bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of the Pneumocystis jirovecii Potential Drug Targets
dhfs
and
abz2
Involved in Folate Biosynthesis
Authors: Luraschi A. , Cissé O. H. , Monod M. , Pagni M. , Hauser P. M. 
Published: February 2015

ABSTRACT

Pneumocystis
species are fungal parasites colonizing mammal lungs with strict host specificity.
Pneumocystis jirovecii
is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the
P. jirovecii
genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model
Pneumocystis carinii
, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of
P. jirovecii
, the dihydrofolate synthase (
dhfs
) and the aminodeoxychorismate lyase (
abz2
). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of
Saccharomyces cerevisiae
.
ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence
In Vivo
Authors: Perdu Caroline , Huber Philippe , Bouillot Stéphanie , Blocker Ariel , Elsen Sylvie , Attrée Ina , Faudry Eric 
Published: February 2015

ABSTRACT

Pseudomonas aeruginosa
is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the
exsB
gene within the
exsCEBA
regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with
yscW
from
Yersinia
spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full
in vivo
virulence of
P. aeruginosa
. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and
in vitro
assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.
Transforming Growth Factor β2 Promotes Transcription of
COX2
and
EP4
, Leading to a Prostaglandin E
2
-Driven Autostimulatory Loop That Enhances Virulence of Theileria annulata-Transformed Macrophages
Authors: Haidar Malak , Echebli Nadia , Ding Ying , Kamau Everlyn , Langsley Gordon 
Published: February 2015

ABSTRACT

Transforming growth factor beta (TGF-β) is a pleiotropic cytokine known to regulate cell growth, differentiation, and motility and is a potent modulator of immune function. TGF-β consequently plays a central role in carcinogenesis, and a dampened TGF-β2 response by
Theileria annulata
-infected monocytes/macrophages underpins disease resistance to tropical theileriosis. Here, we show that concomitant with the loss of TGF-β2 production, there is ablated expression of
COX2
and
EP4
, which leads to a drop in cyclic AMP (cAMP) levels and, consequently, reduced activation of protein kinase A (PKA) and EPAC. This ablated phenotype can be rescued in attenuated macrophages by the addition of exogenous TGF-β2, which reactivates the expression of COX2 and EP4 while repressing that of protein kinase inhibitor gamma (PKIG) to the levels in virulent macrophages. TGF-β2 therefore promotes the adhesion and invasiveness of virulent macrophages by modulating
COX2
,
EP4
, and
PKIG
transcription to initiate a prostaglandin E
2
(PGE2)-driven autostimulatory loop that augments PKA and EPAC activities. A virulence phenotype stemming from the double activation of PKA and EPAC is the induction of a CREB-mediated transcriptional program and the upregulation of JAM-L- and integrin 4αβ1-mediated adhesion of
Theileria
-infected macrophages.
Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts
Authors: Sultan Syed Z. , Sekar Padmapriya , Zhao Xiaowei , Manne Akarsh , Liu Jun , Wooten R. Mark , Motaleb M. A. 
Published: February 2015

ABSTRACTBorrelia burgdorferimust migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotileflaBmutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized amotBmutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotBbacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotBcells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotBcells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts.
Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus
Authors: Sheehan Lauren M. , Budnick James A. , Roop R. Martin , Caswell Clayton C. 
Published: February 2015

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.
Gammaherpesvirus Small Noncoding RNAs Are Bifunctional Elements That Regulate Infection and Contribute to Virulence
In Vivo
Authors: Diebel Kevin W. , Oko Lauren M. , Medina Eva M. , Niemeyer Brian F. , Warren Cody J. , Claypool David J. , Tibbetts Scott A. , Cool Carlyne D. , Clambey Eric T. , van Dyk Linda F. 
Published: February 2015

ABSTRACT

Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi’s sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes
in vitro
, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication
in vitro
and normal establishment of latency in B cells early following acute infection
in vivo
. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis.

IMPORTANCE
The gammaherpesviruses (γHVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection
in vivo
. Here, we generated a mouse γHV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that γHV ncRNAs can directly contribute to virus-induced disease
in vivo
and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs.
Identification of Mammalian Proteins That Collaborate with Type III Secretion System Function: Involvement of a Chemokine Receptor in Supporting Translocon Activity
Authors: Sheahan Kerri-Lynn , Isberg Ralph R. 
Published: February 2015

ABSTRACT

The type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, including
Yersinia pseudotuberculosis
. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from a

Ccr5
−/−

knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis.

IMPORTANCE
Many Gram-negative bacteria require type III secretion systems (T3SS) for host survival, making these highly specialized secretion systems good targets for antimicrobial agents. After the bacterium binds to host cells, T3SS deposit proteins into the cytosol of host cells through a needle-like appendage and a protein translocon channel. Translocation of proteins via this system is highly regulated, and the contribution of the host cell in promoting assembly and insertion of the channel into the plasma membrane, folding of the bacterial proteins, and trafficking of these substrates are all poorly characterized events. In this study, we identified host cell proteins important for activity of YopE, a
Yersinia pseudotuberculosis
T3SS-delivered protein. The results demonstrate that insertion and assembly of the translocon are complex processes, requiring a variety of membrane trafficking and cytoskeletal processes, as well as a surprising role for cell surface signaling molecules in supporting proper function.
E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78
Authors: Ajiro Masahiko , Zheng Zhi-Ming 
Published: February 2015

ABSTRACT
Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.

IMPORTANCE
HPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicing
cis
elements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.
Inflammasome Activation in Response to the Yersinia Type III Secretion System Requires Hyperinjection of Translocon Proteins YopB and YopD
Authors: Zwack Erin E. , Snyder Annelise G. , Wynosky-Dolfi Meghan A. , Ruthel Gordon , Philip Naomi H. , Marketon Melanie M. , Francis Matthew S. , Bliska James B. , Brodsky Igor E. 
Published: February 2015

ABSTRACT

Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with
Yersinia pseudotuberculosis
strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as Δ
yopK
strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS.

IMPORTANCE
The innate immune response is critical to effective clearance of pathogens. Recognition of conserved virulence structures and activities by innate immune receptors such as NLRs constitute one of the first steps in mounting the innate immune response. However, pathogens such as Yersinia actively evade or subvert components of host defense, such as inflammasomes. The T3SS-secreted protein YopK is an essential virulence factor that limits translocation of other Yops, thereby limiting T3SS-induced inflammasome activation. However, what triggers inflammasome activation in cells infected by YopK-deficient Yersinia is not clear. Our findings indicate that hypertranslocation of pore complex proteins promotes inflammasome activation and that YopK prevents inflammasome activation by the T3SS by limiting translocation of YopD and YopB themselves.
Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism
Authors: Maglica Željka , Özdemir Emre , McKinney John D. 
Published: February 2015

ABSTRACT

ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in
Mycobacterium smegmatis
based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening.

IMPORTANCE
New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The method described here for single-cell tracking of intracellular ATP in live bacteria has many advantages compared to conventional ensemble-averaged assays. It provides a continuous real-time readout of bacterial ATP content, cell vitality, and antimicrobial mechanism of action with high temporal resolution at the single-cell level. In combination with high-throughput microfluidic devices and automated microscopy, this method also has the potential to serve as a novel screening tool in antimicrobial drug discovery.