Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1Streptococcus pyogenes

ABSTRACTStreptococcus pyogenes(group AStreptococcus[GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation ofspeBexpression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction ofspeBgene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepOmutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCEDespite the continuing susceptibility ofS. pyogenesto penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that controlspeBgene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator ofspeBgene expression in the globally disseminated M1T1 clone and as being essential for virulence.

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Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02296-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6795-6802 ◽

Cited By ~ 40

Author(s):

Andreas Kaczmarczyk ◽

Julia A. Vorholt ◽

Anne Francez-Charlot

Keyword(s):

Gene Expression ◽

Caulobacter Crescentus ◽

Paracoccus Denitrificans ◽

Expression System ◽

Model Organisms ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Content Type ◽

Wide Range ◽

Inducible Gene

ABSTRACTTunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of thePseudomonas putidaF1cym/cmtsystem. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in otherAlphaproteobacteria, such as the model organismsCaulobacter crescentus,Paracoccus denitrificans, andMethylobacterium extorquens. In the noninduced state, expression from PQ5is low enough to allow gene depletion analysis, as demonstrated with the essential genephyPofSphingomonassp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

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Identification of Bradyrhizobium elkanii Genes Involved in Incompatibility with Soybean Plants Carrying theRj4Allele

Applied and Environmental Microbiology ◽

10.1128/aem.01942-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6710-6717 ◽

Cited By ~ 31

Author(s):

Omar M. Faruque ◽

Hiroki Miwa ◽

Michiko Yasuda ◽

Yoshiharu Fujii ◽

Takakazu Kaneko ◽

...

Keyword(s):

Cysteine Protease ◽

Xanthomonas Campestris ◽

Transcriptional Regulator ◽

Soybean Cultivar ◽

Sequencing Analysis ◽

Content Type ◽

Wide Range ◽

Bradyrhizobium Elkanii ◽

Soybean Plants ◽

Type 3

ABSTRACTSymbioses between leguminous plants and soil bacteria known as rhizobia are of great importance to agricultural production and nitrogen cycling. While these mutualistic symbioses can involve a wide range of rhizobia, some legumes exhibit incompatibility with specific strains, resulting in ineffective nodulation. The formation of nodules in soybean plants (Glycine max) is controlled by several host genes, which are referred to asRjgenes. The soybean cultivar BARC2 carries theRj4gene, which restricts nodulation by specific strains, includingBradyrhizobium elkaniiUSDA61. Here we employed transposon mutagenesis to identify the genetic locus in USDA61 that determines incompatibility with soybean varieties carrying theRj4allele. Introduction of the Tn5transposon into USDA61 resulted in the formation of nitrogen fixation nodules on the roots of soybean cultivar BARC2 (Rj4 Rj4). Sequencing analysis of the sequence flanking the Tn5insertion revealed that six genes encoding a putative histidine kinase, transcriptional regulator, DNA-binding transcriptional activator, helix-turn-helix-type transcriptional regulator, phage shock protein, and cysteine protease were disrupted. The cysteine protease mutant had a high degree of similarity with the type 3 effector protein XopD ofXanthomonas campestris. Our findings shed light on the diverse and complicated mechanisms that underlie these highly host-specific interactions and indicate the involvement of a type 3 effector inRj4nodulation restriction, suggesting thatRj4incompatibility is partly mediated by effector-triggered immunity.

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In VivoProgrammed Gene Expression Based on Artificial Quorum Networks

Applied and Environmental Microbiology ◽

10.1128/aem.01113-15 ◽

2015 ◽

Vol 81 (15) ◽

pp. 4984-4992 ◽

Cited By ~ 1

Author(s):

Teng Chu ◽

Yajun Huang ◽

Mingyu Hou ◽

Qiyao Wang ◽

Jingfan Xiao ◽

...

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Cell Density ◽

Protective Antigen ◽

Expression System ◽

Edwardsiella Tarda ◽

Content Type ◽

Genetic Components ◽

Wide Range

ABSTRACTThe quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of theVibrio fischeri luxI-luxRquorum sensing system. In order to achievein vivoprogrammed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system.In vitroexpression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density.In vivoexpression assays confirmed that the araQS system presented anin vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applicationsin vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuatedEdwardsiella tardastrain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expressionin vivoand might have potential uses, including, but not limited to, bacterial vector vaccines.

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Transcriptome Analysis of Mouse Brain Infected with Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.00439-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3609-3619 ◽

Cited By ~ 55

Author(s):

Sachi Tanaka ◽

Maki Nishimura ◽

Fumiaki Ihara ◽

Junya Yamagishi ◽

Yutaka Suzuki ◽

...

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Immune Responses ◽

Mouse Brain ◽

Transcriptome Analysis ◽

Host Cells ◽

Content Type ◽

Host Immune Responses ◽

Wide Range ◽

The Brain

ABSTRACTToxoplasma gondiiis an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections withT. gondiibecome established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection withT. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In theT. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes duringT. gondiiinfection.

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Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein

mBio ◽

10.1128/mbio.00649-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 10

Author(s):

Keely A. Dulmage ◽

Horia Todor ◽

Amy K. Schmid

Keyword(s):

Gene Expression ◽

Growth Phase ◽

Cell Shape ◽

Regulation Of Gene Expression ◽

Halobacterium Salinarum ◽

Major Protein ◽

Global Regulation ◽

Histone Protein ◽

Content Type ◽

Dna Compaction

ABSTRACTIn all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organismHalobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes,H. salinarumhistone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone inH. salinarumplays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone.IMPORTANCEHistones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeonHalobacterium salinarumis both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather,H. salinarumhistone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis thatH. salinarumhistone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function.

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The MexE/MexF/AmeC Efflux Pump of Agrobacterium tumefaciens and Its Role in Ti Plasmid Virulence Gene Expression

Journal of Bacteriology ◽

10.1128/jb.00609-19 ◽

2020 ◽

Vol 202 (8) ◽

Cited By ~ 2

Author(s):

Andrew N. Binns ◽

Jinlei Zhao

Keyword(s):

Gene Expression ◽

Agrobacterium Tumefaciens ◽

Efflux Pump ◽

Bacterial Pathogen ◽

Virulence Gene ◽

Ti Plasmid ◽

Content Type ◽

Virulence Gene Expression ◽

Extracellular Milieu ◽

Phenol Derivatives

ABSTRACT Expression of the tumor-inducing (Ti) plasmid virulence genes of Agrobacterium tumefaciens is required for the transfer of DNA from the bacterium into plant cells, ultimately resulting in the initiation of plant tumors. The vir genes are induced as a result of exposure to certain phenol derivatives, monosaccharides, and low pH in the extracellular milieu. The soil, as well as wound sites on a plant—the usual site of the virulence activity of this bacterium—can contain these signals, but vir gene expression in the soil would be a wasteful utilization of energy. This suggests that mechanisms may exist to ensure that vir gene expression occurs only at the higher concentrations of inducers typically found at a plant wound site. In a search for transposon-mediated mutations that affect sensitivity for the virulence gene-inducing activity of the phenol, 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone [AS]), an RND-type efflux pump homologous to the MexE/MexF/OprN pump of Pseudomonas aeruginosa was identified. Phenotypes of mutants carrying an insertion or deletion of pump components included hypersensitivity to the vir-inducing effects of AS, hypervirulence in the tobacco leaf explant virulence assay, and hypersensitivity to the toxic effects of chloramphenicol. Furthermore, the methoxy substituents on the phenol ring of AS appear to be critical for recognition as a pump substrate. These results support the hypothesis that the regulation of virulence gene expression is integrated with cellular activities that elevate the level of plant-derived inducers required for induction so that this occurs preferentially, if not exclusively, in a plant environment. IMPORTANCE Expression of genes controlling the virulence activities of a bacterial pathogen is expected to occur preferentially at host sites vulnerable to that pathogen. Host-derived molecules that induce such activities in the plant pathogen Agrobacterium tumefaciens are found in the soil, as well as in the plant. Here, we tested the hypothesis that mechanisms exist to suppress the sensitivity of Agrobacterium species to a virulence gene-inducing molecule by selecting for mutant bacteria that are hypersensitive to its inducing activity. The mutant genes identified encode an efflux pump whose proposed activity increases the concentration of the inducer necessary for vir gene expression; this pump is also involved in antibiotic resistance, demonstrating a relationship between cellular defense activities and the control of virulence in Agrobacterium.

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Regulation of the Peptidoglycan Amidase PGLYRP2 in Epithelial Cells by Interleukin-36γ

Infection and Immunity ◽

10.1128/iai.00384-18 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Glen M. Scholz ◽

Jacqueline E. Heath ◽

Jiamin Aw ◽

Eric C. Reynolds

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Bacterial Pathogen ◽

Oral Bacteria ◽

Oral Epithelium ◽

Content Type ◽

Oral Epithelial Cells ◽

Mitogen Activated Protein ◽

Mucosal Homeostasis ◽

Oral Epithelial

ABSTRACT Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

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Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

Applied and Environmental Microbiology ◽

10.1128/aem.02033-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 7244-7252 ◽

Cited By ~ 14

Author(s):

Katrin Beilharz ◽

Renske van Raaphorst ◽

Morten Kjos ◽

Jan-Willem Veening

Keyword(s):

Gene Expression ◽

Streptococcus Pneumoniae ◽

Cell Biology ◽

Fluorescent Proteins ◽

Protein Localization ◽

Gibson Assembly ◽

Content Type ◽

Assembly Method ◽

Red Fluorescent Proteins ◽

Wide Range

ABSTRACTDuring the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogenStreptococcus pneumoniaeand other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) inS. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP inS. pneumoniaeand is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling inS. pneumoniaeand related organisms.

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Regulation of Virus-Associated Lymphoma Growth and Gene Expression by Bacterial Quorum-Sensing Molecules

Journal of Virology ◽

10.1128/jvi.00478-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 2

Author(s):

Jing Qiao ◽

Yueyu Cao ◽

Jovanny Zabaleta ◽

Liang Yang ◽

Lu Dai ◽

...

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Bacterial Pathogens ◽

Signaling Molecules ◽

Viral Gene ◽

Immunocompromised Patients ◽

Content Type ◽

Kshv Infection ◽

Wide Range ◽

Polymicrobial Infections

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) can cause several human cancers, including primary effusion lymphoma (PEL), which frequently occur in immunocompromised patients. KSHV-infected patients often suffer from polymicrobial infections caused by opportunistic bacterial pathogens. Therefore, it is crucial to understand how these coinfecting microorganisms or their secreted metabolites may affect KSHV infection and the pathogenesis of virus-associated malignancies. Quorum sensing (QS), a cell density-based intercellular communication system, employs extracellular diffusible signaling molecules to regulate bacterial virulence mechanisms in a wide range of bacterial pathogens, such asPseudomonas aeruginosa, which is one of the most common opportunistic microorganisms found in immunocompromised individuals. In this study, we evaluated and compared the influence on PEL growth and the host/viral interactome of the major QS signaling molecules [N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL),N-butyrylhomoserine lactone (BHL), and 2-heptyl-3-hydroxy-4-quinolone (PQS)] in conditioned medium from wild-type (wt) and QS mutant laboratory strains as well as clinical isolates ofP. aeruginosa. Our data indicate thatP. aeruginosacoinfection may facilitate virus dissemination and establishment of new infection and further promote tumor development through effectively inducing viral lytic gene expression by its QS systems.IMPORTANCECurrently, most studies about KSHV infection and/or virus-associated malignancies depend on pure culture systems or immunodeficient animal models. However, the real situation should be much more complicated in KSHV-infected immunocompromised patients due to frequent polymicrobial infections. It is important to understand the interaction of KSHV and coinfecting microorganisms, especially opportunistic bacterial pathogens. Here we report for the first time thatP. aeruginosaand its quorum-sensing signaling molecules display a complicated impact on KSHV-associated lymphoma growth as well as the intracellular host/viral gene expression profile. Our data imply that targeting of coinfecting pathogens is probably necessary during treatment of virus-associated malignancies in these immunocompromised patients.

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