bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chlamydia muridarum Infection of Macrophages Elicits Bactericidal Nitric Oxide Production via Reactive Oxygen Species and Cathepsin B
Authors: Rajaram Krithika , Nelson David E. 
Published: May 2015

ABSTRACTThe ability of certain species ofChlamydiato inhibit the biogenesis of phagolysosomes permits their survival and replication within macrophages. The survival of macrophage-adapted chlamydiae correlates with the multiplicity of infection (MOI), and optimal chlamydial growth occurs in macrophages infected at an MOI of ≤1. In this study, we examined the replicative capacity ofChlamydia muridarumin the RAW 264.7 murine macrophage cell line at different MOIs.C. muridarumproductively infected these macrophages at low MOIs but yielded few viable elementary bodies (EBs) when macrophages were infected at a moderate (10) or high (100) MOI. While high MOIs caused cytotoxicity and irreversible host cell death, macrophages infected at a moderate MOI did not show signs of cytotoxicity until late in the infectious cycle. Inhibition of host protein synthesis rescuedC. muridarumin macrophages infected at a moderate MOI, implying that chlamydial growth was blocked by activated defense mechanisms. Conditioned medium from these macrophages was antichlamydial and contained elevated levels of interleukin 1β (IL-1β), IL-6, IL-10, and beta interferon (IFN-β). Macrophage activation depended on Toll-like receptor 2 (TLR2) signaling, and cytokine production required live, transcriptionally active chlamydiae. A hydroxyl radical scavenger and inhibitors of inducible nitric oxide synthase (iNOS) and cathepsin B also reversed chlamydial killing. High levels of reactive oxygen species (ROS) led to an increase in cathepsin B activity, and pharmacological inhibition of ROS and cathepsin B reduced iNOS expression. Our data demonstrate that MOI-dependent TLR2 activation of macrophages results in iNOS induction via a novel ROS- and cathepsin-dependent mechanism to facilitateC. muridarumclearance.
Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion
Authors: Govind Revathi , Fitzwater Leah , Nichols Rebekah 
Published: May 2015

ABSTRACT

Clostridium difficile
is a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenic
C. difficile
strains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation.
C. difficile
toxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE
142
) and 140 amino acids (TcdE
140
), respectively. TcdE
166
was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms on
C. difficile
cell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE
166
and TcdE
142
had a profound effect on cell viability compared to the TcdE
140
isoform. Similarly, TcdE
166
and TcdE
142
facilitated toxin release more efficiently than did TcdE
140
. The importance of these variations among TcdE isoforms and their role in toxin release are discussed.

IMPORTANCE
C. difficile
is a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains of
C. difficile
produce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are secreted from bacterial cells is not yet clear but involves TcdE, a holin-like protein. In this work, we show that TcdE could be translated from three different start codons, resulting in the production of three TcdE isoforms. Furthermore, we investigated the role of these isoforms in toxin release and cell lysis in
C. difficile
. An understanding of TcdE-dependent toxin secretion may be helpful for the development of strategies for preventing and treating
C. difficile
infections.
Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems
Authors: Burmistrz Michał , Dudek Bartosz , Staniec Dominika , Rodriguez Martinez Jose Ignacio , Bochtler Matthias , Potempa Jan , Pyrc Krzysztof 
Published: May 2015

ABSTRACT

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of
Archaea
and almost half of species of
Bacteria
.
Porphyromonas gingivalis
is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of
P. gingivalis
carry CRISPR arrays, suggesting that these arrays play an important function
in vivo
. Here we show that all four CRISPR arrays present in the
P. gingivalis
W83 genome are transcribed. For one of the arrays, we demonstrate
in vivo
activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3′ end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of
P. gingivalis
W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3′ terminus by the appropriate PAM element.

IMPORTANCE
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of
P. gingivalis
, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial communities and, consequently, the process of disease development and progression.
Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages
Authors: Monk Ian R. , Tree Jai J. , Howden Benjamin P. , Stinear Timothy P. , Foster Timothy J. 
Published: May 2015

ABSTRACT

Staphylococcus aureus
is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of
S. aureus
. Here, we describe the construction and characterization of the IMXXB series of
Escherichia coli
strains that mimic the type I adenine methylation profiles of
S. aureus
clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major
S. aureus
lineages.

IMPORTANCE
The genetic manipulation of clinical
S. aureus
isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of
S. aureus
strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of
E. coli
strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into
S. aureus
. The IMXXB series of
E. coli
strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of
S. aureus
.
Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters
Authors: Oh Hyung Suk , Traktman Paula , Knipe David M. 
Published: May 2015

ABSTRACT
We have shown previously that A-type lamins and intranuclear localization of the herpes simplex virus (HSV) genome are critical for the formation of the VP16 activator complex on HSV immediate-early (IE) gene promoters in murine cells, which implies a critical role for lamin A and its associated proteins in HSV gene expression. Because barrier-to-autointegration factor 1 (BAF/BANF1) has been thought to bridge chromosomes to the nuclear lamina, we hypothesized that BAF might mediate viral genome targeting to the nuclear lamina. We found that overexpression of BAF enhances HSV-1 replication and knockdown of BAF decreases HSV gene expression, delays the kinetics of viral early replication compartment formation, and reduces viral yield compared to those in control small interfering RNA-transfected cells. However, BAF depletion did not affect genome complex targeting to the nuclear periphery. Instead, we found that the levels of a histone-modifying enzyme, SETD1A methyltransferase, and histone H3 lysine 4 trimethylation were reduced on IE and early (E) gene promoters in BAF-depleted cells during HSV lytic infection. Our results demonstrate a novel function of BAF as an epigenetic regulator of HSV lytic infection. We hypothesize that BAF facilitates IE and E gene expression by recruiting the SETD1A methyltransferase to viral IE and E gene promoters.

IMPORTANCE
The nuclear lamina is composed of lamin proteins and numerous lamina-associated proteins. Previously, the chromatin structure of DNA localized proximally to the lamina was thought to be characterized by heterochromatin marks associated with silenced genes. However, recent studies indicate that both heterochromatin- and euchromatin-rich areas coexist on the lamina. This paradigm suggests that lamins and lamina-associated proteins dynamically regulate epigenetic modifications of specific genes in different locations. Our goal is to understand how the lamina and its associated proteins regulate the epigenetics of genes through the study of HSV infection of human cells. We have shown previously that A-type lamins are critical for HSV genome targeting to the nuclear lamina and epigenetic regulation in viral replication. In this study, we found that another lamina-associated protein, BAF, regulates HSV gene expression through an epigenetic mechanism, which provides basic insights into the nuclear lamina and its associated proteins’ roles in epigenetic regulation.
Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds
Authors: Herman-Bausier Philippe , El-Kirat-Chatel Sofiane , Foster Timothy J. , Geoghegan Joan A. , Dufrêne Yves F. 
Published: May 2015

ABSTRACT

Staphylococcus aureus
is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant
S. aureus
(MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during
S. aureus
biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms.

IMPORTANCE
Staphylococcus aureus
is a human pathogen that forms biofilms on indwelling medical devices, such as central venous catheters and prosthetic joints. This leads to biofilm infections that are difficult to treat with antibiotics because many cells within the biofilm matrix are dormant. The fibronectin-binding proteins (FnBPs) FnBPA and FnBPB promote biofilm formation by clinically relevant methicillin-resistant
S. aureus
(MRSA) strains, but the molecular mechanisms involved remain poorly understood. We used atomic force microscopy techniques to demonstrate that FnBPA mediates cell-cell adhesion via multiple, low-affinity homophilic bonds between FnBPA A domains on adjacent cells. Therefore, FnBP-mediated homophilic interactions represent an interesting target to prevent MRSA biofilms. We propose that such homophilic mechanisms may be widespread among staphylococcal cell surface proteins, providing a means to guide intercellular adhesion and biofilm accumulation.
The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
Authors: Montealegre Maria Camila , La Rosa Sabina Leanti , Roh Jung Hyeob , Harvey Barrett R. , Murray Barbara E. 
Published: May 2015

ABSTRACT

The endocarditis and biofilm-associated pili (Ebp) are important in
Enterococcus faecalis
pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on
in silico
analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon
in situ
with ATG, and then to revert this ATG to ATT; translational fusions of
ebpA
to
lacZ
were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen.

IMPORTANCE
Enterococcus faecalis
is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in
E. faecalis
pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in
E. faecalis
. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of
E. faecalis
to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.
Escape of Actively Secreting Shigella flexneri from ATG8/LC3-Positive Vacuoles Formed during Cell-To-Cell Spread Is Facilitated by IcsB and VirA
Authors: Campbell-Valois François-Xavier , Sachse Martin , Sansonetti Philippe J. , Parsot Claude 
Published: May 2015

ABSTRACT

The enteropathogenic bacterium
Shigella flexneri
uses a type 3 secretion apparatus (T3SA) to transfer proteins dubbed translocators and effectors inside host cells, inducing bacterial uptake and subsequent lysis of the entry vacuole. Once in the cytoplasm, the outer membrane protein IcsA induces actin polymerization, enabling cytoplasmic movement and cell-to-cell spread of bacteria. During this infectious process,
S. flexneri
is targeted by ATG8/LC3. The effector IcsB was proposed to inhibit LC3 recruitment by masking a region of IcsA recognized by the autophagy pathway component ATG5. The effector VirA, a GTPase-activating protein (GAP) for Rab1, was also shown to prevent LC3 recruitment. However, the context of LC3 recruitment around
S. flexneri
is not fully understood. Here, we show that LC3 is recruited specifically around secreting bacteria that are still present in vacuoles formed during entry and cell-to-cell spread. While LC3 recruitment occurs around a small proportion of intracellular wild-type bacteria, the
icsB
,
virA
, and
icsB virA
mutants display incremental defaults in escape from LC3-positive vacuoles formed during cell-to-cell spread. Our results indicate that IcsB and VirA act synergistically to allow bacteria to escape from LC3-positive vacuoles by acting at or in the immediate vicinity of the vacuole membrane(s). We also demonstrate that LC3 is recruited around bacteria still present in the single-membrane entry vacuole, in a manner akin to that seen with LC3-associated phagocytosis. Our results indicate that LC3 recruitment occurs around bacteria still, or already, in membrane compartments formed during entry and cell-to-cell spread, and not around bacteria free in the cytoplasm.

IMPORTANCE
The targeting of
S. flexneri
by LC3 is a classic example of the targeting of foreign cytoplasmic particles by autophagy (so-called “xenoautophagy”). It is often assumed that LC3 is recruited around bacteria present in the cytoplasm through the formation of canonical double-membrane autophagosomes. Our results indicate that LC3 is recruited around the entry vacuole composed of a single membrane as in the case of LC3-associated phagocytosis. Effectors IcsB and VirA had been implicated in the blocking of LC3 recruitment, but it was not known if they acted on the same or distinct LC3-recruiting pathways. Our results indicate that LC3 is recruited exclusively around bacteria present in vacuoles formed during entry and cell-to-cell spread and that both IcsB and VirA intervene at the latter stage to facilitate bacterial escape. Our report reconciles several findings and may have broad implications for our understanding of the specific targeting of bacterial pathogens by LC3.
Lower Carboniferous ramp sedimentation of the Central Alborz Basin, northern Iran: integrated sedimentological and rock–magnetic studies
Authors: Sardar Abadi Mehrdad , Da Silva Anne-Christine , Mossadegh Hossein , Spassov Simo , Boulvain Frédéric 
Published: May 2015

Abstract

The Lower Carboniferous Mobarak Formation in the Alborz Basin (northern Iran) was deposited along the northeastern margin of Gondwana in a carbonate ramp setting. This paper focuses on the Tournaisian stratigraphic interval of this formation that crops out at the Jaban section in the southwestern Central Alborz Basin. The following facies associations, representing different ramp palaeoenvironments, have been identified: (1) mudstone–wackestone outer-ramp facies; (2) crinoidal to skeletal grainstone–packstone mid-ramp facies; (3) peloidal to crinoidal grainstone–packstone inner-ramp facies; and (4) coastal facies, which include a variety of microbial laminated to oncoidal grainstones and mudstones with evaporitic pseudomorphs. This ramp profile was affected by frequent storms that were responsible for the formation of several skeletal to non-skeletal shoals in the distal mid-ramp to the most proximal inner-ramp areas. The development of the skeletal to non-skeletal shoals along the sea side of the ramp formed a semi-enclosed lagoon sensitive to the influence of both high tides and storm surges. The magnetic susceptibility (χ
in
) of all the samples was measured and compared with that of the facies from which the sample was taken. There is a clear link between χ
in
and the facies; the average χ
in
values were higher for the distal facies than for the proximal facies. The χ
in
profile of this Lower Carboniferous carbonate sequence reflects stratigraphic variations in response to relative changes in sea level and the input of detrital materials. In the context of the sequence stratigraphic framework, the average χ
in
values for lowstand and transgressive systems tract deposits are higher than for the highstand systems tract deposits. The clear link between χ
in
and facies indicates at least a partly preserved primary χ
in
signal related to the detrital inputs. However, to obtain a better understanding of the nature and origin of the minerals carrying the χ
in
, we performed hysteresis measurements on selected samples. It appears that the χ
in
signal is mainly carried by low-coercivity ferromagnetic minerals such as magnetite, with a mixture of relatively coarse grains (detrital fraction) and ultra-fine grains (probably formed during diagenesis).
On Pointwise Approximation of Conjugate Functions by Some Hump Matrix Means of Conjugate Fourier Series
Authors: Łenski W. , Szal B. 
Published: May 2015

The results generalizing some theorems onN, pnE, γsummability are shown. The same degrees of pointwise approximation as in earlier papers by weaker assumptions on considered functions and examined summability methods are obtained. From presented pointwise results, the estimation on norm approximation is derived. Some special cases as corollaries are also formulated.