Heterogeneous Salmonella-host encounters determine disease progression in a typhoid fever model

An infectious disease is an illness that is caused by invasion and multiplication of pathogenic microorganisms in body tissues of a host. The complex and heterogeneous tissue microenvironments provide different growth opportunities for pathogens with implications for disease outcome. However, the heterogeneous host environment has been largely neglected, because suitable methods to analyze single-cell host-pathogen encounters in vivo were largely lacking. In this work, we addressed the implications of heterogeneous host-Salmonella encounters on disease progression in the well characterized mouse typhoid fever model. Specifically, we combined Salmonella biosensors with high resolution microscopy, flow cytometry and proteomics to reveal the fate of Salmonella in the diverse host microenvironments of spleen. We showed that neutrophils and inflammatory monocytes are recruited to Salmonella infectious foci in spleen where they produce large amounts of reactive oxygen species (ROS), reactive nitrogen species (RNS) and lipids. We revealed that neutrophils and monocytes kill Salmonella with ROS generated through NADPH oxidase and myeloperoxidase (MPO), whereas RNS play a negligible role for infection control. However, ROS can also cause substantial collateral damage in host tissue. We showed that MPO protects the host against self-damage by converting diffusible long-lived ROS into highly reactive ROS with short reach that remain confined to the pathogen microenvironment. Some Salmonella escape to resident red pulp macrophages, which impose only sublethal oxidative bursts on Salmonella. Although macrophages are a primary niche for Salmonella survival, a specific subset of IFN activated macrophages can kill Salmonella with guanylate binding protein associated mechanisms. In addition, we showed that Salmonella growth in spleen is heterogeneous, independently of regional factors or host cell types. Overall, our analysis revealed numerous different Salmonella-host cell encounters in spleen with divergent outcomes. Local failures to control bacterial replication appear next to regions where the host successfully eradicates the pathogen. Together, these data show that disease progression does not necessarily reflect an overall weak host immune response, but rather result from disparate host-pathogen encounters

Myeloperoxidase targets oxidative host attacks to Salmonella and prevents collateral tissue damage

Host control of infections crucially depends on the capability to kill pathogens with reactive oxygen species (ROS). However, these toxic molecules can also readily damage host components and cause severe immunopathology. Here, we show that neutrophils use their most abundant granule protein, myeloperoxidase, to target ROS specifically to pathogens while minimizing collateral tissue damage. A computational model predicted that myeloperoxidase efficiently scavenges diffusible H2O2 at the surface of phagosomal Salmonella and converts it into highly reactive HOCl (bleach), which rapidly damages biomolecules within a radius of less than 0.1 μm. Myeloperoxidase-deficient neutrophils were predicted to accumulate large quantities of H2O2 that still effectively kill Salmonella, but most H2O2 would leak from the phagosome. Salmonella stimulation of neutrophils from normal and myeloperoxidase-deficient human donors experimentally confirmed an inverse relationship between myeloperoxidase activity and extracellular H2O2 release. Myeloperoxidase-deficient mice infected with Salmonella had elevated hydrogen peroxide tissue levels and exacerbated oxidative damage of host lipids and DNA, despite almost normal Salmonella control. These data show that myeloperoxidase has a major function in mitigating collateral tissue damage during antimicrobial oxidative bursts, by converting diffusible long-lived H2O2 into highly reactive, microbicidal and locally confined HOCl at pathogen surfaces

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

We report recessive mutations in the gene for the latent transforming growth factor-β binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-β (TGF-β) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-β signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4

Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol

Phenotypic variation of salmonella in host tissues delays eradication by antimicrobial chemotherapy

Antibiotic therapy often fails to eliminate a fraction of transiently refractory bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance in vitro, but their in vivo relevance remains unclear. Using a fluorescent growth rate reporter, we detected extensive phenotypic variation of Salmonella in host tissues. This included slow-growing subsets as well as well-nourished fast-growing subsets driving disease progression. Monitoring of Salmonella growth and survival during chemotherapy revealed that antibiotic killing correlated with single-cell division rates. Nondividing Salmonella survived best but were rare, limiting their impact. Instead, most survivors originated from abundant moderately growing, partially tolerant Salmonella. These data demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control

Disparate Impact of Oxidative Host Defenses Determines the Fate of Salmonella during Systemic Infection in Mice

Reactive oxygen and nitrogen species function in host defense via mechanisms that remain controversial. Pathogens might encounter varying levels of these species, but bulk measurements cannot resolve such heterogeneity. We used single-cell approaches to determine the impact of oxidative and nitrosative stresses on individual Salmonella during early infection in mouse spleen. Salmonella encounter and respond to both stresses, but the levels and impact vary widely. Neutrophils and inflammatory monocytes kill Salmonella by generating overwhelming oxidative stress through NADPH oxidase and myeloperoxidase. This controls Salmonella within inflammatory lesions but does not prevent their spread to more permissive resident red pulp macrophages, which generate only sublethal oxidative bursts. Regional host expression of inducible nitric oxide synthase exposes some Salmonella to nitrosative stress, triggering effective local Salmonella detoxification through nitric oxide denitrosylase. Thus, reactive oxygen and nitrogen species influence dramatically different outcomes of disparate Salmonella-host cell encounters, which together determine overall disease progression