bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative to Quinine and Quinidine, Their 9-Epimers Exhibit Decreased Cytostatic Activity and Altered Heme Binding but Similar Cytocidal Activity versus Plasmodium falciparum
Authors: Gorka Alexander P. , Sherlach Katy S. , de Dios Angel C. , Roepe Paul D.
Published: November 2012

The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against
P. falciparum
(Karle JM, Karle IL, Gerena L, Milhous WK, Antimicrob. Agents Chemother. 36:1538–1544, 1992). We synthesized 9-
epi
-QN (eQN) and 9-
epi
-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to the cytostatic activity of QN and QD, we observed a large decrease in cytostatic activity (higher 50% inhibitory concentration [IC
50
s]) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observed relatively small changes in cytocidal activity (the 50% lethal dose), similar to observations with chloroquine (CQ) analogues with a wide range of IC
50
s (see the accompanying paper [
A. P. Gorka, J. N. Alumasa, K. S. Sherlach, L. M. Jacobs, K. B. Nickley, J. P. Brower, A. C. de Dios, and P. D. Roepe
, Antimicrob. Agents Chemother. 57:356–364, 2013]). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed that the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-visible (VIS) titrations showed that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicate that binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with a 2:1 stoichiometry (drug to FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic versus cytocidal dosages of quinoline antimalarial drugs and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes.
Characterization of Recombinant Fluoroquinolone-Resistant Pneumococcus-Like Isolates
Authors: Balsalobre Luz , Ortega Montserrat , de la Campa Adela G.
Published: November 2012

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of
Streptococcus pneumoniae
,
Streptococcus mitis
,
Streptococcus oralis
, and
Streptococcus pseudopneumoniae
. Four isolates grouped with
S. pneumoniae
, seven grouped with
S. pseudopneumoniae
, and three grouped with
S. mitis
. These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the
atp
operon chromosomal region, encoding the F
o
F
1
H
+
-ATPase (the target of optochin). All seven isolates grouping with
S. pseudopneumoniae
share the same
spr1368-atpC-atpA
gene order; all four grouping with
S. pneumoniae
share the
spr1368
-IS
1239-atpC-atpA
order, and two out of the three grouping with
S. mitis
share the
spr1284-atpC-atpA
order. In addition, evidence for recombination within the seven housekeeping alleles of the
S. pseudopneumoniae
population was provided by several methods: the index of association (0.4598,
P
< 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and
S. pseudopneumoniae
.
Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections
Authors: Martín-Navarro Carmen María , Lorenzo-Morales Jacob , Machin Rubén P. , López-Arencibia Atteneri , García-Castellano José Manuel , de Fuentes Isabel , Loftus Brendan , Maciver Sutherland K. , Valladares Basilio , Piñero José E.
Published: November 2012

Acanthamoeba
is an opportunistic pathogen in humans, whose infections most commonly manifest as
Acanthamoeba
keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of
Acanthamoeba
, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for
Acanthamoeba
therapeutics. Ergosterol is one of the major sterols in the membrane of
Acanthamoeba
. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in
Acanthamoeba
, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of
Acanthamoeba
HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated
in vitro
against three clinical strains of
Acanthamoeba
by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and
Acanthamoeba
treatment using statins is a novel powerful treatment option against
Acanthamoeba
species in human disease.
Pharmacodynamics of Anidulafungin against Clinical Aspergillus fumigatus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis
Authors: Seyedmousavi Seyedmojtaba , Brüggemann Roger J. M. , Melchers Willem J. G. , Verweij Paul E. , Mouton Johan W.
Published: November 2012

Azole resistance is an emerging increasing problem in
Aspergillus fumigatus
that results in treatment failure. Alternative treatments may improve the therapeutic outcome in patients with azole-resistant invasive aspergillosis (IA). Little is known about the
in vivo
efficacy of the echinocandin anidulafungin (AFG) in IA. The
in vivo
efficacy of 2.5, 5, 10, and 20 mg/kg of body weight AFG was assessed against two clinical
Aspergillus fumigatus
isolates with identical AFG minimum effective concentrations (MECs; 0.03 mg/liter) in a murine model of IA: a wild-type voriconazole (VCZ)-susceptible (VCZ
s
)
A. fumigatus
isolate (AZN 8196) and a VCZ-resistant (VCZ
r
)
A. fumigatus
isolate (V52-35) harboring the TR
34
/L98H resistance mechanism (substitution at codon L98 in combination with a 34-bp tandem repeat in the promoter region of the
CYP51A
gene). The pharmacokinetics of AFG were also assessed for each dose. Increasing doses increased survival for both isolates in a manner dependent on the AFG dose level (
R
2
= 0.99 and 0.95, respectively) up to a maximum of 72.7% and 45.45% for the VCZ
s
and VCZ
r
isolates, respectively. The area under the concentration-time curve (AUC) correlated significantly with the dose in a linear fashion over the entire dosing range (
R
2
= 0.86). The Hill equation with a variable slope fitted the relationship between the 24-h AUC/MEC ratio and 14-day survival well (
R
2
= 0.87;
P
< 0.05). The 50% effective AUC/MEC for total AFG was 126.5 (95% confidence interval, 79.09 to 202.03). AFG treatment improved the survival of mice in a dose-dependent manner; however, a maximal response was not achieved with either isolate even in those treated with the highest AFG dose.
Whole-Genome Analysis of a Daptomycin-Susceptible Enterococcus faecium Strain and Its Daptomycin-Resistant Variant Arising during Therapy
Authors: Tran Truc T. , Panesso Diana , Gao Hongyu , Roh Jung H. , Munita Jose M. , Reyes Jinnethe , Diaz Lorena , Lobos Elizabeth A. , Shamoo Yousif , Mishra Nagendra N. , Bayer Arnold S. , Murray Barbara E. , Weinstock George M. , Arias Cesar A.
Published: November 2012

Development of daptomycin (DAP) resistance in
Enterococcus faecalis
has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [
cls
]). However, the genetic bases for DAP resistance in
Enterococcus faecium
are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible
E. faecium
S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in
E. faecium
R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the “susceptible”
cls
allele from S447 for the “resistant” one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some
E. faecium
strains.
Low-Level Persistence of Drug Resistance Mutations in Hepatitis B Virus-Infected Subjects with a Past History of Lamivudine Treatment
Authors: Margeridon-Thermet Severine , Svarovskaia Evguenia S. , Babrzadeh Farbod , Martin Ross , Liu Tommy F. , Pacold Mary , Reuman Elizabeth C. , Holmes Susan P. , Borroto-Esoda Katyna , Shafer Robert W.
Published: November 2012

We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%;P= 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%;P= 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P< 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.
In Vitro
Activity and Durability of a Combination of an Antibiofilm and an Antibiotic against Vascular Catheter Colonization
Authors: Mansouri Mohammad D. , Hull Richard A. , Stager Charles E. , Cadle Richard M. , Darouiche Rabih O.
Published: November 2012

Catheter-associated infections can cause severe complications and even death. Effective antimicrobial modification of catheters that can prevent device colonization has the potential of preventing clinical infection. We studied
in vitro
the antimicrobial activities of central venous catheters impregnated with
N
-acetylcysteine (NAC), an antibiofilm agent, and a broad-spectrum antibiotic against a range of important clinical pathogens. NAC-levofloxacin-impregnated (NACLEV) catheters were also evaluated for their antiadherence activity. NACLEV catheters produced the most active and durable antimicrobial effect against both Gram-positive and Gram-negative isolates and significantly reduced colonization (
P
< 0.0001) by all tested pathogens compared to control catheters. These
in vitro
results suggest that this antimicrobial combination can potentially be used to combat catheter colonization and catheter-associated infection.
Cytostatic versus Cytocidal Activities of Chloroquine Analogues and Inhibition of Hemozoin Crystal Growth
Authors: Gorka Alexander P. , Alumasa John N. , Sherlach Katy S. , Jacobs Lauren M. , Nickley Katherine B. , Brower Jonathan P. , de Dios Angel C. , Roepe Paul D.
Published: November 2012

We report an improved, nonhazardous, high-throughput assay for
in vitro
quantification of antimalarial drug inhibition of β-hematin (hemozoin) crystallization performed under conditions that are more physiological relative to previous assays. The assay uses the differential detergent solubility of crystalline and noncrystalline forms of heme and is optimized via the use of lipid catalyst. Using this assay, we quantify the effect of pH on the crystal growth-inhibitory activities of current quinoline antimalarials, evaluate the catalytic efficiencies of different lipids, and test for a possible correlation between hemozoin inhibition by drugs versus their antiplasmodial activity. Consistent with several previous reports, we found a good correlation between hemozoin inhibition potency versus cytostatic antiplasmodial potency (50% inhibitory concentration) for a series of chloroquine (CQ) analogues. However, we found no correlation between hemozoin inhibition potency and cytocidal antiplasmodial potency (50% lethal dose) for the same drugs, suggesting that cellular targets for these two layers of 4-aminoquinoline drug activity differ. This important concept is also explored further for QN and its stereoisomers in the accompanying paper (
A. P. Gorka, K. S. Sherlach, A. C. de Dios, and P. D. Roepe
, Antimicrob. Agents Chemother. 57:365–374, 2013).
GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues
Authors: Bebrone Carine , Bogaerts Pierre , Delbrück Heinrich , Bennink Sandra , Kupper Michaël B. , Rezende de Castro Roberta , Glupczynski Youri , Hoffmann Kurt M.
Published: November 2012

A clinical isolate of
Pseudomonas aeruginosa
recovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical β-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1.
Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens
Authors: Berthold Nicole , Czihal Patricia , Fritsche Stefanie , Sauer Ute , Schiffer Guido , Knappe Daniel , Alber Gottfried , Hoffmann Ralf
Published: November 2012

Proline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic
Escherichia coli
strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its
in vitro
characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with
l
-ornithine or
l
-homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is
N
,
N
,
N
′,
N
′-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and
d
-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an
E. coli
strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient
in vivo
than Api88.
Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis
Authors: Gannoun-Zaki Laila , Alibaud Laeticia , Kremer Laurent
Published: November 2012

The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in
Mycobacterium tuberculosis
. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.
Outcome of Debridement and Retention in Prosthetic Joint Infections by Methicillin-Resistant Staphylococci, with Special Reference to Rifampin and Fusidic Acid Combination Therapy
Authors: Peel T. N. , Buising K. L. , Dowsey M. M. , Aboltins C. A. , Daffy J. R. , Stanley P. A. , Choong P. F. M.
Published: November 2012

The management of prosthetic joint infections remains a clinical challenge, particularly infections due to methicillin-resistant staphylococci. Previously, this infection was considered a contraindication to debridement and retention strategies. This retrospective cohort study examined the treatment and outcomes of patients with arthroplasty infection by methicillin-resistant staphylococci managed by debridement and retention in conjunction with rifampin-fusidic acid combination therapy. Over an 11-year period, there were 43 patients with infection by methicillin-resistant staphylococci managed with debridement and retention. This consisted of close-interval repeated arthrotomies with pulsatile lavage. Rifampin was combined with fusidic acid for the majority of patients (88%). Patients were monitored for a median of 33.5 months (interquartile range, 20 to 54 months). Overall, 9 patients experienced treatment failure, with 12- and 24-month estimates of infection-free survival of 86% (95% confidence interval [CI], 71 to 93%) and 77% (95% CI, 60 to 87%), respectively. The following factors were associated with treatment failure: methicillin-resistant
Staphylococcus aureus
(MRSA) arthroplasty infection, a single surgical debridement or ≥4 debridements, and the receipt of less than 90 days of antibiotic therapy. Patients with infection by methicillin-resistant coagulase-negative staphylococci (MR-CNS) were less likely to fail treatment. The overall treatment success rate reported in this study is comparable to those of other treatment modalities for prosthetic joint infections by methicillin-resistant staphylococci. Therefore, the debridement and retention of the prosthesis and rifampin-based antibiotic therapy are a valid treatment option for carefully selected patients.
Additional Diagnostic Yield of Adding Serology to PCR in Diagnosing Viral Acute Respiratory Infections in Kenyan Patients 5 Years of Age and Older
Authors: Feikin Daniel R. , Njenga M. Kariuki , Bigogo Godfrey , Aura Barrack , Gikunju Stella , Balish Amanda , Katz Mark A. , Erdman Dean , Breiman Robert F.
Published: November 2012

The role of serology in the setting of PCR-based diagnosis of acute respiratory infections (ARIs) is unclear. We found that acute- and convalescent-phase paired-sample serologic testing increased the diagnostic yield of naso/oropharyngeal swabs for influenza virus, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, and parainfluenza viruses beyond PCR by 0.4% to 10.7%. Although still limited for clinical use, serology, along with PCR, can maximize etiologic diagnosis in epidemiologic studies.
Study of Avidity of Antigen-Specific Antibody as a Means of Understanding Development of Long-Term Immunological Memory after Vibrio cholerae O1 Infection
Authors: Alam Mohammad Murshid , Arifuzzaman Mohammad , Ahmad Shaikh Meshbahuddin , Hosen M. Ismail , Rahman Mohammad Arif , Rashu Rasheduzzaman , Sheikh Alaullah , Ryan Edward T. , Calderwood Stephen B. , Qadri Firdausi
Published: November 2012

The avidity of antibodies to specific antigens and the relationship of avidity to memory B cell responses to these antigens have not been studied in patients with cholera or those receiving oral cholera vaccines. We measured the avidity of antibodies to cholera toxin B subunit (CTB) andVibrio choleraeO1 lipopolysaccharide (LPS) in Bangladeshi adult cholera patients (n= 30), as well as vaccinees (n= 30) after administration of two doses of a killed oral cholera vaccine. We assessed antibody and memory B cell responses at the acute stage in patients or prior to vaccination in vaccinees and then in follow-up over a year. Both patients and vaccinees mounted CTB-specific IgG and IgA antibodies of high avidity. Patients showed longer persistence of these antibodies than vaccinees, with persistence lasting in patients up to day 270 to 360. The avidity of LPS-specific IgG and IgA antibodies in patients remained elevated up to 180 days of follow-up. Vaccinees mounted highly avid LPS-specific antibodies at day 17 (3 days after the second dose of vaccine), but the avidity waned rapidly to baseline by 30 days. We examined the correlation between antigen-specific memory B cell responses and avidity indices for both antigens. We found that numbers of CTB- and LPS-specific memory B cells significantly correlated with the avidity indices of the corresponding antibodies (P< 0.05; Spearman'sρ= 0.28 to 0.45). These findings suggest that antibody avidity after infection and immunization is a good correlate of the development and maintenance of memory B cell responses toVibrio choleraeO1 antigens.
Rational Design of Membrane Proximal External Region Lipopeptides Containing Chemical Modifications for HIV-1 Vaccination
Authors: Venditto Vincent J. , Watson Douglas S. , Motion Michael , Montefiori David , Szoka Francis C.
Published: November 2012

The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies.
Serologic Evidence of Pandemic Influenza Virus H1N1 2009 Infection in Cats in China
Authors: Su Shuo , Yuan Liguo , Li Huatao , Chen Jidang , Xie Jiexiong , Huang Zhen , Jia Kun , Li Shoujun
Published: November 2012

Infection of domestic cats with (H1N1) pandemic 2009 (pdm09) influenza A virus has recently been documented. In this paper, we report for the first time the sporadically current seroprevalence of (H1N1) pdm09 influenza A virus infection in cats in China. Thirteen of 1,080 sera were found positive by nucleoprotein (NP)-specific enzyme-linked immunosorbent assays (ELISAs) in different cat populations in southern China. It is very important to stress further surveillance of pandemic (H1N1) 2009 influenza A virus in cats in southern China.
Increased Susceptibility of Melanin-Concentrating Hormone-Deficient Mice to Infection with Salmonella enterica Serovar Typhimurium
Authors: Karagiannis Apostolos K. A. , Ziogas Dimitrios C. , Gras-Miralles Beatriz , Geiger Brenda M. , Nagel Jutta , Trebicka Estela , Najarian Robert , Cherayil Bobby J. , Kokkotou Efi
Published: November 2012

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to
Clostridium difficile
toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens,
Salmonella
enterocolitis (using
Salmonella enterica
serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.
Altered Toll-Like Receptor 9 Signaling in Mycobacterium avium subsp. paratuberculosis-Infected Bovine Monocytes Reveals Potential Therapeutic Targets
Authors: Arsenault Ryan J. , Li Yue , Maattanen Pekka , Scruten Erin , Doig Kimberley , Potter Andrew , Griebel Philip , Kusalik Anthony , Napper Scott
Published: November 2012

Mycobacterium aviumsubsp.paratuberculosisis the causative agent of Johne's disease in cattle. The complex, multifaceted interaction ofM. aviumsubsp.paratuberculosiswith its host includes dampening the ability of infected cells to respond to stimuli that promoteM. aviumsubsp.paratuberculosisclearance. By disrupting host defenses,M. aviumsubsp.paratuberculosiscreates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense againstM. aviumsubsp.paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate thatM. aviumsubsp.paratuberculosisinfection changes the responsiveness of bovine monocytes to TLR9 stimulation.M. aviumsubsp.paratuberculosisinhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked byM. aviumsubsp.paratuberculosisinfection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promoteM. aviumsubsp.paratuberculosisclearance. Indeed, Pyk2 signaling appears to be an important aspect ofM. aviumsubsp.paratuberculosisinfection, as Pyk2 inhibitors significantly reduce the number of intracellularM. aviumsubsp.paratuberculosisbacteria. The actions ofM. aviumsubsp.paratuberculosison TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.
Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli
Authors: Donovan Grant T. , Norton J. Paul , Bower Jean M. , Mulvey Matthew A.
Published: November 2012

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenicEscherichia coli(UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, thecyaAandcrpdeletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H2O2) and acid stress. In the mutant strains, H2O2resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.
Beclin 1 Is Required for Starvation-Enhanced, but Not Rapamycin-Enhanced, LC3-Associated Phagocytosis of Burkholderia pseudomallei in RAW 264.7 Cells
Authors: Li Xuelei , Prescott Mark , Adler Ben , Boyce John D. , Devenish Rodney J.
Published: November 2012

LC3-associated phagocytosis (LAP) of
Burkholderia pseudomallei
by murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infecting
B. pseudomallei
. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP of
B. pseudomallei
but that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent.
Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen
Authors: Verma Anita , McNichol Beth , Domínguez-Castillo Rocío I. , Amador-Molina Juan C. , Arciniega Juan L. , Reiter Karine , Meade Bruce D. , Ngundi Miriam M. , Stibitz Scott , Burns Drusilla L.
Published: November 2012

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation anthrax vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.
Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans
Authors: Cruz Melissa R. , Graham Carrie E. , Gagliano Bryce C. , Lorenz Michael C. , Garsin Danielle A.
Published: November 2012

The Gram-positive bacteriumEnterococcus faecalisand the fungusCandida albicansare both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using aCaenorhabditis elegansmodel of polymicrobial infection, we discovered thatE. faecalisandC. albicansnegatively impact each other's virulence. Much of the negative effect ofE. faecalisonC. albicanswas due to the inhibition ofC. albicanshyphal morphogenesis, a developmental program crucial toC. albicanspathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence inE. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibitC. albicansfilamentation in the context of anin vitrobiofilm. Finally, a screen of anE. faecalistransposon mutant library identified other genes required for suppression ofC. albicanshyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.
Identification and Characterization of the First Cholesterol-Dependent Cytolysins from Gram-Negative Bacteria
Authors: Hotze Eileen M. , Le Huynh M. , Sieber Jessica R. , Bruxvoort Christina , McInerney Michael J. , Tweten Rodney K.
Published: November 2012

The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from theFirmicutesandActinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which includeDesulfobulbus propionicustype species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) andEnterobacter lignolyticus(formerlyEnterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neitherD. propionicusnorE. lignolyticusis known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.
Growth of Candida albicans Cells on the Physiologically Relevant Carbon Source Lactate Affects Their Recognition and Phagocytosis by Immune Cells
Authors: Ene Iuliana V. , Cheng Shih-Chin , Netea Mihai G. , Brown Alistair J. P.
Published: November 2012

Candida albicansis a normal resident of the human gastrointestinal and urogenital tracts and also a prevalent fungal pathogen. During both commensalism and infection, it must match the immunological defenses of its host while adapting to environmental cues and the local nutrient status.C. albicansregularly colonizes glucose-poor niches, thereby depending upon alternative carbon sources for growth. However, most studies of host immune responses toC. albicanshave been performed on fungal cells grown on glucose, and the extent to which alternative physiologically relevant carbon sources impact innate immune responses has not been studied. The fungal cell wall is decorated with multifarious pathogen-associated molecular patterns and is the main target for recognition by host innate immune cells. Cell wall architecture is both robust and dynamic, and it is dramatically influenced by growth conditions. We found that growth ofC. albicanscells on lactate, a nonfermentative carbon source available in numerous anatomical niches, modulates their interactions with immune cells and the resultant cytokine profile. Notably, lactate-grownC. albicansstimulated interleukin-10 (IL-10) production while decreasing IL-17 levels, rendering these cells less visible to the immune system than were glucose-grown cells. This trend was observed in clinicalC. albicansisolates from different host niches and from different epidemiological clades. In addition, lactate-grownC. albicanscells were taken up by macrophages less efficiently, but they were more efficient at killing and escaping these phagocytic cells. Our data indicate that carbon source has a major impact upon theC. albicansinteraction with the innate immune system.
Using Major Outer Membrane Protein Typing as an Epidemiological Tool To Investigate Outbreaks Caused by Milk-Borne Campylobacter jejuni Isolates in California
Authors: Jay-Russell Michele T. , Mandrell Robert E. , Yuan Jean , Bates Anna , Manalac Rosa , Mohle-Boetani Janet , Kimura Akiko , Lidgard Janice , Miller William G.
Published: November 2012

We describe using major outer membrane protein (MOMP) typing as a screen to compare the
Campylobacter jejuni porA
gene sequences of clinical outbreak strains from human stool with the
porA
sequences of dairy farm strains isolated during two milk-borne campylobacteriosis outbreak investigations in California. The genetic relatedness of clinical and environmental strains with identical or closely related
porA
sequences was confirmed by multilocus sequence typing and pulsed-field gel electrophoresis analysis. The first outbreak involved 1,644
C. jejuni
infections at 11 state correctional facilities and was associated with consumption of pasteurized milk supplied by an on-site dairy (dairy A) at a prison in the central valley. The second outbreak involved eight confirmed and three suspect
C. jejuni
cases linked to consumption of commercial raw milk and raw chocolate colostrum at another central valley dairy (dairy B). Both dairies bottled fluid milk on the farm and distributed the finished product to off-site locations. Altogether,
C. jejuni
was isolated from 7 of 15 (46.7%) bovine fecal, 12 of 20 (60%) flush alley water, and 1 of 20 (5%) lagoon samples collected on dairy A. At dairy B,
C. jejuni
was cultured from 9 of 26 (34.6%) bovine fecal samples. Environmental strains indistinguishable from the clinical outbreak strains were found in five flush alley water samples (dairy A) and four bovine fecal samples (dairy B). The findings demonstrate that MOMP typing is a useful tool to triage environmental isolates prior to conducting more labor-intensive molecular typing methods.
Global Phylogeny of Shigella sonnei Strains from Limited Single Nucleotide Polymorphisms (SNPs) and Development of a Rapid and Cost-Effective SNP-Typing Scheme for Strain Identification by High-Resolution Melting Analysis
Authors: Sangal Vartul , Holt Kathryn E. , Yuan Jianfeng , Brown Derek J. , Filliol-Toutain Ingrid , Weill François-Xavier , Kim Dong-Wook , da Silveira Wanderley Dias , Pickard Derek , Thomson Nicholas R. , Parkhill Julian , Yu Jun
Published: November 2012

The current
Shigella sonnei
pandemic involves geographically associated, multidrug-resistant clones. This study has demonstrated that
S. sonnei
phylogeny can be accurately defined with limited single nucleotide polymorphisms (SNPs). By typing 6 informative SNPs using a high-resolution melting (HRM) assay, major
S. sonnei
lineages/sublineages can be identified as defined by whole-genome variation.
Acute Right-Sided Heart Failure Caused by Neisseria meningitidis
Authors: Taldir Guillaume , Parize Perrine , Arvis Philippe , Faisy Christophe
Published: November 2012

Meningococcal myocarditis is a rarely diagnosed infection and could be the consequence of primary invasive infection or late immunologic complications. An unusual presentation of meningococcemia in an immunocompetent adult is described, with
Neisseria meningitidis
identified as the cause of selective right-sided heart failure in a case of acute myocarditis.
Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes
Authors: Hussain Mazhar , Lu Guangjin , Torres Shessy , Edmonds Judith H. , Kay Brian H. , Khromykh Alexander A. , Asgari Sassan
Published: November 2012

Wolbachiaas an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown thatwMelpop andwMel strains ofWolbachiainhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g.,Plasmodiumand filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect ofWolbachiaon the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced inWolbachia-infectedAedes aegypticells (Aag2). However, the amount of secreted virus was significantly reduced in the presence ofWolbachia. Intrathoracic injections showed that replication of WNV inA. aegyptimosquitoes infected withwMel strain ofWolbachiawas not inhibited, whereaswMelPop strain ofWolbachiasignificantly reduced the replication of WNV in mosquitoes. Further, whenwMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low inWolbachia-free mosquitoes and were completely inhibited in the presence ofWolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in theWolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain ofWolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited inwMelPop strain ofWolbachia.
Mapping Viral Functional Domains for Genetic Diversity in Plants
Authors: Pita Justin S. , Roossinck Marilyn J.
Published: November 2012

Cucumber mosaic virus
(CMV) comprises numerous isolates with various levels of in-host diversity. Subgroup-distinctive features of the Fny and LS strains provided us with a platform to genetically map the viral control elements for genetic variation
in planta
. We found that both RNAs 1 and 2 controlled levels of genetic diversity, and further fine mapping revealed that the control elements of mutation frequency reside within the first 596 amino acids (aa) of RNA 1. The 2a/2b overlapping region of the 2a protein also contributed to control of viral genetic variation. Furthermore, the 3′ nontranslated region (NTR) of RNA 3 constituted a hot spot of polymorphism, where the majority of fixed mutations found in the population were clustered. The 2b gene of CMV, a viral suppressor of gene silencing, controls the abundance of the fixed mutants in the viral population via a host-dependent mechanism.