Listeria monocytogenes Replicate in Bone Marrow-Derived CD11c+ Cells but Not in Dendritic Cells Isolated from the Murine Gastrointestinal Tract.

Recent fate-mapping studies and gene-expression profiles suggest that commonly used protocols to generate bone marrow-derived cultured dendritic cells yield a heterogeneous mixture, including some CD11chi cells that may not have a bona fide counterpart in vivo. In this study, we provide further evidence of the discordance between ex vivo-isolated and in vitro-cultured CD11c+ cells by analyzing an additional phenotype, the ability to support cytosolic growth of the facultative intracellular bacterial pathogen Listeria monocytogenes Two days after foodborne infection of mice with GFP-expressing L. monocytogenes, a small percentage of CD103neg and CD103+ conventional dendritic cells (cDC) in the intestinal lamina propria and mesenteric lymph nodes were GFP+ However, in vitro infection of the same subsets of cells harvested from naive mice resulted in inefficient invasion by the bacteria (<0.1% of the inoculum). The few intracellular bacteria detected survived for only a few hours. In contrast, cultured CD103negCD11c+ cells induced by GM-CSF readily supported exponential growth of L. monocytogenes Flt3 ligand-induced cultures yielded CD103+CD11c+ cells that more closely resembled cDC, with only a modest level of L. monocytogenes replication. For both culture protocols, the longer the cells were maintained in vitro, the more readily they supported intracellular growth. The results of this study suggest that cDC are not a niche for intracellular growth of L. monocytogenes during intestinal infection of mice.

Monocytes Are the Predominant Cell Type Associated with Listeria monocytogenes in the Gut, but They Do Not Serve as an Intracellular Growth Niche.

After foodborne transmission of the facultative intracellular bacterial pathogen Listeria monocytogenes, most of the bacterial burden in the gut is extracellular. However, we previously demonstrated that intracellular replication in an as yet unidentified cell type was essential for dissemination and systemic spread of L. monocytogenes In this article, we show that the vast majority of cell-associated L. monocytogenes in the gut were adhered to Ly6Chi monocytes, a cell type that inefficiently internalized L. monocytogenes With bone marrow-derived in vitro cultures, high multiplicity of infection or the use of opsonized bacteria enhanced uptake of L. monocytogenes in CD64- monocytes, but very few bacteria reached the cell cytosol. Surprisingly, monocytes that had upregulated CD64 expression in transition toward becoming macrophages fully supported intracellular growth of L. monocytogenes In contrast, inflammatory monocytes that had increased CD64 expression in the bone marrow of BALB/c/By/J mice prior to L. monocytogenes exposure in the gut did not support L. monocytogenes growth. Thus, contrary to the perception that L. monocytogenes can infect virtually all cell types, neither naive nor inflammatory Ly6Chi monocytes served as a productive intracellular growth niche for L. monocytogenes. These results have broad implications for innate immune recognition of L. monocytogenes in the gut and highlight the need for additional studies on the interaction of extracellular, adherent L. monocytogenes with the unique subsets of myeloid-derived inflammatory cells that infiltrate sites of infection.

Intracellular Listeria monocytogenes comprises a minimal but vital fraction of the intestinal burden following foodborne infection.

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlA(m)-expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens of L. monocytogenes were recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication of L. monocytogenes was essential during the intestinal phase of infection. We found that lipoate protein ligase-deficient L. monocytogenes (ΔlplA1) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of the L. monocytogenes burden in the gastrointestinal tract is extracellular, but the small proportion of intracellular L. monocytogenes is essential for dissemination to the MLN and systemic organs.

InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA(m)) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA(m)), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA(m) significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.

Posttranscriptional self-regulation by the Lyme disease bacterium's BpuR DNA/RNA-binding protein.

Bacteria require explicit control over their proteomes in order to compete and survive in dynamic environments. The Lyme disease spirochete Borrelia burgdorferi undergoes substantial protein profile changes during its cycling between vector ticks and vertebrate hosts. In an effort to understand regulation of these transitions, we recently isolated and functionally characterized the borrelial nucleic acid-binding protein BpuR, a PUR domain-containing protein. We now report that this regulatory protein governs its own synthesis through direct interactions with bpuR mRNA. In vitro and in vivo techniques indicate that BpuR binds with high affinity and specificity to the 5' region of its message, thereby inhibiting translation. This negative feedback could permit the bacteria to fine-tune cellular BpuR concentrations. These data add to the understanding of this newly described class of prokaryotic DNA- and RNA-binding regulatory proteins.

Informing the design of clinical decision support services for evaluation of children with minor blunt head trauma in the emergency department: a sociotechnical analysis.

Integration of clinical decision support services (CDSS) into electronic health records (EHRs) may be integral to widespread dissemination and use of clinical prediction rules in the emergency department (ED). However, the best way to design such services to maximize their usefulness in such a complex setting is poorly understood. We conducted a multi-site cross-sectional qualitative study whose aim was to describe the sociotechnical environment in the ED to inform the design of a CDSS intervention to implement the Pediatric Emergency Care Applied Research Network (PECARN) clinical prediction rules for children with minor blunt head trauma. Informed by a sociotechnical model consisting of eight dimensions, we conducted focus groups, individual interviews and workflow observations in 11 EDs, of which 5 were located in academic medical centers and 6 were in community hospitals. A total of 126 ED clinicians, information technology specialists, and administrators participated. We clustered data into 19 categories of sociotechnical factors through a process of thematic analysis and subsequently organized the categories into a sociotechnical matrix consisting of three high-level sociotechnical dimensions (workflow and communication, organizational factors, human factors) and three themes (interdisciplinary assessment processes, clinical practices related to prediction rules, EHR as a decision support tool). Design challenges that emerged from the analysis included the need to use structured data fields to support data capture and re-use while maintaining efficient care processes, supporting interdisciplinary communication, and facilitating family-clinician interaction for decision-making.

Biodiverse Histoplasma Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.

Histoplasma is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative intracellular yeast form. There is substantial diversity among Histoplasma species, but it is not clear how this heterogeneity impacts the progression of pathology and cellular immune responses during acute respiratory infection, which represents the vast majority of histoplasmosis disease burden. After inoculating mice intranasally with a sublethal inoculum, we characterized the immune response to Histoplasma capsulatum (strain G186A) and Histoplasma ohiense (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, H. ohiense induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to H. capsulatum Microscopic analysis of infected lung tissue revealed that although the total number of fungi was similar within inflamed lung lesions, we observed different species-dependent intracellular yeast distribution patterns. Inoculation with gfp-expressing strains indicated that H. ohiense, but not H. capsulatum, was associated primarily with alveolar macrophages early after infection. Interestingly, we observed a significant reduction in the total number of alveolar macrophages 12 to 16 days after H. ohiense, but not H. capsulatum infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages in vitro Together, our data suggest that H. ohiense, but not H. capsulatum, preferentially interacts with alveolar macrophages early after infection, which may lead to a different course of inflammation and resolution despite similar rates of fungal clearance.IMPORTANCE Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen Histoplasma Fungal pneumonia is frequently delayed in diagnosis and treatment due to a prolonged period of quiescence early during infection. In this study, we used a murine respiratory model of histoplasmosis to investigate how different Histoplasma species modulate lung inflammation throughout the complete course of infection. We propose that a relatively low, sublethal inoculum is ideal to model acute pulmonary histoplasmosis in humans, primarily due to the quiescent stage of fungal growth that occurs in the lungs of mice prior to the initiation of inflammation. Our results reveal the unique course of lung immunity associated with divergent species of Histoplasma and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.

Inhibition of Neutrophil Primary Granule Release during Yersinia pestis Pulmonary Infection.

Inhalation of Yersinia pestis causes primary pneumonic plague, the most severe manifestation of plague that is characterized by a dramatic neutrophil influx to the lungs. Neutrophils are ineffective during primary pneumonic plague, failing to control Y. pestis growth in the airways. However, the mechanisms by which Y. pestis resists neutrophil killing are incompletely understood. Here, we show that Y. pestis inhibits neutrophil degranulation, an important line of host innate immune defense. We observed that neutrophils from the lungs of mice infected intranasally with Y. pestis fail to release primary granules throughout the course of disease. Using a type III secretion system (T3SS) injection reporter strain, we determined that Y. pestis directly inhibits neutrophil granule release by a T3SS-dependent mechanism. Combinatorial mutant analysis revealed that a Y. pestis strain lacking both effectors YopE and YopH did not inhibit primary granule release and is killed by neutrophils both in vivo and in vitro Similarly, Y. pestis strains injecting only YopE or YopH are able to inhibit the majority of primary granule release from human neutrophils. We determined that YopE and YopH block Rac2 activation and calcium flux, respectively, to inhibit neutrophil primary granule release in isolated human neutrophils. These results demonstrate that Y. pestis coordinates the inhibition of neutrophil primary granule release through the activities of two distinct effectors, and this inhibition promotes Y. pestis survival during primary pneumonic plague.IMPORTANCEYersinia pestis is the causative agent of plague and is one of the deadliest human pathogens. The pneumonic form of Y. pestis infection has played a critical role in the severity of both historical and modern plague outbreaks, yet the host-pathogen interactions that govern the lethality of Yersinia pestis pulmonary infections are incompletely understood. Here, we report that Yersinia pestis inhibits neutrophil degranulation during infection, rendering neutrophils ineffective and allowing unrestricted growth of Y. pestis in the lungs. This coordinated inhibition of granule release not only demonstrates the pathogenic benefit of "silencing" lung neutrophils but also reveals specific host processes and pathways that could be manipulated to reduce the severity of primary pneumonic plague.

Listeria monocytogenes: cultivation and laboratory maintenance.

This unit describes general procedures for the lab cultivation and storage of the Gram-positive facultative intracellular bacterium Listeria monocytogenes. The basic protocols are relevant for a wide scope of applications including microbial genetics and both in vitro and in vivo infection studies. Commonly used L. monocytogenes strains, serotypes, and growth parameters are also discussed.

Oral transmission of Listeria monocytogenes in mice via ingestion of contaminated food.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.