Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.

A Rapid Caspase-11 Response Induced by IFNγ Priming Is Independent of Guanylate Binding Proteins.

In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFNγ priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFNγ treatment. Indeed, IFNγ priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFNγ priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFNγ.

Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.

Salmonella-Driven Polarization of Granuloma Macrophages Antagonizes TNF-Mediated Pathogen Restriction during Persistent Infection.

Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium (STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11b+CD11c+Ly6C+ macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.

Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization.

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.

Host inflammasome defense mechanisms and bacterial pathogen evasion strategies.

Inflammasomes are a formidable armada of intracellular pattern recognition receptors. They recognize determinants of infection, such as foreign entities or danger signals within the host cell cytosol, rapidly executing innate immune defenses and initiating adaptive immune responses. Although inflammasomes are implicated in many diseases, they are especially critical in host protection against intracellular bacterial pathogens. Given this role, it is not surprising that many pathogens have evolved effective strategies to evade inflammasome activation. In this review, we will provide a brief summary of inflammasome activation during infection with the intent of highlighting recent advances in the field. Additionally, we will review known bacterial evasion strategies and countermeasures that impact pathogenesis.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Effects of sun and freeze-drying techniques on molecular, fatty acid and therapeutic properties of fermented goat milk product.

This study investigated the effect of sun drying (Sd) and freeze drying (Fd) on the chemical, nutritional and biological properties of either unsalted (Us) or salted (Sa) Jameed produced from goat milk. The products were characterized by measuring the chemical, physical and biological properties. SDS-PAGE was used to characterize the effect of processing conditions on protein subunits. Major new bands were found in SDS-PAGE of Jameed prepared by SdUs and FdUs from goat milk but not from that prepared by SdSa and FdSa. Preparation of Jameed by with or without salt treatments of Jameed by sun drying enhances the contents of short chain fatty acids. Result showed that the preparation of Jameed by SdUs decreased the content of caprylic acid. That prepared by sun drying and with or without salt increased the stability, shelf life and inhibitory activities of ACE and α-amylase. The optimum color values were found in Jameed prepared by FdSa. Different processing treatments influenced content of all fatty acids except for margaric and oleic acid.

The hypocholesterolemic and hormone modulation effects of isoflavones alone or co-fermented with probiotic bacteria in hypercholesterolemic rats model.

This study aimed to investigate the beneficial effect of isoflavones alone or probiotics-co-fermented isoflavones on serum and hepatic lipid profile, serum steroid (SHs) and thyroid hormones (THs) of hypercholesterolemic rats (N = 48). Animals were fed for 8 weeks with probiotics-co-fermented isoflavones or isoflavones alone, beside high-fat-high-cholesterol diet. Serum was analyzed for cholesterols, triglycerides (TG), SHs and THs. Results demonstrated that the given treatments significantly decreased serum total-cholesterol (TC), low-density-lipoprotein-cholesterol (LDL-C), high-density-lipoprotein-cholesterol (HDL-C), LDL/HDL ratio, and increased TG, compared to controls. The probiotics-co-fermented isoflavones decreased TC, LDL-C and LDL/HDL ratio more effectively than isoflavones alone. Also, both isoflavones treatments induced a hyperthyroidism state, as the levels of T-T4, T-T3 and fT3 significantly increased. In addition, these treatments decreased testosterone and increased cortisol levels. Thus, isoflavones-containing-treatments, particularly probiotics-co-fermented isoflavones, could reduce CVD incidence by controlling lipid profile; and this control could in part be due to modulation of SHs and THs.

Development of a novel anti-canine CD20 monoclonal antibody with diagnostic and therapeutic potential.

In humans, passive immunotherapy with anti-CD20 monoclonal antibodies (mAbs) has created immeasurable improvements in outcomes of patients with B-cell malignancies. However, the lack of comparable reagents has precluded development of this approach in dogs. We developed a novel anti-canine CD20 mAb designated as 6C8. 6C8 recognized the extracellular domain of canine CD20 and showed high-affinity binding to canine CD20 in solution, as well as in its native conformation on canine B-cells. The 6C8 target was expressed invariably in B-cell lineage cells, but not in T-cells or myeloid cells. 6C8 promoted phagocytosis of B-cell lymphoma cells by macrophages, but in its current framework, it did not induce direct cytotoxicity or complement dependent cytotoxicity. In summary, we have established a novel anti-canine CD20 mAb that is useful as a diagnostic tool to phenotype B-cells, and which could be integrated as a tool for passive immunotherapy to treat dogs with B-cell disorders.

Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification.

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Evaluation of the nutraceutical, physiochemical and sensory properties of raisin jam.

This objective of this study was to evaluate the effect of jam processing of grape and raisin on the nutraceutical, physiochemical, and sensory properties. The results showed that fresh grape had the highest antioxidant activity, and total phenolic and anthocyanin content followed by grape jam, raisin, and raisin jam, respectively. No significant differences existed in soluble solids, pH, or firmness between grape and raisin jams. No significant differences in color parameters, ΔE, and chroma existed between grape and raisin jam. Descriptive sensory results showed minor differences in some sensory attributes between grape and raisin jams. In terms of consumer evaluation (9-point verbal hedonic scale and a 5-point just-about-right scale) the jams made from local raisins were parity with those from grape, despite small differences especially in whole raisin jam. Although raisin and other dried products are not traditionally considered as a raw material for jam processing, they have the same potential as fresh fruits.

Lingual leishmaniasis complicating visceral disease.

Leishmania species are obligate intracellular parasites transmitted by various types of female sand flies. The clinical syndrome that results depends on a number of factors including the Leishmania species and immune response of the host. Here, we report successful treatment of lingual leishmaniasis complicating visceral disease in an immunocompetent patient.

Rhodococcus equi pneumonia in a patient with fludarabine-treated chronic lymphocytic leukemia and CD4-lymphopenia.

Cavitary pneumonia caused by Rhodococcus equi may occur in patients with defective cell-mediated immunity. Prolonged CD4 lymphopenia may develop in patients with chronic lymphocytic leukemia who receive fludarabine. The authors report the first case of a patient with chronic lymphocytic leukemia who developed R equi pneumonia after fludarabine therapy.

Primary gluteal abscess due to Salmonella typhimurium: a case report and review of the literature.

Infections due to nontyphoidal salmonellae are common, and their incidence has been increasing in the last few years. In this case report, we document the unusual finding of a gluteal abscess caused by Salmonella typhimurium in the absence of detectable bacteremia. Fluoroquinolones remain an important treatment option for salmonellosis, and to avoid potential treatment failures, we underscore the importance of routine screening for nalidixic acid susceptibility with all extraintestinal Salmonella isolates. To our knowledge, this is the first published case of S typhimurium gluteal abscess in the absence of bacteremia and risk factors for salmonellosis.

NMR and mutagenesis studies on the phosphorylation region of human cardiac troponin I.

Phosphorylation of the cardiac troponin complex by PKA at S22 and S23 of troponin I (TnI) accelerates Ca(2+) release from troponin C (TnC). The region of TnI around the bisphosphorylation site binds to, and stabilizes, the Ca(2+) bound N-terminal domain of TnC. Phosphorylation interferes with this interaction between TnI and TnC resulting in weaker Ca(2+) binding. In this study, we used (1)H-(15)N-HSQC NMR to investigate at the atomic level the interaction between an N-terminal fragment of TnI consisting of residues 1-64 of TnI (I1-64) and TnC. We produced several mutants of I1-64, TnI, and TnC to test the contribution of certain residues to the transmission of the phosphorylation signal in both NMR experiments and functional assays. We also investigated how phosphorylation of the PKC sites in I1-64 (S41 and S43) affects the interaction of I1-64 with TnC. We found that phosphorylation of S22 and S23 produced only localized effects in the structure of I1-64 between residues 24 and 34. Residues 1-17 of I1-64 did not bind to TnC, and residues 38-64 bound tightly to the C-terminal domain of TnC regardless of phosphorylation. Residues 22-34 bound weakly to TnC in a phosphorylation sensitive manner. Bisphosphorylation prevented this phosphorylation switch region from interacting with TnC. Systematic mutation of residues in the phosphorylation switch did not prevent PKA phosphorylation from accelerating Ca(2+) release from troponin. We conclude that the phosphorylation switch binds to TnC via an extended interaction site spanning residues R19 to A34.

Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C.

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.

Irradiation effects on meat color - a review.

Color changes in irradiated fresh meat occur because of the susceptibility of the myoglobin molecule, especially the iron, to alterations in the chemical environment and to energy input. The potential for iron electrons to exist in various states makes the environment adjacent to the iron atom particularly vulnerable to the presence of electron-donating compounds and high energy inputs (irradiation). Initial condition of the myoglobin (Fe(++)O(2), Fe(+++)), modification of oxidation-reduction potential of the tissue, and generation of ligand-forming compounds (CO) from endogenous organic compounds and water are enhanced or suppressed depending on the gas atmosphere, temperature, pH and myoglobin concentration of the system. Generation of stable red pigments or brown pigments which become red over time appears to be due to binding of irradiation-generated reactive oxygen species (()O(2)(-)) or gasses (CO) which become ligands bound by iron under altered reducing conditions. Rapid generation of large amounts of metmyoglobin when irradiation is conducted in an oxygen-containing environment appears to be an acceleration of the normal process by which myoglobin undergoes oxidation. Generation of green pigments appears to be due to breakdown of the porphyrin integrity and/or formation of sulfmyoglobin. Maintenance of ideal meat color during irradiation can be enhanced by various combinations of pre-slaughter feeding of antioxidants to livestock, optimizing the condition of the meat prior to irradiation, addition of antioxidants, gas atmosphere (MAP), packaging, and temperature control.