of sterile PBS and vortexed at high speed for 5 min. In this step, the flagella were sheared from the bacterial surface into the supernatant (41). After centrifugation, the supernatant was transferred into a fresh tube. The bacterial pellet was resolved in SDS-PAGE sample buffer. The supernatant was subjected to trichloroacetic acid (TCA) precipitation at a final concentration of 6% on ice for 15 min. The TCA-precipitated proteins were collected by centrifugation at 4°C for 10 min at 13,000 rpm in an Eppendorf 5415R centrifuge. The resulting pellet was washed twice with cold acetone. Finally, the precipitated proteins were resuspended in SDS-PAGE sample buffer. Whole-cell lysates and sheared (TCA-precipitated) proteins were analyzed by SDS-PAGE and Western blotting with anti-Salmonella type H-i serum (Denka Seiken Co., Ltd.).

Statistical analysis.

Unpaired Student's t test, one-sample t test, and the exact Mann-Whitney U test were performed using the software GraphPad Prism. P values of less than 0.05 were considered statistically significant.

Ethics statement.

All animal experiments were reviewed and approved by the Kitasato University Institutional Animal Care and Use Committee (permit numbers 17-52, 17-54, and 17-55).

Supplementary Material

Supplemental file 1

IAI.00187-20-s0001.pdf^{(414.3KB, pdf)}

ACKNOWLEDGMENTS

We are grateful to Ryosuke Goto and Mayuka Fujimoto for construction of the S. Typhimurium mutants. We also thank Tomomi Ishihara, Yuya Kishino, Sayaka Kinto, Shiori Kikuchi, and Nanami Suda for technical assistance.

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants JP16K08783 (to T.M.), JP19K07543 (to T.H.), and JP18K07119 (to N.O.).

The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

Footnotes

Supplemental material is available online only.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

IAI.00187-20-s0001.pdf^{(414.3KB, pdf)}

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PERMALINK

A Peptidoglycan Amidase Activator Impacts Salmonella enterica Serovar Typhimurium Gut Infection

Nao Nakamura

Yusuke Hoshino

Takuro Shiga

Takeshi Haneda

Nobuhiko Okada

Tsuyoshi Miki

Roles

ABSTRACT

INTRODUCTION

RESULTS

The S. Typhimurium activators NlpD and EnvC for PGN amidases are very similar to those in E. coli.

S. Typhimurium EnvC is the main activator of PGN amidases, contributing to cell division.

FIG 1.

S. Typhimurium EnvC contributes to gut colonization.

FIG 2.

Reduced colonization of the envC mutant is partly attributable to the hypersensitivity to bile acids.

TABLE 1.

FIG 3.

The envC mutant displays the impairment in gut colonization in the streptomycin-untreated mice, in which the complex microbiota is present with no inflammation.

FIG 4.

A colonization defect of the envC mutant is conferred by reduced chemotactic movement.

FIG 5.

The S. Typhimurium envC mutant exhibits attenuated triggering of enterocolitis.

FIG 6.

DISCUSSION

FIG 7.

MATERIALS AND METHODS

Bioinformatic analysis.

Bacterial strains and plasmids.

TABLE 2.

Bacterial morphology analysis.

Mouse infection experiments.

Plasmid construction.

Determination of MICs.

Quantification of total bile acids in feces.

Swimming motility and swarming assay.

Analysis of flagellin production and transport.

Statistical analysis.

Ethics statement.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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