Transposon Sequencing ofBrucella abortusUncovers Essential Genes for GrowthIn Vitroand Inside Macrophages

ABSTRACTBrucella abortusis a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we report a multidimensional transposon sequencing analysis to identify genes essential forBrucella abortusgrowth in rich medium and replication in RAW 264.7 macrophages. The construction of a dense transposon mutant library and mapping of 929,769 unique mini-Tn5insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in theB. abortusgenome. Chromosome II carries a lower proportion (5%) of essential genes than chromosome I (19%), supporting the hypothesis of a recent acquisition of a megaplasmid as the origin of chromosome II. Temporally resolved transposon sequencing analysis as a function of macrophage infection stages identified 79 genes with a specific attenuation phenotype in macrophages, at either 2, 5, or 24 h postinfection, and 86 genes for which the attenuated mutant phenotype correlated with a growth defect on plates. We identified 48 genes required for intracellular growth, including thevirBoperon, encoding the type IV secretion system, which supports the validity of the screen. The remaining genes encode amino acid and pyrimidine biosynthesis, electron transfer systems, transcriptional regulators, and transporters. In particular, we report the need of an intact pyrimidine nucleotide biosynthesis pathway in order forB. abortusto proliferate inside RAW 264.7 macrophages.

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Intracellular Growth and Cell Cycle Progression are Dependent on (p)ppGpp Synthetase/Hydrolase in Brucella abortus

Pathogens ◽

10.3390/pathogens9070571 ◽

2020 ◽

Vol 9 (7) ◽

pp. 571

Author(s):

Mathilde Van der Henst ◽

Elodie Carlier ◽

Xavier De Bolle

Keyword(s):

Cell Cycle ◽

Brucella Abortus ◽

Cell Cycle Progression ◽

Growth Conditions ◽

Host Cells ◽

G1 Arrest ◽

Raw 264.7 ◽

Rich Medium ◽

Intracellular Growth ◽

Raw 264.7 Macrophages

Brucella abortus is a pathogenic bacterium able to proliferate inside host cells. During the first steps of its trafficking, it is able to block the progression of its cell cycle, remaining at the G1 stage for several hours, before it reaches its replication niche. We hypothesized that starvation mediated by guanosine tetra- or penta-phosphate, (p)ppGpp, could be involved in the cell cycle arrest. Rsh is the (p)ppGpp synthetase/hydrolase. A B. abortus ∆rsh mutant is unable to grow in minimal medium, it is unable to survive in stationary phase in rich medium and it is unable to proliferate inside RAW 264.7 macrophages. A strain producing the heterologous constitutive (p)ppGpp hydrolase Mesh1b is also unable to proliferate inside these macrophages. Altogether, these data suggest that (p)ppGpp is necessary to allow B. abortus to adapt to its intracellular growth conditions. The deletion of dksA, proposed to mediate a part of the effect of (p)ppGpp on transcription, does not affect B. abortus growth in culture or inside macrophages. Expression of a gene coding for a constitutively active (p)ppGpp synthetase slows down growth in rich medium and inside macrophages. Using an mCherry–ParB fusion able to bind to the replication origin of the main chromosome of B. abortus, we observed that expression of the constitutive (p)ppGpp synthetase gene generates an accumulation of bacteria at the G1 phase. We thus propose that (p)ppGpp accumulation could be one of the factors contributing to the G1 arrest observed for B. abortus in RAW 264.7 macrophages.

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SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages

Infection and Immunity ◽

10.1128/iai.00010-17 ◽

2017 ◽

Vol 85 (3) ◽

Cited By ~ 15

Author(s):

Regina A. Günster ◽

Sophie A. Matthews ◽

David W. Holden ◽

Teresa L. M. Thurston

Keyword(s):

Cell Death ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Macrophage Infection ◽

Content Type ◽

Type Iii ◽

Tnf Α ◽

Arginine Residues

ABSTRACT Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of N-acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3.

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Temporal Kinetics and Quantitative Analysis of Cryptococcus neoformans Nonlytic Exocytosis

Infection and Immunity ◽

10.1128/iai.01503-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2059-2067 ◽

Cited By ~ 15

Author(s):

Sabriya A. Stukes ◽

Hillel W. Cohen ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Time Lapse ◽

Host Cells ◽

Type I ◽

Phagocytic Cells ◽

Macrophage Infection ◽

Content Type ◽

Type Iii ◽

Cell To Cell Transfer ◽

Extracellular Environment

ABSTRACTCryptococcus neoformansis a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems.C. neoformanshas the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies ofC. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellularC. neoformansand identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P< 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection.

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Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica

Infection and Immunity ◽

10.1128/iai.00063-09 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3227-3233 ◽

Cited By ~ 37

Author(s):

Liang Shi ◽

Saiful M. Chowdhury ◽

Heather S. Smallwood ◽

Hyunjin Yoon ◽

Heather M. Mottaz-Brewer ◽

...

Keyword(s):

Nitric Oxide ◽

Salmonella Enterica ◽

Time Course ◽

Negative Impact ◽

Raw 264.7 ◽

Bacterial Cells ◽

Macrophage Infection ◽

Raw 264.7 Macrophages ◽

Macrophage Response ◽

Proteomic Approach

ABSTRACT To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H2 (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella-affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella, infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6.

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Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

Infection and Immunity ◽

10.1128/iai.00565-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 11

Author(s):

Mariette Barbier ◽

Dylan T. Boehm ◽

Emel Sen-Kilic ◽

Claire Bonnin ◽

Theo Pinheiro ◽

...

Keyword(s):

Adenylate Cyclase ◽

Mutant Strain ◽

Pertussis Toxin ◽

Bordetella Pertussis ◽

Sigma Factor ◽

Negative Regulator ◽

Host Cells ◽

Sequencing Analysis ◽

Western Blots ◽

Content Type

ABSTRACT Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

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Toll-Like Receptor 4-Linked Janus Kinase 2 Signaling Contributes to Internalization of Brucella abortus by Macrophages

Infection and Immunity ◽

10.1128/iai.00403-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2448-2458 ◽

Cited By ~ 22

Author(s):

Jin Ju Lee ◽

Dong Hyeok Kim ◽

Dae Geun Kim ◽

Hu Jang Lee ◽

Wongi Min ◽

...

Keyword(s):

Brucella Abortus ◽

Actin Polymerization ◽

Small Gtpase ◽

Janus Kinase ◽

Host Cells ◽

Raw 264.7 Cells ◽

Janus Kinase 2 ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Content Type

ABSTRACTBrucella abortusis an intracellular pathogen that uses a crafty strategy to invade and proliferate within host cells, but the distinct signaling pathways associated with phagocytic mechanisms ofB. abortusremain unclear. The present study was performed to test the hypothesis that Toll-like receptor 4 (TLR4)-linked signaling interacting with Janus kinase 2 (JAK2) plays an essential role inB. abortusphagocytosis by macrophages. The effects of TLR4-JAK2 signaling onB. abortusphagocytosis in murine macrophage RAW 264.7 cells were observed through an infection assay and confocal microscopy. We determined that the uptake ofB. abortuswas negatively affected by the dysfunction of TLR4 and JAK2. F-actin polymerization detected by flow cytometry and F-actin assay was amplified forB. abortusentry, whereas that event was attenuated by the disruption of TLR4 and JAK2. Importantly, JAK2 phosphorylation and actin skeleton reorganization were suppressed immediately afterB. abortusinfection in bone marrow-derived macrophages (BMDMs) from TLR4−/−mice, showing the cooperation of JAK2 with TLR4. Furthermore, small GTPase Cdc42 participated in the intermediate pathway of TLR4-JAK2 signaling onB. abortusphagocytosis. Consequently, TLR4-associated JAK2 activation in the early cellular signaling events plays a pivotal role inB. abortus-induced phagocytic processes in macrophages, implying the pathogenic significance of JAK2-mediated entry. Here, we elucidate that this specific phagocytic mechanism ofB. abortusmight provide achievable strategies for inhibitingB. abortusinvasion.

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The Endoribonuclease RNase E Coordinates Expression of mRNAs and Small Regulatory RNAs and Is Critical for the Virulence of Brucella abortus

Journal of Bacteriology ◽

10.1128/jb.00240-20 ◽

2020 ◽

Vol 202 (20) ◽

Author(s):

Lauren M. Sheehan ◽

James A. Budnick ◽

Jaquille Fyffe-Blair ◽

Kellie A. King ◽

Robert E. Settlage ◽

...

Keyword(s):

Brucella Abortus ◽

Human Infection ◽

Regulatory Rna ◽

Sequencing Analysis ◽

Mrna Levels ◽

Rnase E ◽

Content Type ◽

Regulatory Pathways ◽

Small Regulatory Rna ◽

The Impact

ABSTRACT RNases are key regulatory components in prokaryotes, responsible for the degradation and maturation of specific RNA molecules at precise times. Specifically, RNases allow cells to cope with changes in their environment through rapid alteration of gene expression. To date, few RNases have been characterized in the mammalian pathogen Brucella abortus. In the present work, we sought to investigate several RNases in B. abortus and determine what role, if any, they have in pathogenesis. Of the 4 RNases reported in this study, the highly conserved endoribonuclease, RNase E, was found to play an integral role in the virulence of B. abortus. Although rne, which encodes RNase E, is essential in B. abortus, we were able to generate a strain encoding a defective version of RNase E lacking the C-terminal portion of the protein, and this strain (rne-tnc) was attenuated in a mouse model of Brucella infection. RNA-sequencing analysis revealed massive RNA dysregulation in B. abortus rne-tnc, with 122 upregulated and 161 downregulated transcripts compared to the parental strain. Interestingly, several mRNAs related to metal homeostasis were significantly decreased in the rne-tnc strain. We also identified a small regulatory RNA (sRNA), called Bsr4, that exhibited significantly elevated levels in rne-tnc, demonstrating an important role for RNase E in sRNA-mediated regulatory pathways in Brucella. Overall, these data highlight the importance of RNase E in B. abortus, including the role of RNase E in properly controlling mRNA levels and contributing to virulence in an animal model of infection. IMPORTANCE Brucellosis is a debilitating disease of humans and animals globally, and there is currently no vaccine to combat human infection by Brucella spp. Moreover, effective antibiotic treatment in humans is extremely difficult and can lead to disease relapse. Therefore, it is imperative that systems and pathways be identified and characterized in the brucellae so new vaccines and therapies can be generated. In this study, we describe the impact of the endoribonuclease RNase E on the control of mRNA and small regulatory RNA (sRNA) levels in B. abortus, as well as the importance of RNase E for the full virulence of B. abortus. This work greatly enhances our understanding of ribonucleases in the biology and pathogenesis of Brucella spp.

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Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae

Infection and Immunity ◽

10.1128/iai.00374-19 ◽

2019 ◽

Vol 87 (11) ◽

Cited By ~ 1

Author(s):

Richard C. White ◽

Hilary K. Truchan ◽

Huaixin Zheng ◽

Jessica Y. Tyson ◽

Nicholas P. Cianciotto

Keyword(s):

Legionella Pneumophila ◽

Fluorescent Protein ◽

Host Cells ◽

Recent Analysis ◽

Type Ii Secretion ◽

Type Ii ◽

Wild Type ◽

Macrophage Infection ◽

Content Type ◽

Intracellular Infection

ABSTRACT It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii. Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant’s defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

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Epistatic Interplay between Type IV Secretion Effectors Engages the Small GTPase Rab2 in the Brucella Intracellular Cycle

mBio ◽

10.1128/mbio.03350-19 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Erin P. Smith ◽

Alexis Cotto-Rosario ◽

Elizabeth Borghesan ◽

Kiara Held ◽

Cheryl N. Miller ◽

...

Keyword(s):

Brucella Abortus ◽

Bacterial Pathogens ◽

Small Gtpase ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Secretion Systems ◽

Content Type ◽

Type Iv ◽

Infectious Cycle

ABSTRACT Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors. IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

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Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

Infection and Immunity ◽

10.1128/iai.01125-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 521-530 ◽

Cited By ~ 16

Author(s):

Mara S. Roset ◽

Lucía García Fernández ◽

Vito G. DelVecchio ◽

Gabriel Briones

Keyword(s):

Host Cell ◽

Brucella Abortus ◽

Intracellular Localization ◽

Bacterial Pathogen ◽

Host Cells ◽

Content Type ◽

Double Deletion ◽

Proteomic Investigation ◽

Increased Sensitivity ◽

Lower Temperature

ABSTRACTBrucellais an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis.Brucellavirulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant forBrucellaintracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grownBrucella. To define the potential role of cyclophilins inBrucellavirulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. TheBrucella abortusΔcypABmutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, theB. abortusΔcypABmutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. TheB. abortusΔcypABmutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important forBrucellavirulence and survival in the host cells.

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