RECON gene disruption enhances host resistance to enable genome-wide evaluation of intracellular pathogen fitness during infection.

Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq in vivo is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in Akr1c13 that renders the resulting RECON-/- mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen Listeria monocytogenes in vivo. We identified 135 genes which were required for L. monocytogenes growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for L. monocytogenes survival and investigated the role of the folate enzyme FolD in L. monocytogenes liver pathogenesis. A mutant lacking folD was impaired for growth in murine livers by 2.5-log10 compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in alsR, which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log10 and 3-log10, respectively, but showed modest phenotypes in in vitro models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the in vivo growth defect, as deletion of the operon in the ∆alsR background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for L. monocytogenes host infection, which highlights the utility of the RECON-/- mouse model for future screening efforts. IMPORTANCE: Listeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via transposon sequencing. We identified the liver-specific gene folD and a repressor, alsR, that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.

Mycobacterium tuberculosis Sulfolipid-1 Activates Nociceptive Neurons and Induces Cough.

Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid.

Screening Mycobacterium tuberculosis Secreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection.

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.

Heme Oxygenase-1 Regulates Inflammation and Mycobacterial Survival in Human Macrophages during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth.

Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.

Sensing of Mycobacterium tuberculosis and consequences to both host and bacillus.

Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis, has killed more people than any other bacterial pathogen in human history and remains one of the most important transmissible diseases worldwide. Because of the long-standing interaction of Mtb with humans, it is no surprise that human mucosal and innate immune cells have evolved multiple mechanisms to detect Mtb during initial contact. To that end, the cell surface of human cells is decorated with numerous pattern recognition receptors for a variety of mycobacterial ligands. Furthermore, once Mtb is ingested into professional phagocytes, other host molecules are engaged to report on the presence of an intracellular pathogen. In this review, we discuss the role of specific mycobacterial products in modulating the host's ability to detect Mtb. In addition, we describe the specific host receptors that mediate the detection of mycobacterial infection and the role of individual receptors in mycobacterial pathogenesis in humans and model organisms.

Enhanced toxicity of the protein cross-linkers divinyl sulfone and diethyl acetylenedicarboxylate in comparison to related monofunctional electrophiles.

Previously, we determined that diethyl acetylenedicarboxylate (DAD), a protein cross-linker, was significantly more toxic than analogous monofunctional electrophiles. We hypothesized that other protein cross-linkers enhance toxicity similarly. In agreement with this hypothesis, the bifunctional electrophile divinyl sulfone (DVSF) was 6-fold more toxic than ethyl vinyl sulfone (EVSF) in colorectal carcinoma cells and greater than 10-fold more toxic in Saccharomyces cerevisiae. DVSF and DAD caused oligomerization of yeast thioredoxin 2 (Trx2p) in vitro and promoted Trx2p cross-linking to other proteins in yeast at cytotoxic doses. Our results suggest that protein cross-linking is considerably more detrimental to cellular homeostasis than simple alkylation.

Structure-activity comparison of the cytotoxic properties of diethyl maleate and related molecules: identification of diethyl acetylenedicarboxylate as a thiol cross-linking agent.

Many α,ß-unsaturated carbonyl compounds are used in biochemical and medical research. Their biological effects are due in large part to their electrophilic properties, whereby they undergo reaction with nucleophilic sites in proteins and nucleic acids. Here, we describe a structure-activity comparison of the cytotoxic properties of diethyl maleate (DEM) and closely related chemical analogs. All molecules that contained an α,ß-unsaturated carbonyl group were cytotoxic to human colorectal carcinoma cells, causing apoptotic cell death. However, related molecules lacking this chemical moiety were not cytotoxic. One of the molecules screened, diethyl acetylenedicarboxylate (DAD), was considerably more cytotoxic than DEM and other analogues. Induction of cell death by DAD was significantly decreased following preincubation of cells with N-acetylcysteine, suggesting that its reactivity with thiols in cells might account for its cytotoxicity. By use of a model thiol compound, it was found that DAD can undergo addition reactions with two equivalents of thiol. When the reactivity of DAD with proteins was explored, it was determined that DAD induces oligomerization of Gpx3p, a yeast glutathione peroxidase with highly reactive cysteine residues in its active site. Our results suggest that DAD functions as a protein-thiol cross-linker, providing a potential chemical explanation for its cytotoxic potency.