Enhancing the Bioavailability of Silver Through Nanotechnology Approaches Could Overcome Efflux Pump Mediated Silver Resistance in Methicillin Resistant Staphylococcus aureus.

While the wide-spectrum antimicrobial properties and stability of silver nanomaterials have been copiously utilized in many medical and consumer products, we found that Methicillin Resistant Staphylococcus aureus (MRSA) is less susceptible to silver in comparison to Methicillin Sensitive Staphylococcus aureus (MSSA). Pre-exposure of MRSA to sub-lethal concentrations of AgNO3 caused 2.5-fold increase in LD50 of silver suggesting an inducible resistance mechanism. Studies involving gene expression profiling and efflux pump blockers showed the induction of P-type efflux pumps (Cop A, Cop Z and Nor B) as the principle mechanism conferring silver resistance in MRSA. Chlorpromazine-an efflux pump blocker increased sensitivity of MRSA to silver. Leveraging on these observations, silver resistance in MRSA was circumvented by enhancing the bioavailability of silver by cationic functioning of silver nanoparticles or by co-delivering silver together with chlorpromazine. Atomic Force Microscopy showed that poly-ethylene imine (PEI) functionalized silver nanoparticles adhere to bacterial cells which was found to increase the bioavailability, membrane rupture and cell death. The strategy of co-delivery of AgNO3 and chlorpromazine using chitosan-functionalized wormhole silica nanoparticles caused 12 log reduction in bacterial count which was 1000 times higher than bacterial reduction by AgNO3 alone. In short, these studies showed that circumventing antimicrobial resistance in pathogenic bacteria is possible by designed silver nanotechnology.

Nod2 is required for the early innate immune clearance of Acinetobacter baumannii from the lungs.

Acinetobacter baumannii (A. baumannii) is a significant cause of severe nosocomial pneumonia in immunocompromised individuals world-wide. With limited treatment options available, a better understanding of host immnity to A. baumannii infection is critical to devise alternative control strategies. Our previous study has identified that intracellular Nod1/Nod2 signaling pathway is required for the immune control of A. baumannii in airway epithelial cells in vitro. In the current study, using Nod2-/- mice and an in vivo sublethal model of pulmonary infection, we show that Nod2 contributes to the early lung defense against A. baumannii infection through reactive oxygen species (ROS)/reactive nitrogen species (RNS) production as Nod2-/- mice showed significantly reduced production of ROS/RNS in the lungs following A. baumannii infection. Consistent with the higher bacterial load, A. baumannii-induced neutrophil recruitment, cytokine/chemokine response and lung pathology was also exacerbated in Nod2-/- mice at early time points post-infection. Finally, we show that administration of Nod2 ligand muramyl dipeptide (MDP) prior to infection protected the wild- type mice from A. baumannii pulmonary challenge. Collectively, Nod2 is an important player in the early lung immunity against A. baumannii and modulating Nod2 pathway could be considered as a viable therapeutic strategy to control A. baumannii pulmonary infection.

Erratum: E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

This corrects the article DOI: 10.1038/ncomms15865.

E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria.

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

Bruton's tyrosine kinase phosphorylates DDX41 and activates its binding of dsDNA and STING to initiate type 1 interferon response.

The innate immune system senses cytosolic dsDNA and bacterial cyclic dinucleotides and initiates signaling via the adaptor STING to induce type 1 interferon (IFN) response. We demonstrate here that BTK-deficient cells have impaired IFN-ß production and TBK1/IRF3 activation when stimulated with agonists or infected with pathogens that activate STING signaling. BTK interacts with STING and DDX41 helicase. The kinase and SH3/SH2 interaction domains of BTK bind, respectively, the DEAD-box domain of DDX41 and transmembrane region of STING. BTK phosphorylates DDX41, and its kinase activities are critical for STING-mediated IFN-ß production. We show that Tyr364 and Tyr414 of DDX41 are critical for its recognition of AT-rich DNA and binding to STING, and tandem mass spectrometry identifies Tyr414 as the BTK phosphorylation site. Modeling studies further indicate that phospho-Tyr414 strengthens DDX41's interaction with STING. Hence, BTK plays a critical role in the activation of DDX41 helicase and STING signaling.

The Nod1, Nod2, and Rip2 axis contributes to host immune defense against intracellular Acinetobacter baumannii infection.

Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of ß-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii.

Scrub typhus with sepsis and acute respiratory distress syndrome.

Scrub typhus is a major infectious threat in the Asia-Pacific region. We report an unusual case of scrub typhus in a patient in Singapore who presented with sepsis and acute respiratory distress syndrome but lacked the pathognomonic eschar. The patient recovered after appropriate diagnosis and doxycycline treatment. Rickettsial diseases should be included in the differential diagnosis of febrile illnesses in regions where the diseases are endemic, and absence of eschar should not be the criterion used to rule out scrub typhus.

Receptor interacting protein-2 contributes to host defense against Anaplasma phagocytophilum infection.

The Gram-negative obligate intracellular bacterium Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease occurring worldwide. HGA is generally self-limiting; however, the underlying mechanisms, particularly the innate immune pathways that mediate the immune clearance of A. phagocytophilum, are less understood. We herein report an unexpected role for Receptor interacting protein-2 (Rip2), the adaptor protein for the Nod-like receptors (NLRs), Nod1/Nod2, in the host immune response against A. phagocytophilum infection. Although A. phagocytophilum genome is reported to lack the genes encoding the known ligands of Nod1 and Nod2, its infection upregulated the transcription of Rip2 in human primary neutrophils. Our results revealed that Rip2-deficient mice had significantly higher bacterial load than wild-type controls throughout the infection period. In addition, the Rip2-deficient mice took strikingly longer duration to clear A. phagocytophilum infection. Detailed analysis identified that interferon gamma (IFNγ) and interleukin (IL)-18 but not IL-12, macrophage inflammatory protein-2, and KC response were diminished in A. phagocytophilum-challenged Rip2-deficient mice. Together, these results revealed that Rip2 plays important roles in the immune control of A. phagocytophilum and may contribute to our understanding of the host response to Rickettsiales.

Anaplasma phagocytophilum AptA modulates Erk1/2 signalling.

Anaplasma phagocytophilum causes human granulocytic anaplasmosis, one of the most common tick-borne diseases in North America. This unusual obligate intracellular pathogen selectively persists within polymorphonuclear leucocytes. In this study, using the yeast surrogate model we identified an A. phagocytophilum virulence protein, AptA (A. phagocytophilum toxin A), that activates mammalian Erk1/2 mitogen-activated protein kinase. This activation is important for A. phagocytophilum survival within human neutrophils. AptA interacts with the intermediate filament protein vimentin, which is essential for A. phagocytophilum-induced Erk1/2 activation and infection. A. phagocytophilum infection reorganizes vimentin around the bacterial inclusion, thereby contributing to intracellular survival. These observations reveal a major role for the bacterial protein, AptA, and the host protein, vimentin, in the activation of Erk1/2 during A. phagocytophilum infection.

RNA interference screen for human genes associated with West Nile virus infection.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.

The tick Ixodes scapularis is an efficient vector for microbes, including the Lyme disease agent Borrelia burgdorferi. Ticks engorging on vertebrates induce recruitment of inflammatory cells to the bite site. For efficient transmission to the vector, pathogens have to traffic through this complex feeding site while avoiding the deleterious effects of immune cells. We show that a tick protein, Salp25D, plays a critical role-in the mammalian host-for acquisition of Borrelia burgdorferi by the vector. Silencing salp25D in tick salivary glands impaired spirochete acquisition by ticks engorging on B. burgdorferi-infected mice. Immunizing mice against Salp25D also decreased Borrelia acquisition by I. scapularis. Salp25D detoxified reactive oxygen species at the vector-pathogen-host interface, thereby providing a survival advantage to B. burgdorferi at the tick feeding site in mice. These data demonstrate that pathogens can exploit arthropod molecules to defuse mammalian responses in order to successfully enter the vector.

ASC/PYCARD and caspase-1 regulate the IL-18/IFN-gamma axis during Anaplasma phagocytophilum infection.

Anaplasma phagocytophilum is an obligate intracellular pathogen that resides within neutrophils and can cause fever, pancytopenia, or death. IFN-gamma plays a critical role in the control of A. phagocytophilum; however, the mechanisms that regulate IFN-gamma production remain unclear. In this study, we demonstrate that apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, a central adaptor molecule in the Nod-like receptor (NLR) pathway, regulates the IL-18/IFN-gamma axis during A. phagocytophilum infection through its effect on caspase-1. Caspase-1- and asc-null mice were more susceptible than control animals to A. phagocytophilum infection due to the absence of IL-18 secretion and reduced IFN-gamma levels in the peripheral blood. Moreover, caspase-1 and ASC deficiency reduced CD4+ T cell-mediated IFN-gamma after in vitro restimulation with A. phagocytophilum. The NLR family member IPAF/NLRC4, but not NALP3/NLRP3, was partially required for IFN-gamma production in response to A. phagocytophilum. Taken together, our data demonstrate that ASC and caspase-1 are critical for IFN-gamma-mediated control of A. phagocytophilum infection.

IL-12/23p40-dependent clearance of Anaplasma phagocytophilum in the murine model of human anaplasmosis.

Human anaplasmosis is an emerging infectious disease transmitted by ticks that can be potentially fatal in the immunocompromised and the elderly. The mechanisms of defense against the causative agent, Anaplasma phagocytophilum, are not completely understood; however, interferon (IFN)-gamma plays an important role in pathogen clearance. Here, we show that IFN-gamma is regulated through an early IL-12/23p40-dependent mechanism. Interleukin (IL)-12/23p40 is regulated in macrophages and dendritic cells after activation by microbial agonists and cytokines and constitutes a subunit of IL-12 and IL-23. IL-12/23p40-deficient mice displayed an increased A. phagocytophilum burden, accelerated thrombocytopenia and increased neutrophil numbers in the spleen at day 6 postinfection. Infection of MyD88- and mitogen-activated kinase kinase 3 (MKK3)-deficient mice suggested that the early susceptibility due to IL-12/23p40 deficiency was not dependent on signaling through MyD88 or MKK3. The lack of IL-12/23p40 reduced IFN-gamma production in both CD4(+) and CD8(+) T cells although the effect was more pronounced in CD4(+) T cells. Our data suggest that the immune response against A. phagocytophilum is a multifactorial and cooperative process. The IL-12/23p40 subunit drives the CD4(+) Th1 immune response in the early phase of infection and IL-12/23p40-independent mechanisms ultimately contribute to pathogen elimination from the host.

Rab 5 is required for the cellular entry of dengue and West Nile viruses.

The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Early transcriptional response of human neutrophils to Anaplasma phagocytophilum infection.

Anaplasma phagocytophilum, an unusual obligate intracellular pathogen that persists within neutrophils, causes human anaplasmosis (previously known as human granulocytic ehrlichiosis). To study the effects of this pathogen on the transcriptional profile of its host cell, we performed a comprehensive DNA microarray analysis of the early (4-h) transcriptional response of human neutrophils to A. phagocytophilum infection. A. phagocytophilum infection resulted in the up- and down-regulation of 177 and 67 neutrophil genes, respectively. These data were verified by quantitative reverse transcription-PCR of selected genes. Notably, the up-regulation of many antiapoptotic genes, including the BCL2A1, BIRC3, and CFLAR genes, and the down-regulation of the proapoptotic TNFSF10 gene were observed. Genes involved in inflammation, innate immunity, cytoskeletal remodeling, and vesicular transport also exhibited differential expression. Vascular endothelial growth factor was also induced. These data suggest that A. phagocytophilum may alter selected host pathways in order to facilitate its survival within human neutrophils. To gain further insight into the bacterium's influence on host cell gene expression, this report presents a detailed comparative analysis of our data and other gene expression profiling studies of A. phagocytophilum-infected neutrophils and promyelocytic cell lines.

Modulation of NB4 promyelocytic leukemic cell machinery by Anaplasma phagocytophilum.

Anaplasma phagocytophilum is a gram-negative obligate intracellular bacterium that persists within neutrophils. We assessed the impact of A. phagocytophilum infection in NB4 promyelocytic leukemic cells using high-density oligoarray, two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry. Our Affymetrix data revealed that A. phagocytophilum altered the expression of transcription factors, cell adhesion molecules, signal transduction genes, and proinflammatory cytokines. However, the expression of Toll-like receptors, MYD88, RNF36, IRF3, and TBK1 and inhibitors of the NF-kappaB gene was not altered. A. phagocytophilum infection also altered the apoptotic program of NB4 cells and resulted in increased transcription of antiapoptotic genes (MCL1 and BFL1). The transcription and translation of iron-metabolism genes (light polypeptide ferritin chain, transferrin, and the transferrin receptor) were significantly altered, suggesting a possible link between A. phagocytophilum infection and iron metabolism. Our study clearly demonstrates multifactorial effects of A. phagocytophilum infection on NB4 promyelocytic leukemic cell machinery.

Leishmaniasis: current status of vaccine development.

Leishmaniasis, a spectrum of diseases caused by various forms of Leishmania has become a major health problem all over the world. Vaccination against leishmaniasis has passed through many developmental stages beginning with the ancient practice of 'leishmanization'. Due to various problems and difficulties associated with traditional vaccines, the interest has been shifted to novel approaches of vaccination like DNA vaccination, vaccination with live vectors encoding leishmanial antigens and finally to designer vaccines. In an effort towards developing an anti-leishmanial vaccine, our laboratory has been working on various genes present in an amplified locus of Leishmania known as the 'LD1 locus'. Two genes, ORFF and BT1 (previously ORFG), are part of the multigenic LD1 locus on chromosome 35. BT1 encodes a biopterin transporter, while the function of ORFF gene product is unknown. Immunization of mice with recombinant ORFF (rORFF) and BT1 proteins, individually, or in combination, conferred partial protection against challenge with Leishmania donovani. We also tested the protective efficacy of ORFF DNA vaccine in BALB/c mice model and found that the level of protection was significantly higher than that of ORFF protein. Protection conferred by ORFF DNA vaccine correlated with significant levels of in vitro splenocyte proliferation and low levels of antigen-specific antibodies. There was a preferential production of IFN-gamma compared to IL-4, which indicated the induction of a protective Th1 response, by the DNA vaccine. Thus, DNA immunization may offer an attractive alternative strategy against leishmaniasis. We present here the current status of vaccine development against leishmaniasis.