Replication In Human Macrophages Research Articles

Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice.

Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S. Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S. Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflict with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR–Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

Legionella pneumophila Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans.

Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER–Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella’s intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Salmonella enterica Serovar Typhi Induces Host Metabolic Reprogramming to Increase Glucose Availability for Intracellular Replication.

Salmonella enterica serovar Typhi (S. Typhi) is a human-limited intracellular pathogen and the cause of typhoid fever, a severe systemic disease. Pathogen–host interaction at the metabolic level affects the pathogenicity of intracellular pathogens, but it remains unclear how S. Typhi infection influences host metabolism for its own benefit. Herein, using metabolomics and transcriptomics analyses, combined with in vitro and in vivo infection assays, we investigated metabolic responses in human macrophages during S. Typhi infection, and the impact of these responses on S. Typhi intracellular replication and systemic pathogenicity. We observed increased glucose content, higher rates of glucose uptake and glycolysis, and decreased oxidative phosphorylation in S. Typhi-infected human primary macrophages. Replication in human macrophages and the bacterial burden in systemic organs of humanized mice were reduced by either the inhibition of host glucose uptake or a mutation of the bacterial glucose uptake system, indicating that S. Typhi utilizes host-derived glucose to enhance intracellular replication and virulence. Thus, S. Typhi promotes its pathogenicity by inducing metabolic changes in host macrophages and utilizing the glucose that subsequently accumulates as a nutrient for intracellular replication. Our findings provide the first metabolic signature of S. Typhi-infected host cells and identifies a new strategy utilized by S. Typhi for intracellular replication.

Salmonella enterica serovar Typhi genomic regions involved in low pH resistance and in invasion and replication in human macrophages

PurposeSalmonella enterica serovar Typhi, the etiological agent of typhoid fever, causes a systemic life-threatening disease. To carry out a successful infection process, this bacterium needs to survive alkaline and acid pH conditions presented in the mouth, stomach, small intestine, and gallbladder. Therefore, in this work, a genetic screening to identify S. Typhi genes involved in acid and circumneutral pH resistance was performed.MethodsA collection of S. Typhi mutants deleted of fragments ranging from 6 to 80 kb were obtained by the Datsenko and Wanner method. Bacterial growth rate assays of each mutant were performed to identify S. Typhi genes involved in circumneutral and acid pH resistance. S. Typhi mutants deficient to growth at specific pH were evaluated in their capacity to invade and replicate in phagocytic cells.ResultsIn this work, it is reported that S. Typhi ∆F4 (pH 4.5), S. Typhi ∆F44 (pH 4.5, 5.5, and 6.5), and S. Typhi ∆F73 (pH 4.5, 5.5, 6.5, and 7.5) were deficient to grow in the pH indicated. These three mutant strains were also affected in their ability to invade and replicate in human macrophages.ConclusionsS. Typhi contains defined genomic regions that influence the survival at specific pH values, as well as the invasion and replication inside human cells. Thus, this genetic information probably allows the bacteria to survive in different human compartments for an efficient infection cycle.

HIV-1 Vpr antagonizes innate immune activation by targeting karyopherin-mediated NF-κB/IRF3 nuclear transport.

HIV-1 must replicate in cells that are equipped to defend themselves from infection through intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here, we show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr-dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import and inhibition of innate immune activation to promote HIV-1 replication and transmission.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing)

The human immunodeficiency virus (HIV) pandemic is driving the re-emergence of tuberculosis (TB) as a global health threat, both by increasing the susceptibility of HIV-infected people to infection with Mycobacterium tuberculosis (Mtb), and increasing the rate of emergence of drug-resistant Mtb. There are several other clinical challenges for treatment of co-infected patients including: expense, pill burden, toxicity, and malabsorption that further necessitate the search for new drugs that may be effective against both pathogens simultaneously. The anti-helminthic niclosamide has been shown to have activity against a laboratory strain of Mtb in liquid culture while bacteriostatic activity against non-replicating M. abscessus was also recently described. Here we extend these findings to further demonstrate that niclosamide inhibits mycobacterial growth in infected human macrophages and mediates potent bacteriostatic activity against the virulent Mtb Beijing strain. Importantly, we provide the first evidence that niclosamide inhibits HIV replication in human macrophages and Jurkat T cells through post-integration effects on pro-virus transcription. The dual antiviral and anti-mycobacterial activity was further observed in an in vitro model of HIV and Mtb co-infection using human primary monocyte-derived macrophages. These results support further investigation of niclosamide and derivatives as anti-retroviral/anti-mycobacterial agents that may reduce clinical challenges associated with multi-drug regimens and drug resistance.

A Legionella pneumophila amylase is essential for intracellular replication in human macrophages and amoebae

Legionella pneumophila invades protozoa with an “accidental” ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.

Lactate oxidation facilitates growth of Mycobacterium tuberculosis in human macrophages

Mycobacterium tuberculosis (Mtb) uses alveolar macrophages as primary host cells during infection. In response to an infection, macrophages switch from pyruvate oxidation to reduction of pyruvate into lactate. Lactate might present an additional carbon substrate for Mtb. Here, we demonstrate that Mtb can utilize L-lactate as sole carbon source for in vitro growth. Lactate conversion is strictly dependent on one of two potential L-lactate dehydrogenases. A knock-out mutant lacking lldD2 (Rv1872c) was unable to utilize L-lactate. In contrast, the lldD1 (Rv0694) knock-out strain was not affected in growth on lactate and retained full enzymatic activity. On the basis of labelling experiments using [U-13C3]-L-lactate as a tracer the efficient uptake of lactate by Mtb and its conversion into pyruvate could be demonstrated. Moreover, carbon flux from lactate into the TCA cycle, and through gluconeogenesis was observed. Gluconeogenesis during lactate consumption depended on the phosphoenolpyruvate carboxykinase, a key enzyme for intracellular survival, showing that lactate utilization requires essential metabolic pathways. We observed that the ΔlldD2 mutant was impaired in replication in human macrophages, indicating a critical role for lactate oxidation during intracellular growth.

The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense.

During antibacterial autophagy, ubiquitination of intracellular bacteria recruits proteins that mediate bacterial delivery to the lysosome for degradation. Smurf1 is an E3 ubiquitin ligase whose role in selective bacterial autophagy is unknown. We show that Smurf1 facilitates selective autophagy of the human pathogen Mycobacterium tuberculosis (Mtb). Smurf1-/- macrophages are defective in recruiting polyubiquitin, the proteasome, the ubiquitin-binding autophagy adaptor NBR1, the autophagy protein LC3, and the lysosomal marker LAMP1 to Mtb-associated structures and are more permissive for Mtb growth. This function of Smurf1 requires both its ubiquitin-ligase and C2 phospholipid-binding domains, and involves K48- rather than K63-linked ubiquitination. Chronically infected Smurf1-/- mice have increased bacterial load, increased lung inflammation, and accelerated mortality. SMURF1 controls Mtb replication in human macrophages and associates with bacteria in lungs of patients with pulmonary tuberculosis. Thus, Smurf1 is required for selective autophagy of Mtb and host defense against tuberculosis infection.

The Janus kinase inhibitor ruxolitinib reduces HIV replication in human macrophages and ameliorates HIV encephalitis in a murine model

A hallmark of persistent HIV-1 infection in the central nervous system is increased activation of mononuclear phagocytes and surrounding astrogliosis, conferring persistent HIV-induced inflammation. This inflammation is believed to result in neuronal dysfunction and the clinical manifestations of HIV-associated neurocognitive disorders (HAND). The Jak/STAT pathway is activated in macrophages/myeloid cells upon HIV-1 infection, modulating many pro-inflammatory pathways that result in HAND, thereby representing an attractive cellular target. Thus, the impact of ruxolitinib, a Janus Kinase (Jak) 1/2 inhibitor that is FDA approved for myelofibrosis and polycythemia vera, was assessed for its potential to inhibit HIV-1 replication in macrophages and HIV-induced activation in monocytes/macrophages in culture. In addition, a murine model of HIV encephalitis (HIVE) was used to assess the impact of ruxolitinib on histopathological features of HIVE, brain viral load, as well as its ability to penetrate the blood–brain-barrier (BBB). Ruxolitinib was found to inhibit HIV-1 replication in macrophages, HIV-induced activation of monocytes (CD14/CD16) and macrophages (HLA-DR, CCR5, and CD163) without apparent toxicity. In vivo, systemically administered ruxolitinib was detected in the brain during HIVE in SCID mice and markedly inhibited astrogliosis. Together, these data indicate that ruxolitinib reduces HIV-induced activation and infiltration of monocytes/macrophages in vitro, reduces the replication of HIV in vitro, penetrates the BBB when systemically administered in mice and reduces astrogliosis in the brains of mice with HIVE. These data suggest that ruxolitinib will be useful as a novel therapeutic to treat humans with HAND.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.

Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages.

BackgroundHIV-1 Tat is essential for HIV replication and is also a well-known neurotoxic factor causing HIV-associated neurocognitive disorder (HAND). Currently, combined antiretroviral therapy targeting HIV reverse transcriptase or protease cannot prevent the production of early viral proteins, especially Tat, once HIV infection has been established. HIV-infected macrophages and glial cells in the brain still release Tat into the extracellular space where it can exert direct and indirect neurotoxicity. Therefore, stable production of anti-Tat antibodies in the brain would neutralize HIV-1 Tat and thus provide an effective approach to protect neurons.MethodsWe constructed a humanized anti-Tat Hutat2:Fc fusion protein with the goal of antagonizing HIV-1 Tat and delivered the gene into cell lines and primary human monocyte-derived macrophages (hMDM) by an HIV-based lentiviral vector. The function of the anti-Tat Hutat2:Fc fusion protein and the potential side effects of lentiviral vector-mediated gene transfer were evaluated in vitro.ResultsOur study demonstrated that HIV-1-based lentiviral vector-mediated gene transduction resulted in a high-level, stable expression of anti-HIV-1 Tat Hutat2:Fc in human neuronal and monocytic cell lines, as well as in primary hMDM. Hutat2:Fc was detectable in both cells and supernatants and continued to accumulate to high levels within the supernatant. Hutat2:Fc protected mouse cortical neurons against HIV-1 Tat86-induced neurotoxicity. In addition, both secreted Hutat2:Fc and transduced hMDM led to reducing HIV-1BaL viral replication in human macrophages. Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability. Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc.ConclusionsOur study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary hMDM and does not lead to any significant changes in hMDM immune-activation. The neuroprotective and HIV-1 suppressive effects produced by Hutat2:Fc were comparable to that of a full-length anti-Tat antibody. This study provides the foundation and insights for future research on the potential use of Hutat2:Fc as a novel gene therapy approach for HAND through utilizing monocytes/macrophages, which naturally cross the blood-brain barrier, for gene delivery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0195-2) contains supplementary material, which is available to authorized users.

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.

Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012.

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.