To investigate the relevance of zinc in host-pathogen interactions, we have constructed Salmonella enterica mutant strains in which the znuA gene, which encodes the periplasmic component of the ZnuABC high-affinity Zn 2؉ transporter, was deleted. This mutation does not alter the ability of Salmonella to grow in rich media but drastically reduces its ability to multiply in media deprived of zinc. In agreement with this phenotype, ZnuA accumulates only in bacteria cultivated in environments poor in zinc. In spite of the nearly millimolar intracellular concentration of zinc, we have found that znuA is highly expressed in intracellular salmonellae recovered either from cultivated cells or from the spleens of infected mice. We have also observed that znuA mutants are impaired in their ability to grow in Caco-2 epithelial cells and that bacteria starved for zinc display decreased ability to multiply in phagocytes. A dramatic reduction in the pathogenicity of the znuA mutants was observed in Salmonella-susceptible (BALB/c) or Salmonella-resistant (DBA-2) mice infected intraperitoneally or orally. This study shows that the amount of free metals available for bacterial growth within the infected animal is limited, despite the apparent elevated concentration of free metals within cells and in plasma and suggests that Salmonella exploits the ZnuABC zinc transporter to maximize zinc availability in such conditions. These results shed new light on the complex functions of zinc in vertebrate and bacterial physiology and pave the way for a better comprehension of pathogenic mechanisms in Salmonella infections.The ability of bacteria to colonize specific environments relies on their ability to obtain adequate supplies of the nutrients that are indispensable for their growth. Of particular relevance for human and animal health is to understand how bacterial pathogens face the problem of nutrient limitation in the infected host, an environment where several essential elements are not freely available for infectious microorganisms (44). Well-studied examples are the strategies adopted by pathogens to obtain iron within their host. In fact, iron availability in eukaryotes is strictly controlled by metal-binding proteins (i.e., ferritin, transferrin, and lactoferrin) which prevent its reactivity and limit the uptake ability by invasive microorganisms (40,42,43). Moreover, growth of intracellular bacteria is also controlled by specific pumps which remove iron from the bacterium-containing phagosomes (19, 48). As iron plays crucial catalytic roles in a large number of bacterial proteins, an adequate supply of this transition metal is necessary for bacterial survival and multiplication. Therefore, different pathogenic bacteria have evolved sophisticated strategies to acquire and utilize host iron, including the production of molecules (siderophores, hemophores, and membrane-associated pumps) characterized by an extraordinarily elevated iron affinity (40,42,43). The outcome of the competition for iron between the host cell and the micro...

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.

This study indicated that mice immunized with Brucella abortus RB51 bacteria and subsequently challenged with B. abortus 2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.

Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.Brucellosis is a major zoonotic disease, widely distributed in humans and domestic and wild animals, especially in developing countries. Among the different species of the Brucella genus, B. abortus and B. melitensis are the most pathogenic and virulent, not only for cattle or for sheep and goats, respectively, but also for other animal species. The occurrence of the disease in humans is largely dependent on the animal reservoir, with the highest rate of human infection in areas where rates of brucellosis in sheep and goats are high (6,35).Brucellosis vaccines are essential elements in control programs. Attenuated B. abortus strain 19 and B. melitensis strain Rev1 are proven effective vaccines; they induce good levels of protection against B. abortus in cattle and against B. melitensis in sheep and goats, preventing premature abortions (7, 30, 31). However, both vaccines have the drawback of inducing Opolysaccharide-specific antibodies that interfere with the discrimination between vaccinated and infected animals during serological screening (7, 31). In addition, they retain pathogenicity and sometimes cause abortion in vaccinated animals (10, 18, 41) and remain infectious for humans (3,5,26). The use of the conjunctival route when administering B. melit...

Brucellae are gram-negative, facultative intracellular bacteria which are one of the most common causes of abortion in animals. In addition, they are the source of a severe zoonosis. In this trial, we evaluated the effect of oral inoculation of Brucella abortus RB51 in mice against a challenge infection with B. abortus 2308. First, we showed that a gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both vaccine strain B. abortus RB51 and virulent strain B. abortus 2308. Successively, we assessed the clearance and the immune response following an oral infection with B. abortus RB51. Oral inoculation gave a mild infection which was cleared 42 days after infection, and it induced a delayed humoral and cell-mediated immune response. Finally, we immunized mice by oral inoculation with B. abortus RB51, and we challenged them with the virulent strain B. abortus 2308 by an oral or intraperitoneal route 42 days after vaccination. Oral inoculation of B. abortus RB51 was able to give protection to mice infected with the virulent strain B. abortus 2308 by the oral route but not to mice infected intraperitoneally. Our results indicate that oral inoculation of mice with B. abortus RB51 is able to give a protective immunity against an oral infection with virulent strains, and this protection seems to rely on an immune response at the mucosal level.Brucellae are gram-negative, facultative intracellular bacteria which are one of the most common causes of abortion in animals. In addition, they are the source of a severe zoonosis, which is characterized in human beings by a multitude of somatic complaints, including fever, sweats, anorexia, fatigue, malaise, weight loss, and depression (15). Immune response to Brucella spp. have been studied mainly with mice. In this animal model, protective immunity seems to be mediated by both humoral and cellular immune responses (2). In particular, in vivo studies indicate that an immune response mediated by both CD4 ϩ and CD8 ϩ T lymphocytes is important in controlling infection. Within these subsets, gamma interferon-producing CD4 ϩ T cells play a prominent role in recovery from infection (23). Vaccination against Brucella infections in animals is usually performed by parenteral administration of live attenuated smooth Brucella abortus S19. This vaccine is effective in conferring protection against virulent strains of B. abortus, but it also has a number of disadvantages, including causing abortion in pregnant animals, being pathogenic for humans, and inducing a humoral response which is similar to the response after natural infection, thus hampering procedures for the detection of infected animals by serological testing. In particular, the similarity in humoral responses is of great importance in field conditions because it is impossible, with B. abortus as the antigen, to serologically discriminate between vaccinated and infected animals (11). Recently, a stable, lipopolysaccharide O-antigen-deficient mutant of virulent...

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Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens.We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.

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