An Oral Fluorouracil Prodrug, Capecitabine, Mitigates a Gram-Positive Systemic Infection in Mice.

New classes of antibiotics are needed to fight bacterial infections, and repurposing existing drugs as antibiotics may enable rapid deployment of new treatments. Screens for antibacterials have been traditionally performed in standard laboratory media, but bacterial pathogens experience very different environmental conditions during infection, including nutrient limitation. To introduce the next generation of researchers to modern drug discovery methods, we developed a course-based undergraduate research experience (CURE) in which undergraduate students screened a library of FDA-approved drugs for their ability, in a nutrient-poor medium, to prevent the growth of the human Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium. The nine drugs identified all disrupt DNA metabolism in bacteria and eukaryotes. One of the hit compounds, capecitabine, is a well-tolerated oncology drug that is administered orally, a preferred treatment route. We demonstrated that capecitabine is more effective at inhibiting S. Typhimurium growth in nutrient-limited than in standard rich microbiological broth, an explanation for why the antibiotic activity of this compound has not been previously recognized. Capecitabine is enzymatically converted to the active pyrimidine analogue, fluorouracil (5-FU), and Gram-positive bacteria, including Staphylococcus aureus, are significantly more sensitive to 5-FU than Gram-negative bacteria. We therefore tested capecitabine for efficacy in a murine model of S. aureus peritonitis. Oral capecitabine administration reduced the colonization of tissues and increased animal survival in a dose-responsive manner. Since capecitabine is inexpensive, orally available, and relatively safe, it may have utility for treatment of intractable Gram-positive bacterial infections. IMPORTANCE As bacterial infections become increasingly insensitive to antibiotics, whether established, off-patent drugs could treat infections becomes an important question. At the same time, basic research has revealed that during infection, mammals starve pathogens for nutrients and, in response, bacteria dramatically alter their biology. Therefore, it may be fruitful to search for drugs that could be repurposed as antibiotics using bacteria grown with limited nutrients. This approach, executed with undergraduate student researchers, identified nine drugs known to interfere with the production and/or function of DNA. We further explored one of these drugs, capecitabine, a well-tolerated human oncology drug. Oral administration of capecitabine reduced infection with the human pathogen Staphylococcus aureus and increased survival in mice. These data suggest that capecitabine has potential as a therapy for patients with otherwise untreatable bacterial infections.

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

Clofazimine Reduces the Survival of Salmonella enterica in Macrophages and Mice.

Drug resistant pathogens are on the rise, and new treatments are needed for bacterial infections. Efforts toward antimicrobial discovery typically identify compounds that prevent bacterial growth in microbiological media. However, the microenvironments to which pathogens are exposed during infection differ from rich media and alter the biology of the pathogen. We and others have therefore developed screening platforms that identify compounds that disrupt pathogen growth within cultured mammalian cells. Our platform focuses on Gram-negative bacterial pathogens, which are of particular clinical concern. We screened a panel of 707 drugs to identify those with efficacy against Salmonella enterica Typhimurium growth within macrophages. One of the drugs identified, clofazimine (CFZ), is an antibiotic used to treat mycobacterial infections that is not recognized for potency against Gram-negative bacteria. We demonstrated that in macrophages CFZ enabled the killing of S. Typhimurium at single digit micromolar concentrations, and in mice, CFZ reduced tissue colonization. We confirmed that CFZ does not inhibit the growth of S. Typhimurium and E. coli in standard microbiological media. However, CFZ prevents bacterial replication under conditions consistent with the microenvironment of macrophage phagosomes, in which S. Typhimurium resides during infection: low pH, low magnesium and phosphate, and the presence of certain cationic antimicrobial peptides. These observations suggest that in macrophages and mice the efficacy of CFZ against S. Typhimurium is facilitated by multiple aspects of soluble innate immunity. Thus, systematic screens of existing drugs for infection-based potency are likely to identify unexpected opportunities for repurposing drugs to treat difficult pathogens.

Infection-based chemical screens uncover host-pathogen interactions.

Bacterial pathogens must resist host innate immunity to cause disease. While Gram-negative bacteria have a protective outer membrane, this membrane is subject to host-induced damage that makes these pathogens vulnerable. We developed a high content screening platform that identifies compounds that cause the killing of the bacterial pathogen Salmonella enterica in macrophages. This platform enables the rapid discovery of compounds that work in concert with the macrophage to prevent pathogen survival, as most hit compounds are not active in standard microbiological media and are not pro-drugs. We describe within the platform and the compounds it has found, and consider how they may help us discover new ways to fight infection.

Salmonella enterica Requires Lipid Metabolism Genes To Replicate in Proinflammatory Macrophages and Mice.

To survive and replicate during infection, pathogens utilize different carbon and energy sources depending on the nutritional landscape of their host microenvironment. Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen that occupies diverse cellular niches. While it is clear that Salmonella Typhimurium requires access to glucose during systemic infection, data on the need for lipid metabolism are mixed. We report that Salmonella Typhimurium strains lacking lipid metabolism genes were defective for systemic infection of mice. Bacterial lipid import, ß-oxidation, and glyoxylate shunt genes were required for tissue colonization upon oral or intraperitoneal inoculation. In cultured macrophages, lipid import and ß-oxidation genes were required for bacterial replication and/or survival only when the cell culture medium was supplemented with nonessential amino acids. Removal of glucose from tissue culture medium further enhanced these phenotypes and, in addition, conferred a requirement for glyoxylate shunt genes. We also observed that Salmonella Typhimurium needs lipid metabolism genes in proinflammatory but not anti-inflammatory macrophages. These results suggest that during systemic infection, the Salmonella Typhimurium that relies upon host lipids to replicate is within proinflammatory macrophages that have access to amino acids but not glucose. An improved understanding of the host microenvironments in which pathogens have specific metabolic requirements may facilitate the development of targeted approaches to treatment.

Salmonella Typhimurium Infection of Human Monocyte-Derived Macrophages.

The successful infection of macrophages by non-typhoidal serovars of Salmonella enterica is likely essential to the establishment of the systemic disease they sometimes cause in susceptible human populations. However, the interactions between Salmonella and human macrophages are not widely studied, with mouse macrophages being a much more common model system. Fundamental differences between mouse and human macrophages make this less than ideal. Additionally, the inability of human macrophage-like cell lines to replicate some properties of primary macrophages makes the use of primary cells desirable. Here we present protocols to study the infection of human monocyte-derived macrophages with Salmonella Typhimurium. These include a method for differentiating monocyte-derived macrophages in vitro and protocols for infecting them with Salmonella Typhimurium, as well as assays to measure the extent of infection, replication, and death. These protocols are useful for the investigation of both bacterial and host factors that determine the outcome of infection. © 2018 by John Wiley & Sons, Inc.

A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAßN (Phe-Arg ß-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models.

Salmonella Meningitis Associated with Monocyte Infiltration in Mice.

In the current study, we examined the ability of Salmonella enterica serovar Typhimurium to infect the central nervous system and cause meningitis following the natural route of infection in mice. C57BL/6J mice are extremely susceptible to systemic infection by Salmonella Typhimurium because of loss-of-function mutations in Nramp1 (SLC11A1), a phagosomal membrane protein that controls iron export from vacuoles and inhibits Salmonella growth in macrophages. Therefore, we assessed the ability of Salmonella to disseminate to the central nervous system (CNS) after oral infection in C57BL/6J mice expressing either wild-type (resistant) or mutant (susceptible) alleles of Nramp1. In both strains, oral infection resulted in focal meningitis and ventriculitis with recruitment of inflammatory monocytes to the CNS. In susceptible Nramp1-/- mice, there was a direct correlation between bacteremia and the number of bacteria in the brain, which was not observed in resistant Nramp1+/+ mice. A small percentage of Nramp1+/+ mice developed severe ataxia, which was associated with high bacterial loads in the CNS as well as clear histopathology of necrotizing vasculitis and hemorrhage in the brain. Thus, Nramp1 is not essential for Salmonella entry into the CNS or neuroinflammation, but may influence the mechanisms of CNS entry as well as the severity of meningitis.

Bacterial Stimulation of Toll-Like Receptor 4 Drives Macrophages To Hemophagocytose.

During acute infection with bacteria, viruses or parasites, a fraction of macrophages engulf large numbers of red and white blood cells, a process called hemophagocytosis. Hemophagocytes persist into the chronic stage of infection and have an anti-inflammatory phenotype. Salmonella enterica serovar Typhimurium infection of immunocompetent mice results in acute followed by chronic infection, with the accumulation of hemophagocytes. The mechanism(s) that triggers a macrophage to become hemophagocytic is unknown, but it has been reported that the proinflammatory cytokine gamma interferon (IFN-γ) is responsible. We show that primary macrophages become hemophagocytic in the absence or presence of IFN-γ upon infection with Gram-negative bacterial pathogens or prolonged exposure to heat-killed Salmonella enterica, the Gram-positive bacterium Bacillus subtilis, or Mycobacterium marinum. Moreover, conserved microbe-associated molecular patterns are sufficient to stimulate macrophages to hemophagocytose. Purified bacterial lipopolysaccharide (LPS) induced hemophagocytosis in resting and IFN-γ-pretreated macrophages, whereas lipoteichoic acid and synthetic unmethylated deoxycytidine-deoxyguanosine dinucleotides, which mimic bacterial DNA, induced hemophagocytosis only in IFN-γ-pretreated macrophages. Chemical inhibition or genetic deletion of Toll-like receptor 4, a pattern recognition receptor responsive to LPS, prevented both Salmonella- and LPS-stimulated hemophagocytosis. Inhibition of NF-κB also prevented hemophagocytosis. These results indicate that recognition of microbial products by Toll-like receptors stimulates hemophagocytosis, a novel outcome of prolonged Toll-like receptor signaling, suggesting hemophagocytosis is a highly conserved innate immune response.

Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by Salmonella enterica Serovar Typhimurium infection in mice.

The Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium causes persistent systemic inflammatory disease in immunocompetent mice. Following oral inoculation with S. Typhimurium, mice develop a hematopathological syndrome akin to typhoid fever with splenomegaly, microcytic anemia, extramedullary erythropoiesis, and increased hemophagocytic macrophages in the bone marrow, liver, and spleen. Additionally, there is marked loss of iron from the spleen, an unanticipated result, given the iron sequestration reported in anemia of inflammatory disease. To establish why tissue iron does not accumulate, we evaluated multiple measures of pathology for 4 weeks following oral infection in mice. We demonstrate a quantitative decrease in splenic iron following infection despite increased numbers of splenic phagocytes. Infected mice have increased duodenal expression of the iron exporter ferroportin-1, consistent with increased uptake of dietary iron. Liver and splenic macrophages also express high levels of ferroportin-1. These observations indicate that splenic and hepatic macrophages export iron during S. Typhimurium infection, in contrast to macrophage iron sequestration observed in anemia of inflammatory disease. Tissue macrophage export of iron occurs concurrent with high serum concentrations of interferon gamma (IFN-γ) and interleukin 12 (IL-12). In individual mice, high concentrations of both proinflammatory (tumor necrosis factor alpha [TNF-α]) and anti-inflammatory (IL-10) cytokines in serum correlate with increased tissue bacterial loads throughout 4 weeks of infection. These in vivo observations are consistent with previous cell culture studies and suggest that the relocation of iron from tissue macrophages during infection may contribute to anemia and also to host survival of acute S. Typhimurium infection.

The biomechanisms of metal and metal-oxide nanoparticles' interactions with cells.

Humans are increasingly exposed to nanoparticles (NPs) in medicine and in industrial settings, where significant concentrations of NPs are common. However, NP interactions with and effects on biomolecules and organisms have only recently been addressed. Within we review the literature regarding proposed modes of action for metal and metal-oxide NPs, two of the most prevalent types manufactured. Iron-oxide NPs, for instance, are used as tracers for magnetic resonance imaging of oncological tumors and as vehicles for therapeutic drug delivery. Factors and theories that determine the physicochemical and biokinetic behaviors of NPs are discussed, along with the observed toxicological effects of NPs on cells. Key thermodynamic and kinetic models that explain the sources of energy transfer from NPs to biological targets are summarized, in addition to quantitative structural activity relationship (QSAR) modeling efforts. Future challenges for nanotoxicological research are discussed. We conclude that NP studies based on cell culture are often inconsistent and underestimate the toxicity of NPs. Thus, the effect of NPs needs to be examined in whole animal systems.

Salmonella enterica infection stimulates macrophages to hemophagocytose.

UNLABELLED: Hemophagocytes are cells of the monocyte lineage that have engulfed erythrocytes and leukocytes. Hemophagocytes frequently accumulate in patients with severe acute bacterial infections, such as those caused by Salmonella enterica, Brucella abortus, and Mycobacterium tuberculosis. The relationship between hemophagocytosis and infection is not well understood. In the murine liver, S. enterica serovar Typhimurium resides within hemophagocytic macrophages containing leukocytes. Here we show that S. Typhimurium also resides within hemophagocytes containing erythrocytes. In cell culture, S. Typhimurium benefits from residence within hemophagocytes by accessing iron, but why macrophages hemophagocytose is unknown. We show that treatment of macrophages with a cocktail of the proinflammatory cytokine interferon gamma (IFN-γ) and lipopolysaccharide (LPS) stimulates engulfment of nonsenescent erythrocytes. Exposure of resting or IFN-γ-treated macrophages to live, but not to heat-killed, S. Typhimurium cells also stimulates erythrocyte engulfment. Single-cell analyses show that S. Typhimurium-infected macrophages are more likely to erythrophagocytose and that infected macrophages engulf more erythrocytes than uninfected macrophages within the same culture well. In addition, macrophages containing erythrocytes harbor more bacteria. However, S. Typhimurium does not promote macrophage engulfment of polystyrene beads, suggesting a role for a ligand on the target cell. Finally, neither of the two S. Typhimurium type 3 secretion systems, T3SS1 or T3SS2, is fully required for hemophagocytosis. These results indicate that infection of macrophages with live S. Typhimurium cells stimulates hemophagocytosis. IMPORTANCE: Macrophages are white blood cells (leukocytes) that engulf and destroy pathogens. Hemophagocytes, a subset of macrophages, are characteristic of severe acute infection in patients with, for instance, typhoid fever, brucellosis, tuberculosis, and leishmaniasis. Each of these diseases has the potential to become chronic. Hemophagocytes (blood-eating cells) engulf and degrade red and white blood cells for unknown reasons. The bacterial pathogen Salmonella acquires the essential nutrient iron from murine hemophagocytes. We report that Salmonella stimulates macrophages to engulf blood cells, indicating that cells of this bacterium actively promote the formation of a specialized cellular niche in which they can acquire nutrients, evade killing by the host immune system, and potentially transition to chronic infection.

Salmonella acquires ferrous iron from haemophagocytic macrophages.

Bacteria harbour both ferrous and ferric iron transporters. We now report that infection of macrophages and mice with a Salmonella enterica Typhimurium strain containing an inactivated feoB-encoded ferrous iron transporter results in increased bacterial replication, compared to infection with wild type. Inactivation of other cation transporters, SitABCD or MntH, did not increase bacterial replication. The feoB mutant strain does not have an intrinsically faster growth rate. Instead, increased replication correlated with increased expression in macrophages of the fepB-encoded bacterial ferric iron transporter and also required siderophores, which capture ferric iron. Co-infection of mice with wild type and a feoB mutant strain yielded a different outcome: FeoB is clearly required for tissue colonization. In co-infected primary mouse macrophages, FeoB is required for S. Typhimurium replication if the macrophages were IFNγ treated and contain phagocytosed erythrocytes, a model for haemophagocytosis. Haemophagocytes are macrophages that have engulfed erythrocytes and/or leucocytes and can harbour Salmonella in mice. These observations suggest that Salmonella acquires ferrous iron from haemophagocytic macrophages.

The ferric enterobactin transporter Fep is required for persistent Salmonella enterica serovar typhimurium infection.

Most bacterial pathogens require iron to grow and colonize host tissues. The Gram-negative bacterium Salmonella enterica serovar Typhimurium causes a natural systemic infection of mice that models acute and chronic human typhoid fever. S. Typhimurium resides in tissues within cells of the monocyte lineage, which limit pathogen access to iron, a mechanism of nutritional immunity. The primary ferric iron import system encoded by Salmonella is the siderophore ABC transporter FepBDGC. The Fep system has a known role in acute infection, but it is unclear whether ferric iron uptake or the ferric iron binding siderophores enterobactin and salmochelin are required for persistent infection. We defined the role of the Fep iron transporter and siderophores in the replication of Salmonella in macrophages and in mice that develop acute followed by persistent infections. Replication of wild-type and iron transporter mutant Salmonella strains was quantified in cultured macrophages, fecal pellets, and host tissues in mixed- and single-infection experiments. We show that deletion of fepB attenuated Salmonella replication and colonization within macrophages and mice. Additionally, the genes required to produce and transport enterobactin and salmochelin across the outer membrane receptors, fepA and iroN, are needed for colonization of all tissues examined. However, salmochelin appears to be more important than enterobactin in the colonization of the spleen and liver, both sites of dissemination. Thus, the FepBDGC ferric iron transporter and the siderophores enterobactin and salmochelin are required by Salmonella to evade nutritional immunity in macrophages and cause persistent infection in mice.

Hemophagocytic macrophages in murine typhoid fever have an anti-inflammatory phenotype.

Histiocytes are white blood cells of the monocytic lineage and include macrophages and dendritic cells. In patients with a variety of infectious and noninfectious inflammatory disorders, histiocytes can engulf nonapoptotic leukocytes and nonsenescent erythrocytes and thus become hemophagocytes. We report here the identification and characterization of splenic hemophagocytes in a natural model of murine typhoid fever. The development of a flow-cytometric method allowed us to identify hemophagocytes based on their greater than 6N (termed 6N+) DNA content. Characterization of the 6N+ population from infected mice showed that these cells consist primarily of macrophages rather than dendritic cells and contain T lymphocytes, consistent with hemophagocytosis. Most 6N+ macrophages from Salmonella enterica serovar Typhimurium-infected mice contain intact DNA, consistent with hemophagocytosis. In contrast, most 6N+ macrophages from control mice or mice infected with a different bacterial pathogen, Yersinia pseudotuberculosis, contain damaged DNA. Finally, 6N+ splenic macrophages from S. Typhimurium-infected mice express markers consistent with an anti-inflammatory M2 activation state rather than a classical M1 activation state. We conclude that macrophages are the predominant splenic hemophagocyte in this disease model but not in Y. pseudotuberculosis-infected mice. The anti-inflammatory phenotype of hemophagocytic macrophages suggests that these cells contribute to the shift from acute to chronic infection.

A glycine betaine importer limits Salmonella stress resistance and tissue colonization by reducing trehalose production.

Mechanisms by which Salmonella establish chronic infections are not well understood. Microbes respond to stress by importing or producing compatible solutes, small molecules that stabilize proteins and lipids. The Salmonella locus opuABCD (also called OpuC) encodes a predicted importer of the compatible solute glycine betaine. Under stress conditions, if glycine betaine cannot be imported, Salmonella enterica produce the disaccharide trehalose, a highly effective compatible solute. We demonstrate that strains lacking opuABCD accumulate more trehalose under stress conditions than wild-type strains. ΔopuABCD mutant strains are more resistant to high-salt, low-pH and -hydrogen peroxide, conditions that mimic aspects of innate immunity, in a trehalose-dependent manner. In addition, ΔopuABCD mutant strains require the trehalose production genes to out-compete wild-type strains in mice and macrophages. These data suggest that in the absence of opuABCD, trehalose accumulation increases bacterial resistance to stress in broth and mice. Thus, opuABCD reduces bacterial colonization via a mechanism that limits trehalose production. Mechanisms by which microbes limit disease may reveal novel pathways as therapeutic targets.

Salmonella enterica replication in hemophagocytic macrophages requires two type three secretion systems.

Salmonella enterica serotype Typhimurium is a natural pathogen of mice, which acquire the bacteria orally and develop systemic acute infections that can become subacute to chronic infections. S. Typhimurium can reside within hemophagocytic macrophages (HMs) in SV129S6 mice, an Slc11a1/Nramp1(+/+) inbred strain. HMs are activated macrophages which have ingested viable hematopoietic cells and are a key characteristic of infectious and inflammatory diseases. Here we show that modest S. Typhimurium replication in HMs begins at 18 h postinfection, while activated macrophages kill the bacteria. For bacterial replication to occur, the phagocytosed viable cells must be grown to a low cell density and the multiplicity of infection must be low. HMs are able to kill phagocytosed Escherichia coli, produce reactive nitrogen species, and retain S. Typhimurium within membrane-bound vesicles. S. Typhimurium does not rescue E. coli upon coinfection of HMs. This indicates that S. Typhimurium does not cause HMs to become permissive for other microbes; rather, S. Typhimurium is especially equipped to survive within HMs. Two type three secretion systems (T3SS) encoded by S. Typhimurium are required for replication within HMs. While the T3SS within Salmonella pathogenicity island 2 (SPI-2) has been previously shown to be important for bacterial survival in cells, a role for SPI-1 in replication in macrophages has not been reported. The requirement for SPI-1 in HMs may help explain the role of SPI-1 during long-term colonization of mice.

Chronic murine typhoid fever is a natural model of secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using Salmonella enterica serotype Typhimurium by oral gavage to mimic naturally-occurring infection in Sv129S6 mice, we characterized the clinical, hematologic and morphologic host responses to disease thereby describing an animal model with the clinico-pathologic features of secondary HLH as set forth by the Histiocyte Society: fever, splenomegaly, cytopenias (anemia, thrombocytopenia), hemophagocytosis in bone marrow and spleen, hyperferritinemia, and hypofibrinogenemia. Disease severity correlates with high splenic and hepatic bacterial load, and we show disease course can be monitored and tracked in live animals. Whereby secondary HLH is known to occur in human patients with typhoid fever and other infectious diseases, our characterization of a viable natural disease model of secondary HLH offers an important means to elucidate pathogenesis of poorly understood mechanisms of secondary HLH and investigation of novel therapies. We characterize previously unreported secondary HLH in a chronic mouse model of typhoid fever, and novel changes in hematology including decreased tissue ferric iron storage that differs from classically described anemia of chronic disease. Our studies demonstrate S. Typhimurium infection of mice is a natural infectious disease model of secondary HLH that may have utility for elucidating disease pathogenesis and developing novel therapies.

Intracellular microbes and haemophagocytosis.

Haemophagocytosis (hemophagocytosis) is the phenomenon of activated macrophage consumption of red and white blood cells, including professional phagocytes and lymphocytes. It can occur in patients with severe cases of intracellular microbial infection, including avian influenza, leishmaniasis, tuberculosis and typhoid fever. While well-known to physicians since at least the mid-1800s, haemophagocytosis has been little studied due to a paucity of tractable animal and cell culture models. Recently, haemophagocytosis has been described in a mouse model of typhoid fever, and it was noted that the infectious agent, Salmonella enterica, resides within haemophagocytic macrophages in mice. In addition, a cell culture model for haemophagocytosis revealed that S. enterica preferentially replicate in haemophagocytic macrophages. This review describes how, at the molecular and cellular levels, S. enterica may promote and take advantage of haemophagocytosis to establish long-term systemic infections in mammals. The role, relevance and possible molecular mechanisms of haemophagocytosis are discussed within the context of other microbial infections and of genetic deficiencies in which haemophagocytosis occurs and is associated with morbidity.