bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterosubtypic Protection Conferred by the Human Monoclonal Antibody PN-SIA28 against Influenza A Virus Lethal Infections in Mice
Authors: Retamal Miguel , Abed Yacine , Rhéaume Chantal , Cappelletti Francesca , Clementi Nicola , Mancini Nicasio , Clementi Massimo , Burioni Roberto , Boivin Guy
Published: February 2015

PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011,http://dx.doi.org/10.1371/journal.pone.0028001). Previousin vitrostudies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypesin vitrobut also, more importantly, protect from a lethal influenza virus challengein vivo.
Characterization of Tn
6238
with a New Allele of
aac(6′)-Ib-cr
Authors: Quiroga María P. , Orman Betina , Errecalde Laura , Kaufman Sara , Centrón Daniela
Published: February 2015

Here, we report that the genetic structure of Tn
1331
remained conserved in Argentina from 1989 to 2013 (72 of 73 isolates), with the exception being the plasmid-borne Tn
1331
-like transposon Tn
6238
containing a new
aac(6′)-Ib-cr
allele recovered from a colistin-resistant
Klebsiella pneumoniae
clinical isolate. A bioinformatic analysis of
aac(6′)-Ib
-like gene cassettes suggests that this new
aac(6′)-Ib-cr
allele emerged through mutation or homologous recombination in the Tn
1331
genetic platform. Tn
6238
is a novel platform for the dissemination of aminoglycoside and fluoroquinolone resistance determinants.
In VitroPharmacodynamics of Various Antibiotics in Combination against Extensively Drug-Resistant Klebsiella pneumoniae
Authors: Lim Tze-Peng , Cai Yiying , Hong Yanjun , Chan Eric Chun Yong , Suranthran Sasikala , Teo Jocelyn Qi-Min , Lee Winnie Huiling , Tan Thean-Yen , Hsu Li-Yang , Koh Tse-Hsien , Tan Thuan-Tong , Kwa Andrea Lay-Hoon
Published: February 2015

Extensively drug-resistant (XDR)Klebsiella pneumoniaeis an emerging pathogen in Singapore. With limited therapeutic options available, combination antibiotics may be the only viable option. In this study, we aimed to elucidate effective antibiotic combinations against XDRK. pneumoniaeisolates. Six NDM-1-producing and two OXA-181-producingK. pneumoniaestrains were exposed to 12 antibiotics alone and in combination via time-kill studies. A hollow-fiber infection model (HFIM) with pharmacokinetic validation was used to simulate clinically relevant tigecycline-plus-meropenem dosing regimens against 2 XDRK. pneumoniaeisolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. Thein vitrogrowth rates were determined and serial passages on drug-free and selective media were carried out on resistant isolates obtained at 240 h. Both the polymyxin B and tigecycline MICs ranged from 1 to 4 mg/liter. In single time-kill studies, all antibiotics alone demonstrated regrowth at 24 h, except for polymyxin B against 2 isolates. Tigecycline plus meropenem was found to be bactericidal in 50% of the isolates. For the isolates that produced OXA-181-like carbapenemases, none of the 55 tested antibiotic combinations was bactericidal. Against 2 isolates in the HFIM, tigecycline plus meropenem achieved a >90% reduction in bacterial burden for 96 h before regrowth was observed until 109CFU/ml at 240 h. Phenotypically stable and resistant isolates, which were recovered from tigecycline-supplemented plates post-HFIM studies, had lower growth rates than those of their respective parent isolates, possibly implying a substantial biofitness deficit in this population. We found that tigecycline plus meropenem may be a potential antibiotic combination for XDRK. pneumoniaeinfections, but its efficacy was strain specific.
Surveillance of Artemisinin Resistance in Plasmodium falciparum in India Using the kelch13 Molecular Marker
Authors: Mishra Neelima , Prajapati Surendra Kumar , Kaitholia Kamlesh , Bharti Ram Suresh , Srivastava Bina , Phookan Sobhan , Anvikar Anupkumar R. , Dev Vas , Sonal Gagan Singh , Dhariwal Akshay Chandra , White Nicholas J. , Valecha Neena
Published: February 2015

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant
Plasmodium falciparum
. Genome association studies have strongly linked a locus on
P. falciparum
chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (
Pfk-13
) were strongly linked to resistance. To date, this information has not been shown in Indian samples.
Pfk-13
mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of
Pfk-13
point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.
Ambush of Clostridium difficile Spores by Ramoplanin: Activity in an
In Vitro
Model
Authors: Kraus Carl N. , Lyerly Matthew W. , Carman Robert J.
Published: February 2015

Clostridium difficile
infection (CDI) is a gastrointestinal disease caused by
C. difficile
, a spore-forming bacterium that in its spore form is tolerant to standard antimicrobials. Ramoplanin is a glycolipodepsipeptide antibiotic that is active against
C. difficile
with MICs ranging from 0.25 to 0.50 μg/ml. The activity of ramoplanin against the spores of
C. difficile
has not been well characterized; such activity, however, may hold promise, since posttreatment residual intraluminal spores are likely elements of disease relapse, which can impact more than 20% of patients who are successfully treated.
C. difficile
spores were found to be stable in deionized water for 6 days.
In vitro
spore counts were consistently below the level of detection for 28 days after even brief (30-min) exposure to ramoplanin at concentrations found in feces (300 μg/ml). In contrast, suppression of spore counts was not observed for metronidazole or vancomycin at human fecal concentrations during treatment (10 μg/ml and 500 μg/ml, respectively). Removal of the
C. difficile
exosporium resulted in an increase in spore counts after exposure to 300 μg/ml of ramoplanin. Therefore, we propose that rather than being directly sporicidal, ramoplanin adheres to the exosporium for a prolonged period, during which time it is available to attack germinating cells. This action, in conjunction with its already established bactericidal activity against vegetative
C. difficile
forms, supports further evaluation of ramoplanin for the prevention of relapse after
C. difficile
infection in patients.
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

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

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

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

Borrelia 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

Metal 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

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

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

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

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

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.
Differential Expression and Roles of Staphylococcus aureus Virulence Determinants during Colonization and Disease
Authors: Jenkins Amy , Diep Binh An , Mai Thuy T. , Vo Nhung H. , Warrener Paul , Suzich Joann , Stover C. Kendall , Sellman Bret R.
Published: February 2015

Staphylococcus aureus
is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of
sdrC
,
fnbA
,
fhuD
,
sstD
, and
hla
was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens.

IMPORTANCE
Many bacteria, such as
Staphylococcus aureus
, asymptomatically colonize human skin and nasal passages but can also cause invasive diseases, such as bacteremia, pneumonia, sepsis, and osteomyelitis. The goal of this study was to analyze differences in the expression of selected S. aureus genes during a commensal lifestyle and as an invasive pathogen to gain insight into the commensal-to-pathogen transition and how a bacterial pathogen adapts to different environments within a host (e.g., from nasal colonization to invasive pathogen). The gene expression data were also used to select genes for which to construct knockout mutants to assess the role of several proteins in nasal colonization and lethal bacteremia. These results not only provide insight into the factors involved in S. aureus disease pathogenesis but also provide potential therapeutic targets.
In Vivo
Transcriptional Profiling of Yersinia pestis Reveals a Novel Bacterial Mediator of Pulmonary Inflammation
Authors: Pechous Roger D. , Broberg Christopher A. , Stasulli Nikolas M. , Miller Virginia L. , Goldman William E.
Published: February 2015

Inhalation of
Yersinia pestis
results in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed the
in vivo
transcription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes,
ybtX
, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion of
ybtX
in another lethal respiratory pathogen,
Klebsiella pneumoniae
, also resulted in diminished host inflammation during infection. Thus, our
in vivo
transcriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia.

IMPORTANCE
Yersinia pestis is responsible for at least three major pandemics, most notably the Black Death of the Middle Ages. Due to its pandemic potential, ease of dissemination by aerosolization, and a history of its weaponization, Y. pestis is categorized by the Centers for Disease Control and Prevention as a tier 1 select agent most likely to be used as a biological weapon. To date, there is no licensed vaccine against Y. pestis. Importantly, an early “silent” phase followed by the rapid onset of nondescript influenza-like symptoms makes timely treatment of pneumonic plague difficult. A more detailed understanding of the bacterial and host factors that contribute to pathogenesis is essential to understanding the progression of pneumonic plague and developing or enhancing treatment options.
Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
Authors: Chen Allan L. , Kim Elliot W. , Toh Justin Y. , Vashisht Ajay A. , Rashoff Andrew Q. , Van Christina , Huang Amy S. , Moon Andy S. , Bell Hannah N. , Bentolila Laurent A. , Wohlschlegel James A. , Bradley Peter J.
Published: February 2015

The inner membrane complex (IMC) of
Toxoplasma gondii
is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii.

IMPORTANCE
The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an
in vivo
biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.
The Short-Term Effects of Acupuncture on Patients with Diabetic Gastroparesis: A Randomised Crossover Study
Authors: Li Guomin , Huang Chengfei , Zhang Xuezhu , Xie Hui , Cheng Haiyan , Tang Yinshan , Li Zhigang
Published: February 2015

Background

Acupuncture may improve gastrointestinal symptoms in patients with various disorders, but its efficacy in diabetic gastroparesis is unclear.

Objective

To evaluate the short-term effects of acupuncture on gastric emptying and gastroparesis symptoms in patients with diabetic gastroparesis.

Methods

In a single-blind controlled crossover trial, 25 patients with diabetic gastroparesis were randomly assigned to undergo 1 week of real acupuncture (RA) treatment followed by 1 week of sham acupuncture (SA) treatment, or vice versa, with a washout of 1 month in between. Before and after each intervention, gastric retention, the Gastroparesis Cardinal Symptom Index (GCSI), fasting blood glucose (FBG) and HbA1c levels were measured.

Results

21 patients completed the study. Compared with SA treatment, RA treatment was associated with significantly greater reductions in gastric retention at 2 h (−11.1±7.0%; 95% CI −13.6% to −6.2%; p<0.01) and at 4 h (−5.0±2.8%; 95% CI −6.0% to −0.2%; p=0.04) and in GCSI score (−8.0±3.4; 95% CI −8.4 to −2.8; p<0.01). There were no significant differences in FBG and HbA1c levels between RA and SA treatments.

Conclusions

In patients with diabetic gastroparesis, 1 week of short-term manual acupuncture reduces gastric retention and improves gastroparesis symptoms.
Napping, development and health from 0 to 5 years: a systematic review
Authors: Thorpe Karen , Staton Sally , Sawyer Emily , Pattinson Cassandra , Haden Catherine , Smith Simon
Published: February 2015

Background

Duration and quality of sleep affect child development and health. Encouragement of napping in preschool children has been suggested as a health-promoting strategy.

Objectives

The aim of this study is to assess evidence regarding the effects of napping on measures of child development and health.

Design

This study is a systematic review of published, original research articles of any design.

Subjects

Children aged 0–5 years.

Method

Electronic database search was performed following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and assessment of research quality was carried out following a Grading of Recommendations, Assessment, Development and Evaluations (GRADE) protocol.

Results

Twenty-six articles met inclusion criteria. These were of heterogeneous quality; all had observational designs (GRADE-low). Development and health outcomes included salivary cortisol, night sleep, cognition, behaviour, obesity and accidents. The findings regarding cognition, behaviour and health impacts were inconsistent, probably because of variation in age and habitual napping status of the samples. The most consistent finding was an association between napping and later onset, shorter duration and poorer quality of night sleep, with evidence strongest beyond the age of 2 years.

Limitations

Studies were not randomised. Most did not obtain data on the children's habitual napping status or the context of napping. Many were reliant on parent report rather than direct observation or physiological measurement of sleep behaviour.

Conclusions

The evidence indicates that beyond the age of 2 years napping is associated with later night sleep onset and both reduced sleep quality and duration. The evidence regarding behaviour, health and cognition is less certain. There is a need for more systematic studies that use stronger designs. In preschool children presenting with sleep problems clinicians should investigate napping patterns.
Pulmonary function as a risk factor for dementia death: an individual participant meta-analysis of six UK general population cohort studies
Authors: Russ Tom C , Starr John M , Stamatakis Emmanuel , Kivimäki Mika , Batty G David
Published: February 2015

Background

In addition to being associated with all-cause mortality and cardiovascular disease mortality, lung function has been linked with dementia. However, existing studies typically provide imprecise estimates due to small numbers of outcome events and are based on unrepresentative samples of the general population.

Methods

Individual participant meta-analysis of six cohort studies from the Health Survey for England and the Scottish Health Survey (total N=54 671). Dementia-related mortality was identified by mention of dementia on any part of the death certificate (mean follow-up 11.7 years). Study-specific Cox proportional hazard models of the association between lung function and dementia-related death were pooled using random effect meta-analysis to produce overall results.

Results

There was a dose–response association between poorer lung function and a higher risk of dementia-related death (age- and sex-adjusted HR compared to highest quartile of forced expiratory volume in 1 s (FEV1), 95% CI: second quartile 1.32, 0.99 to 1.76; third quartile 1.78, 1.30 to 2.43; fourth (lowest) quartile 2.74, 1.73 to 4.32). There was no significant heterogeneity in study-specific estimates (I2=0%). Controlling for height, socioeconomic status, smoking and general health attenuated but did not remove the association (second quartile 1.15, 0.82 to 1.62; third quartile 1.37, 0.96 to 1.94; fourth quartile 2.09, 1.17 to 3.71). Results for forced vital capacity and peak flow were similar.

Conclusions

In these general population samples, the relation between three measures of lung function and dementia death followed a dose–response gradient. Being in the bottom quartile of lung function was associated with a doubling of the risk.
Outcome of oesophagogastric carcinoma in young patients
Authors: Bowrey D J , Clark G W B , Rees B I , Williams G T , Carey P D
Published: February 2015

The survival of young patients (⩽ 50 years of age) with carcinoma of the oesophagus or stomach has been reported to be poorer than that of their older counterparts. The aim of the current study was to review the outcome of such young patients with oesophagogastric cancer and to compare the outcome in patients with carcinoma of the oesophagus/cardia with patients with carcinoma of the more distal stomach. The study population was 50 patients. Tumour location was oesophagus/cardia (n=33) and gastric body/antrum (n=17). The most common presenting symptoms were weight loss (66%), epigastric pain (54%), dysphagia (50%), and heartburn (40%). Seventeen patients had experienced foregut symptoms for a period of ⩾6 months. These patients were more likely to have symptoms of gastro-oesophageal reflux disease and to have received acid suppression therapy than patients with shorter symptom durations. Only 20 patients underwent a potentially curative resection, while 10 underwent open and close laparotomy. The overall median survival was 7 months and the 5-year survival was 8%. Multivariate analysis revealed that surgical resection and UICC stage were the only factors that significantly influenced survival. There was no difference in the survival of patients with proximally situated tumours compared to those with distally located tumours. Wide variations in clinical practice were seen between different surgeons. Consequently, a multidisciplinary team designed to manage all patients with oesophagogastric cancer according to nationally agreed protocols has been established in our hospital. Earlier diagnosis of these tumours is to be encouraged, even if this necessitates the more liberal use of endoscopy in the evaluation of young patients with persistent foregut symptoms.
Hyponatraemia and seizures after ecstasy use
Authors: Holmes Simon B , Banerjee Anindo K , Alexander William D
Published: February 2015

A patient presented to our unit with seizures and profound hyponatraemia after ingestion of a single tablet of ecstasy. The seizures proved resistant to therapy and ventilation on the intensive care unit was required. Resolution of the seizures occurred on correction of the metabolic abnormalities. The pathogenesis of seizures and hyponatraemia after ecstasy use is discussed. Ecstasy use should be considered in any young patient presenting with unexplained seizures and attention should be directed towards electrolyte levels, particularly sodium.
A case of ureteric obstruction, retroperitoneal fibrosis, and carcinoid tumour
Authors: Dev S , Al-Dujaily S , Subbuswamy S G
Published: February 2015

We report the incidental finding at surgery for retroperitoneal fibrosis of a carcinoid tumour causing complete right ureteric obstruction. Retroperitoneal fibrosis is an uncommon inflammatory disease that leads to extensive fibrosis throughout the retroperitoneum. It can occur at any age, peak incidence being in patients between 40 and 60 years of age. Carcinoid tumours arise from enterochromaffin or amine precursor uptake and decarboxylation cells that occur in gastrointestinal tract. Carcinoid tumours are an uncommon clinical entity and incidence varies with gender and age. No association between retroperitoneal fibrosis and carcinoid tumour has been previously reported in the English literature, although one case has been reported in a French journal.