Pietro Mastroeni

Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family

The intracellular bacterium Listeria monocytogenes can invade several types of normally non‐phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800‐amino‐acid protein, and inIB, which encodes a 630‐amino‐acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB In invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.

Role of T cells, TNFα and IFNγ in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro− salmonella vaccines☆

Abstract The SL3261 Salmonella typhimurium aro A live vaccine strain confers solid protection against oral challenge with virulent salmonellae, immunity persisting long after the vaccine has been cleared from the tissues. BALB/c mice immunized with SL3261 and later subjected to in vivo depletion of both CD4 + and CD8 + T cells had impaired recall of immunity to oral challenge with the virulent S. typhimurium C5, with increased mortality and higher bacterial loads in the reticuloendothelial system (RES). Selective depletion of CD4 + cells alone significantly impaired resistance both 8 and 14 weeks after vaccination as determined by estimation of bacterial numbers in organ homogenates. Depletion of CD8 + cells alone had less effect on immunity when performed at 8 weeks than at 14 weeks after immunization. Administration of anti-IFNγ or anti-TNFα antibodies also impaired recall of immunity, exacerbating a secondary infection in vaccinated mice. Challenge of T cell-depleted immune mice with virulent salmonellae caused hepatosplenomegaly with minute grossly visible focal lesions, and a marked increase in the number and severity of necrotic foci in spleen, liver and lymph nodes. A widespread mononuclear cell infiltrate was present. The histopathology in anti-IFNγ-treated mice was qualitatively similar to that seen in T-cell depleted mice. In contrast, in the anti-TNFα-treated mice splenomegaly was much less than in T cell-depleted mice. Granulomas were absent, no mononuclear infiltration was observed and there was severe necrosis; the lesions appeared similar to or worse than those seen in naive mice. Surprisingly, IFNγ was detectable in sera of both controls and T cell-depleted mice on day 8 of the secondary infection, as well as in sera of anti-TNFα-treated mice on day 6 of infection. The results indicate that T cells, IFNγ and TNFα are all important in the specific recall of immunity to virulent salmonellae conferred by immunization with live vaccines, with the effect of T cell and IFNγ depletion (marked macrophage infiltration) being qualitatively very different from that of TNFα neutralization (no mononuclear infiltrate or granuloma formation).

Rapid Expression of Chemokines and Proinflammatory Cytokines in Newly Hatched Chickens Infected with Salmonella enterica Serovar Typhimurium

ABSTRACT Poultry meat and eggs contaminated with Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective against Salmonella infection in chickens has not been characterized at the molecular level. In the present study, bacterial colonization, development of pathological lesions, and proinflammatory cytokine and chemokine gene expression were investigated in the liver, spleen, jejunum, ileum, and cecal tonsils in newly hatched chickens 6, 12, 24, and 48 h after oral infection with Salmonella serovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereas Salmonella started to appear in the liver only from 24 h postinfection. Large numbers of heterophils, equivalent to neutrophils in mammals, and inflammatory edema could be seen in the lamina propria of the intestinal villi and in the liver. Interleukin 8 (IL-8), K60 (a CXC chemokine), macrophage inflammatory protein 1 β, and IL-1β levels were significantly upregulated in the intestinal tissues and in the livers of the infected birds. However, the spleens of the infected birds show little or no change in the expression levels of these cytokines and chemokines. Increased expression of the proinflammatory cytokines and chemokines (up to several hundred-fold) correlated with the presence of inflammatory signs in those tissues. This is the first description of in vivo expression of chemokines and proinflammatory cytokines in response to oral infection with Salmonella in newly hatched chickens.

Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens.

ABSTRACT In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6−/−).Igh-6−/− mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5.Igh-6−/− mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6−/− mice, unlikeIgh-6+/+ C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance inIgh-6−/− mice. Total splenocytes and purified CD4+ T cells obtained fromIgh-6−/− mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained fromIgh-6+/+ C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.

Immunity to salmonellosis

Summary:  Salmonella enterica is a genetically broad species harboring isolates that display considerable antigenic heterogeneity and significant differences in virulence potential. Salmonella generally exhibit an invasive potential and they can survive for extended periods within cells of the immune system. They cause acute or chronic infections that can be local (e.g. gastroenteritis) or systemic (e.g. typhoid). In vivo Salmonella infections are complex with multiple arms of the immune system being engaged. Both humoral and cellular responses can be detected and characterized, but full protective immunity is not always induced, even following natural infection. The murine model has proven to be a fertile ground for exploring immune mechanisms and observations in the mouse have often, although not always, correlated with those in other infectable species, including humans. Host genetic studies have identified a number of mammalian genes that are central to controlling infection, operating both in innate and acquired immune pathways. Vaccines, both oral and parenteral, are available or under development, and these have been used with some success to explore immunity in both model systems and clinically in humans.

Cytokine and Chemokine Responses Associated with Clearance of a Primary Salmonella enterica Serovar Typhimurium Infection in the Chicken and in Protective Immunity to Rechallenge

ABSTRACT Infection of poultry with Salmonella enterica serovar Typhimurium poses a significant risk to public health through contamination of meat from infected animals. Vaccination has been proposed to control infections in chickens. However, the vaccines are currently largely empirical, and our understanding of the mechanisms that underpin immune clearance and protection in avian salmonellosis is not complete. In this study we describe the cytokine, chemokine, and antibody responses and cellular changes in primary and secondary infections of chickens with Salmonella serovar Typhimurium. Infection of 1-week-old chickens induced early expression of a macrophage inflammatory protein (MIP) family chemokine in the spleen and liver, followed by increased expression of gamma interferon accompanied by increased numbers of both CD4+ and CD8+ T cells and the formation of granuloma-like follicular lesions. This response correlated with a Th1-mediated clearance of the systemic infection. Primary infection also induced specific immunoglobulin M (IgM), IgG, and IgA antibody responses. In contrast to previously published studies performed with newly hatched chicks, the expression levels of proinflammatory cytokines in the gastrointestinal tract were not greatly increased following infection. However, significant expression of the anti-inflammatory cytokine transforming growth factor β4 was detected in the gut early in infection. Following secondary challenge, the birds were fully protected against systemic infection and showed a high level of protection against gastrointestinal colonization. Rapid expression of the MIP family chemokine and interleukin-6 was detected in the guts of these birds and was accompanied by an influx of lymphocytes. Increased levels of serum IgA-specific antibodies were also found following rechallenge. These findings suggest that cellular responses, particularly Th1 responses, play a crucial role in immune clearance in avian salmonellosis and that protection against rechallenge involves the rapid recruitment of cells to the gastrointestinal tract. Additionally, the high levels of inflammatory response found following Salmonella serovar Typhimurium infection of newly hatched chicks were not observed following infection of older birds (1 week old), in which the expression of regulatory cytokines appeared to limit inflammation.

Role of antibody to lipopolysaccharide in protection against low-and high-virulence strains of Francisella tularensis

Mice immunised with lipopolysaccharide (LPS) from Francisella tularensis were protected against challenge with the live vaccine strain (LVS). However, when similarly immunised mice were challenged using the fully virulent F. tularensis strain Schu4, only an increase in the time to death was observed. Passive transfer of serum from LPS-immunised mice to naive mice afforded protection against F. tularensis LVS. LPS-immunised mice depleted of either CD4+ or CD8+ T-cells survived a F. tularensis LVS challenge although the rate of clearance of bacteria from the spleen was significantly reduced in the CD8+ depleted group. LPS-immunised mice boosted with F. tularensis LVS were re-challenged with F. tularensis Schu4. This cohort was significantly protected (LD(50) increased from <1 to >1000 CFU). However, passive transfer of serum did not confer protection and mice depleted of CD4+ or CD8+ T-cells did not survive.

Modelling within-host spatiotemporal dynamics of invasive bacterial disease.

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host–pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host–pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics.

Dynamics of bacterial growth and distribution within the liver during Salmonella infection.

Salmonella enterica causes severe systemic diseases in humans and animals and grows intracellularly within discrete tissue foci that become pathological lesions. Because of its lifestyle Salmonella is a superb model for studying the in vivo dynamics of bacterial distribution. Using multicolour fluorescence microscopy in the mouse typhoid model we have studied the interaction between different bacterial populations in the same host as well as the dynamic evolution of foci of infection in relation to bacterial growth and localization. We showed that the growth of Salmonella in the liver results in the spread of the microorganisms to new foci of infection rather than simply in the expansion of the initial ones. These foci were associated with independently segregating bacterial populations and with low numbers of bacteria in each infected phagocyte. Using fast‐growing and slow‐growing bacteria we also showed that the increase in the number of infected phagocytes parallels the net rate of bacterial growth of the microorganisms in the tissues

Copper Homeostasis in Salmonella Is Atypical and Copper-CueP Is a Major Periplasmic Metal Complex

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P1-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P1-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.