Michele Pesciaroli

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.

The ZupT transporter plays an important role in zinc homeostasis and contributes to Salmonella enterica virulence

Zinc is an essential metal for cellular homeostasis and function in both eukaryotes and prokaryotes. To acquire this essential nutrient, bacteria employ transporters characterized by different affinity for the metal. Several studies have investigated the role of the high affinity transporter ZnuABC in the bacterial response to zinc shortage, showing that this transporter has a key role in adapting bacteria to zinc starvation. In contrast, the role of the low affinity zinc importer ZupT has been the subject of limited investigations. Here we show that a Salmonella strain lacking ZupT is impaired in its ability to grow in metal devoid environments and that a znuABC zupT strain exhibits a severe growth defect in zinc devoid media, is hypersensitive to oxidative stress and contains reduced levels of intracellular free zinc. Moreover, we show that ZupT also plays a role in the ability of S. Typhimurium to colonize the host tissues. During systemic infections, the single zupT mutant strain was attenuated only in Nramp1(+/+) mice, but competition experiments between znuABC and znuABC zupT mutants revealed that ZupT contributes to metal uptake in vivo independently of the presence of a functional Nramp1 transporter. Altogether, the here reported results show that ZupT plays an important role in Salmonella zinc homeostasis, being involved in metal import both in vitro and in infected animals.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter: an efficacious orally-administered mucosal vaccine against salmonellosis in pigs.

We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ΔznuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ΔznuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ΔznuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ΔznuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ΔznuABC represents a promising candidate vaccine against salmonellosis in pigs.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Salmonella Typhimurium lacking the Znuabc transporter is attenuated and immunogenic in pigs

Meat contamination by Salmonella spp. is emerging as a major cause of human enteric infections in industrialized countries. The attempts to reduce human cases of salmonellosis encompass pre- and post-harvest interventions. In this context, vaccination of pigs may represent an effective instrument in eliminating/reducing Salmonella burden through the food chain. We have previously demonstrated that Salmonella Typhimurium lacking the ZnuABC transporter (S. Typhimurium ΔznuABC) is a promising candidate live vaccine in different mouse models of Salmonella Typhimurium infection. In this study, we confirmed in pigs the attenuation of S. Typhimurium ΔznuABC. Moreover, we evaluated the safety and immunogenicity of S. Typhimurium ΔznuABC administered to pigs by the oral route. We monitored clinical conditions of animals and we conducted a microbiological culture and a quantification of the humoral and cellular immune response, respectively, on fecal and blood samples of pigs. After vaccination with attenuated S. Typhimurium ΔznuABC, pigs showed a modest degree of hyperthermia. In addition, fecal shedding of S. Typhimurium ΔznuABC could not be detected 28 days after the inoculum. Furthermore, vaccination with S. Typhimurium ΔznuABC elicited a distinct production of anti-Salmonella antibodies and IFN-γ. Taken together, these results suggest that S. Typhimurium ΔznuABC is attenuated and immunogenic in pigs. Although the vaccine dosages do not guarantee complete safety there is ample margin to set up better conditions of use, suggesting that S. Typhimurium ΔznuABC could be a promising attenuated strain to be used as live mucosal vaccine for oral delivery.

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice.

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4+CD25+T regulatory cells are able to limit the effectiveness of CD4+T cells and are able to favor the maintenance and the progression of B. abortus infection.

Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice.

It has been demonstrated that antibodies specific for O-PS antigen of Brucella smooth strains are involved in the protective immunity of brucellosis. Since the rough strain Brucella melitensis B115 was able to protect mice against wild Brucella strains brucellosis despite the lack of anti-OPS antibodies, in this study we evaluated the biological significance of antibodies induced by this strain, directed to antigens other than O-PS, passively tranferred to untreated mice prior to infection with Brucella abortus 2308 and B. melitensis 16M virulent strains. The protective ability of specific antisera collected from mice vaccinated with B. melitensis B115, B. abortus RB51 and B. abortus S19 strains was compared. The results indicated that antibodies induced by B115 were able to confer a satisfactory protection, especially against B. abortus 2308, similar to that conferred by the antiserum S19, while the RB51 antiserum was ineffective. These findings suggest that antibodies induced by B115 could act as opsonins as well as antibodies anti-O-PS, thus triggering more efficient internalization and degradation of bacteria within phagocytes. This is the first study assessing the efficacy of antibodies directed to antigens other than O-PS in the course of brucellosis infection.

Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis

BackgroundSalmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed.ResultsThe microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria.ConclusionsThese results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes.

B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections

Brucellosis is one of the most serious zoonoses all over the world, with B. melitensis, B. abortus and B. suis being the most pathogenic species for humans. Vaccination of domesticated livestock still represents the most efficient way to prevent human infection. However, the available Brucella vaccines retain an important residual virulence and induce antibodies interfering with surveillance programs. Moreover, each vaccine shows different protective effects versus different Brucella species and different animal hosts. Nowadays, while B. melitensis and B. suis infections in cattle are emerging as a significant problem, there are no available vaccines to overcome such issue. B. melitensis strain B115, a natural, attenuated rough strain in our previous studies proved to be highly protective against B. melitensis and B. ovis infections in mice, without inducing interfering antibodies. In this study, we tested the efficiency of B115 as vaccine against B. abortus and B. suis. Vaccination of mice with 10(8) CFU/mouse of B. melitensis B115 conferred a satisfactory protection against B. abortus 2308. On the contrary, mice vaccinated once with 10(8) or 10(9) CFU/mouse of B115 were weakly protected against B. suis infection. Conversely, when mice were vaccinated twice with 10(9) CFU B115/mouse, the protective activity significantly increased. Unlike its rough phenotype, B115 showed an adequate persistence in mice accompanied to a solid humoral and cell-mediated immunity. All together, these findings suggest the potential usefulness of B115 to control brucellosis in animal hosts due to heterologous challenges.