Publications by authors named "Paul de Figueiredo"

47 Publications

Live attenuated bacterium limits cancer resistance to CAR-T therapy by remodeling the tumor microenvironment.

J Immunother Cancer 2022 01;10(1)

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77802, USA

The tumor microenvironment (TME) is characterized by the activation of immune checkpoints, which limit the ability of immune cells to attack the growing cancer. To overcome immune suppression in the clinic, antigen-expressing viruses and bacteria have been developed to induce antitumor immunity. However, the safety and targeting specificity are the main concerns of using bacteria in clinical practice as antitumor agents. In our previous studies, we have developed an attenuated bacterial strain ( 16M ∆, henceforth Bm∆) for clinical use, which is safe in all tested animal models and has been removed from the select agent list by the Centers for Disease Control and Prevention. In this study, we demonstrated that Bm∆ homed to tumor tissue and improved the TME in a murine model of solid cancer. In addition, live Bm∆ promoted proinflammatory M1 polarization of tumor macrophages and increased the number and activity of CD8 T cells in the tumor. In a murine colon adenocarcinoma model, when combined with adoptive transfer of tumor-specific carcinoembryonic antigen chimeric antigen receptor CD8 T cells, tumor cell growth and proliferation was almost completely abrogated, and host survival was 100%. Taken together, these findings demonstrate that the live attenuated bacterial treatment can defeat cancer resistance to chimeric antigen receptor T-cell therapy by remodeling the TME to promote macrophage and T cell-mediated antitumor immunity.
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http://dx.doi.org/10.1136/jitc-2021-003760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8734016PMC
January 2022

Host Protease Activity on Bacterial Pathogens Promotes Complement and Antibiotic-Directed Killing.

Pathogens 2021 Nov 18;10(11). Epub 2021 Nov 18.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX 77807, USA.

Our understanding of how the host immune system thwarts bacterial evasive mechanisms remains incomplete. Here, we show that host protease neutrophil elastase acts on and to destroy factors that prevent serum-associated, complement-directed killing. The protease activity also enhances bacterial susceptibility to antibiotics in sera. These findings implicate a new paradigm where host protease activity on bacteria acts combinatorially with the host complement system and antibiotics to defeat bacterial pathogens.
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http://dx.doi.org/10.3390/pathogens10111506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618517PMC
November 2021

SARS-CoV-2 inhibits induction of the MHC class I pathway by targeting the STAT1-IRF1-NLRC5 axis.

Nat Commun 2021 11 15;12(1):6602. Epub 2021 Nov 15.

Department of Immunology, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.

The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.
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http://dx.doi.org/10.1038/s41467-021-26910-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594428PMC
November 2021

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells.

J Vis Exp 2021 09 17(175). Epub 2021 Sep 17.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center; College of Veterinary Medicine, Texas A&M University;

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.
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http://dx.doi.org/10.3791/62764DOI Listing
September 2021

Interactions between fungal hyaluronic acid and host CD44 promote internalization by recruiting host autophagy proteins to forming phagosomes.

iScience 2021 Mar 12;24(3):102192. Epub 2021 Feb 12.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX 77807, USA.

Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.
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http://dx.doi.org/10.1016/j.isci.2021.102192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920835PMC
March 2021

Sub-second heat inactivation of coronavirus using a betacoronavirus model.

Biotechnol Bioeng 2021 05 3;118(5):2067-2075. Epub 2021 Mar 3.

Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, USA.

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub-second heat inactivation of CoV. The system is composed of a stainless-steel capillary immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log reduction in viral titer (starting titer: 5 × 10 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log reduction).
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http://dx.doi.org/10.1002/bit.27720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013827PMC
May 2021

The candidate vaccine strain Brucella ovis ∆abcBA is protective against Brucella melitensis infection in mice.

Microbiol Immunol 2020 Nov 8;64(11):730-736. Epub 2020 Oct 8.

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.
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http://dx.doi.org/10.1111/1348-0421.12850DOI Listing
November 2020

Bayesian gamma-negative binomial modeling of single-cell RNA sequencing data.

BMC Genomics 2020 Sep 9;21(Suppl 9):585. Epub 2020 Sep 9.

Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, USA.

Background: Single-cell RNA sequencing (scRNA-seq) is a powerful profiling technique at the single-cell resolution. Appropriate analysis of scRNA-seq data can characterize molecular heterogeneity and shed light into the underlying cellular process to better understand development and disease mechanisms. The unique analytic challenge is to appropriately model highly over-dispersed scRNA-seq count data with prevalent dropouts (zero counts), making zero-inflated dimensionality reduction techniques popular for scRNA-seq data analyses. Employing zero-inflated distributions, however, may place extra emphasis on zero counts, leading to potential bias when identifying the latent structure of the data.

Results: In this paper, we propose a fully generative hierarchical gamma-negative binomial (hGNB) model of scRNA-seq data, obviating the need for explicitly modeling zero inflation. At the same time, hGNB can naturally account for covariate effects at both the gene and cell levels to identify complex latent representations of scRNA-seq data, without the need for commonly adopted pre-processing steps such as normalization. Efficient Bayesian model inference is derived by exploiting conditional conjugacy via novel data augmentation techniques.

Conclusion: Experimental results on both simulated data and several real-world scRNA-seq datasets suggest that hGNB is a powerful tool for cell cluster discovery as well as cell lineage inference.
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http://dx.doi.org/10.1186/s12864-020-06938-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487589PMC
September 2020

Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HBV Replication through Production of IFN-γ and TNF-⍺.

iScience 2020 Jul 1;23(7):101333. Epub 2020 Jul 1.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, MREB II, Room 3344, 8447 Riverside Pkwy, Bryan, TX 77807, USA. Electronic address:

The viral antigen (Ag)-specific CD8 cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed HBV replication and prevented HBV surface Ag expression. We further demonstrate that the adoptive transfer significantly increased T cell accumulation and production of antiviral cytokines. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the liver and suppress HBV replication through producing antiviral cytokines.
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http://dx.doi.org/10.1016/j.isci.2020.101333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364173PMC
July 2020

A Tractable Cell System Enables Rapid Identification of Host Factors.

Front Cell Infect Microbiol 2020 26;10:240. Epub 2020 May 26.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan TX, United States.

is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported. is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.
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http://dx.doi.org/10.3389/fcimb.2020.00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264411PMC
June 2021

PRESCIENT: platform for the rapid evaluation of antibody success using integrated microfluidics enabled technology.

Lab Chip 2020 05 20;20(9):1628-1638. Epub 2020 Mar 20.

Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA.

Identifying antibodies (Abs) that neutralize infectious agents is the first step for developing therapeutics, vaccines, and diagnostic tools for these infectious agents. However, current approaches for identifying neutralizing Abs (nAbs) typically rely on dilution-based assays that are costly, inefficient, and only survey a small subset of the entire repertoire. There are also intrinsic biases in many steps of conventional nAb identification processes. More importantly, conventional assays rely on simple Ab-antigen binding assays, which may not result in identifying the most potent nAbs, as the strongest binder may not be the most potent nAb. Droplet microfluidic systems have the capability to overcome such limitations by conducting complex multi-step assays with high reliability, resolution, and throughput in a pico-liter volume water-in-oil emulsion droplet format. Here, we describe the development of PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated microfluidics ENabled Technology), a droplet microfluidic system that can enable high-throughput single-cell resolution identification of nAb repertoires elicited in response to viral infection. We demonstrate PRESCIENT's ability to identify Abs that neutralize a model viral agent, Murine coronavirus (murine hepatitis virus), which causes high mortality rates in experimentally infected mice. In-droplet infection of host cells by the virus was first demonstrated, followed by demonstration of in-droplet neutralization by nAbs produced from a single Ab-producing hybridoma cell. Finally, fluorescence intensity analyses of two populations of hybridoma cell lines (nAb-producing and non-nAb-producing hybridoma cell lines) successfully discriminated between the two populations. The presented strategy and platform have the potential to identify and investigate neutralizing activities against a broad range of potential infectious agents for which nAbs have yet to be discovered, significantly advancing the nAb identification process as well as reinvigorating the field of Ab discovery, characterization, and development.
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http://dx.doi.org/10.1039/c9lc01165jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269184PMC
May 2020

Quantitative Yeast Genetic Interaction Profiling of Bacterial Effector Proteins Uncovers a Role for the Human Retromer in Salmonella Infection.

Cell Syst 2018 09 1;7(3):323-338.e6. Epub 2018 Aug 1.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77802, USA. Electronic address:

Intracellular bacterial pathogens secrete a repertoire of effector proteins into host cells that are required to hijack cellular pathways and cause disease. Despite decades of research, the molecular functions of most bacterial effectors remain unclear. To address this gap, we generated quantitative genetic interaction profiles between 36 validated and putative effectors from three evolutionarily divergent human bacterial pathogens and 4,190 yeast deletion strains. Correlating effector-generated profiles with those of yeast mutants, we recapitulated known biology for several effectors with remarkable specificity and predicted previously unknown functions for others. Biochemical and functional validation in human cells revealed a role for an uncharacterized component of the Salmonella SPI-2 translocon, SseC, in regulating maintenance of the Salmonella vacuole through interactions with components of the host retromer complex. These results exhibit the power of genetic interaction profiling to discover and dissect complex biology at the host-pathogen interface.
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http://dx.doi.org/10.1016/j.cels.2018.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160342PMC
September 2018

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of .

Front Cell Infect Microbiol 2018 20;8:103. Epub 2018 Apr 20.

Key Laboratory of Zoonosis Research, Ministry of Education, College of Plant Sciences, Jilin University, Changchun, China.

spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to and intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of .
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http://dx.doi.org/10.3389/fcimb.2018.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919948PMC
April 2019

The pre-rRNA processing factor Nop53 regulates fungal development and pathogenesis via mediating production of reactive oxygen species.

Environ Microbiol 2018 04 26;20(4):1531-1549. Epub 2018 Mar 26.

College of Plant Sciences, Jilin University, Changchun 130062, China.

Botrytis cinerea is a necrotrophic plant fungal pathogen that annually causes enormous economic losses worldwide. The ribosome is an organelle for cellular protein biosynthesis. However, little is known about how the ribosome operates as a machine to mediate microbial pathogenesis. Here, we demonstrate that Nop53, a late-acting factor for 60S ribosomal subunit maturation, is crucial for the pathogen's development and virulence. BcNop53 is functionally equivalent to yeast nop53p. Complementation of BcNOP53 completely restored the growth defect of the yeast Δnop53 mutant. BcNop53 is located in nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth. Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen, possibly due to reduced production of reactive oxygen species. Moreover, loss of BcNOP53 impaired pathogen conidiation and stress adaptation, altered conidial and sclerotial morphology, retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes. Sclerotium production was, however, increased. Complementation with the wild-type BcNOP53 allele rescued defects found in the ΔBcnop53 mutant. Our work establishes a systematic elucidation of Nop53 in regulating microbial development and pathogenesis, provides novel insights into ribosomal processes that regulate fungal pathogenesis, and may open up new targets for addressing fungal diseases.
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http://dx.doi.org/10.1111/1462-2920.14082DOI Listing
April 2018

Publisher Correction: Reassessing apoptosis in plants.

Nat Plants 2017 11;3(11):906

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, 97331, USA.

In the version of this Perspective originally published, the name of co-author Paul de Figueiredo was incorrect. This has now been corrected.
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http://dx.doi.org/10.1038/s41477-017-0045-1DOI Listing
November 2017

Reassessing apoptosis in plants.

Nat Plants 2017 Oct 25;3(10):773-779. Epub 2017 Sep 25.

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, 97331, USA.

Cell death can be driven by a genetically programmed signalling pathway known as programmed cell death (PCD). In plants, PCD occurs during development as well as in response to environmental and biotic stimuli. Our understanding of PCD regulation in plants has advanced significantly over the past two decades; however, the molecular machinery responsible for driving the system remains elusive. Thus, whether conserved PCD regulatory mechanisms include plant apoptosis remains enigmatic. Animal apoptotic regulators, including Bcl-2 family members, have not been identified in plants but expression of such regulators can trigger or suppress plant PCD. Moreover, plants exhibit nearly all of the biochemical and morphological features of apoptosis. One difference between plant and animal PCD is the absence of phagocytosis in plants. Evidence is emerging that the vacuole may be key to removal of unwanted plant cells, and may carry out functions that are analogous to animal phagocytosis. Here, we provide context for the argument that apoptotic-like cell death occurs in plants.
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http://dx.doi.org/10.1038/s41477-017-0020-xDOI Listing
October 2017

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection.

Cell Host Microbe 2017 May;21(5):637-649.e6

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, College Station, Texas 77843, USA; Norman Borlaug Center, Texas A&M University, College Station, Texas 77843, USA; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA. Electronic address:

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.
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http://dx.doi.org/10.1016/j.chom.2017.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538893PMC
May 2017

The septin protein Sep4 facilitates host infection by plant fungal pathogens via mediating initiation of infection structure formation.

Environ Microbiol 2017 05 23;19(5):1730-1749. Epub 2017 Jan 23.

College of Plant Sciences, Jilin University, Changchun, 130062, China.

Many phytopathogenic fungi use infection structures (IFSs, i.e., appressoria and infection cushions) to penetrate host cuticles. However, the conserved mechanisms that mediate initiation of IFS formation in divergent pathogens upon sensing the presence of host plants remain obscure. Here, we demonstrate that a conserved septin gene SEP4 plays crucial roles in this process. Disruption of SEP4 in the plant grey mould fungus Botrytis cinerea completely blocked IFS formation and abolished the virulence of ΔBcsep4 mutants on unwounded hosts. During IFS formation, mutants lacking SEP4 could produce reactive oxygen species (ROS) normally. Inhibition of ROS production in strains harbouring the SEP4 gene resulted in disordered assembly of Sep4 and the subsequent failure to form infection cushions, suggesting that proper Sep4 assembly regulated by ROS is required for initiation of IFS formation and infection. Moreover, loss of SEP4 severely impaired mutant conidiation, melanin and chitin accumulation in hyphal tips and lesion expansion on wounded hosts, but significantly promoted germ tube elongation and sclerotium production. SEP4-mediated fungal pathogenic development, including IFS formation, was validated in the hemibiotroph Magnaporthe oryzae. Our findings indicate that Sep4 plays pleiotropic roles in B. cinerea development and specifically facilities host infection by mediating initiation of IFS formation in divergent plant fungal pathogens in response to ROS signaling.
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http://dx.doi.org/10.1111/1462-2920.13613DOI Listing
May 2017

Reconstitution of Phospholipase A2-Dependent Golgi Membrane Tubules.

Methods Mol Biol 2016 ;1496:75-90

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14850, USA.

The Golgi complex is the Grand Central Station of intracellular membrane trafficking in the secretory and endocytic pathways. Anterograde and retrograde export of cargo from the Golgi complex involves a complex interplay between the formation of coated vesicles and membrane tubules, although much less is known about tubule-mediated trafficking. Recent advances using in vitro assays have identified several cytoplasmic phospholipase A2 (PLA2) enzymes that are required for the biogenesis of membrane tubules and their roles in the functional organization of the Golgi complex. In this chapter we describe methods for the cell-free reconstitution of PLA2-dependent Golgi membrane tubule formation. These methods should facilitate the identification of other proteins that regulate this process.
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http://dx.doi.org/10.1007/978-1-4939-6463-5_7DOI Listing
January 2018

One Health: Addressing Global Challenges at the Nexus of Human, Animal, and Environmental Health.

PLoS Pathog 2016 09 15;12(9):e1005731. Epub 2016 Sep 15.

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1005731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025119PMC
September 2016

The Case for Live Attenuated Vaccines against the Neglected Zoonotic Diseases Brucellosis and Bovine Tuberculosis.

PLoS Negl Trop Dis 2016 08 18;10(8):e0004572. Epub 2016 Aug 18.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, Texas, United States of America.

Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms.
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http://dx.doi.org/10.1371/journal.pntd.0004572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990199PMC
August 2016

Autophagy under attack.

Elife 2016 Feb 23;5. Epub 2016 Feb 23.

Norman Borlaug Institute and the Department of Plant Pathology and Microbiology, Texas A&M University, College Station, United States.

Pathogens target proteins involved in autophagy to inhibit immune responses in plants.
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http://dx.doi.org/10.7554/eLife.14447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775212PMC
February 2016

Space: A Final Frontier for Vacuolar Pathogens.

Traffic 2016 May 24;17(5):461-74. Epub 2016 Feb 24.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX, USA.

There is a fundamental gap in our understanding of how a eukaryotic cell apportions the limited space within its cell membrane. Upon infection, a cell competes with intracellular pathogens for control of this same precious resource. The struggle between pathogen and host provides us with an opportunity to uncover the mechanisms regulating subcellular space by understanding how pathogens modulate vesicular traffic and membrane fusion events to create a specialized compartment for replication. By comparing several important intracellular pathogens, we review the molecular mechanisms and trafficking pathways that drive two space allocation strategies, the formation of tight and spacious pathogen-containing vacuoles. Additionally, we discuss the potential advantages of each pathogenic lifestyle, the broader implications these lifestyles might have for cellular biology and outline exciting opportunities for future investigation.
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http://dx.doi.org/10.1111/tra.12382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048968PMC
May 2016

Methods for analyzing the role of phospholipase A₂ enzymes in endosome membrane tubule formation.

Methods Cell Biol 2015 7;130:157-80. Epub 2015 Jul 7.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Cargo export from mammalian endosomal compartments often involves membrane tubules, into which soluble and membrane-bound cargos are segregated for subsequent intracellular transport. These membrane tubules are highly dynamic and their formation is mediated by a variety of endosome-associated proteins. However, little is known about how these membrane tubules are temporally or spatially regulated, so other tubule-associated proteins are likely to be discovered and analyzed. Therefore, methods to examine the biogenesis and regulation of endosome membrane tubules will prove to be valuable for cell biologists. In this chapter, we describe methods for studying this process using both cell-free, in vitro reconstitution assays, and in vivo image analysis tools.
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http://dx.doi.org/10.1016/bs.mcb.2015.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876852PMC
June 2016

Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

Am J Pathol 2015 Jun 17;185(6):1505-17. Epub 2015 Apr 17.

Department of Veterinary Pathobiology, Texas A&M University and Texas AgriLife Research, College Station, Texas. Electronic address:

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics.
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http://dx.doi.org/10.1016/j.ajpath.2015.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450313PMC
June 2015

A three-dimensional electrode for highly efficient electrocoalescence-based droplet merging.

Biomed Microdevices 2015 Apr;17(2):35

Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, 77843, USA.

Droplet merging is one of the key functions in the ever-widening applications of droplet microfluidics. Enhancing the efficiency of electric field-based droplet merging, namely electrocoalescence, can lead to an increase in platform stability and overcome one of the major bottlenecks in further improving throughputs of droplet microfluidic systems. In this work, a paired three-dimensional (3D) electrode design that can provide a uniform electric field within a droplet merging region, which is also properly aligned with the droplet dipole moments for highly efficient electrocoalescence is presented. A systematic study was conducted to compare the droplet merging performance of the presented 3D electrode design to other commonly used planar electrode, coplanar electrode, dual-coplanar electrode, and liquid metal 3D electrode designs. The presented 3D electrode design reduced the threshold input voltage required to obtain droplet fusion by up to 75%. In addition, a droplet merging efficiency of higher than 95% was consistently observed, compared to less than 85% merging efficiency for the conventionally used electrode designs. We expect that this droplet electrocoalescence design will improve the overall throughput and merging success rate in droplet microfluidic based high-throughput assays.
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http://dx.doi.org/10.1007/s10544-014-9921-xDOI Listing
April 2015

SiRNA screens using Drosophila cells to identify host factors required for infection.

Methods Mol Biol 2014 ;1197:229-44

Department of Veterinary Pathobiology, Texas A&M University, 4474 TAMU, College Station, TX, 77843, USA,

Drosophila melanogaster offers a powerful model system for interrogating interactions between host cells and human bacterial pathogens. Brucella, a gram-negative, facultative intracellular bacterium is the causative agent of brucellosis, a zoonotic disease of global consequence. Over the past several decades, pathogen factors that mediate Brucella infection have been identified. However, host factors that mediate infection have remained obscure. We have used the power of the Drosophila S2 cell system to identify and characterize host factors that support infection by Brucella melitensis. Host protein inositol-requiring enzyme 1 (IRE1α), a transmembrane kinase and master regulator of the eukaryotic unfolded protein response, was shown to play an important role in regulating Brucella infection, thereby providing the first glimpse of host mechanisms that are subverted by the pathogen to support its intracellular lifestyle. Furthermore, our study also established the Drosophila S2 cell as a powerful system for elucidating Brucella host factors. Here, we describe a protocol for using the Drosophila S2 cell system for studying the Brucella-host interaction.
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http://dx.doi.org/10.1007/978-1-4939-1261-2_13DOI Listing
May 2015

Death be not proud--cell death control in plant fungal interactions.

PLoS Pathog 2013 Sep 12;9(9):e1003542. Epub 2013 Sep 12.

Norman Borlaug Center, Texas A&M University, College Station, Texas, United States of America ; Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, Texas, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1003542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771904PMC
September 2013
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