bluuebunny/crossref_metadata_2025_split huggingface dataset:
Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition
Authors: Mitsui Takaaki , Watanabe-Takahashi Miho , Shimizu Eiko , Zhang Baihao , Funamoto Satoru , Yamasaki Shinji , Nishikawa Kiyotaka 
Published: July 2016

ABSTRACT

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic
Escherichia coli
(EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.
A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain
Authors: González-Rivera Christian , Campbell Anne M. , Rutherford Stacey A. , Pyburn Tasia M. , Foegeding Nora J. , Barke Theresa L. , Spiller Benjamin W. , McClain Mark S. , Ohi Melanie D. , Lacy D. Borden , Cover Timothy L. 
Published: July 2016

ABSTRACT

Helicobacter pylori
secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by
H. pylori
. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.
The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract
Authors: Yang Zhangsheng , Tang Lingli , Shao Lili , Zhang Yuyang , Zhang Tianyuan , Schenken Robert , Valdivia Raphael , Zhong Guangming 
Published: July 2016

ABSTRACTDespite the extensivein vitrocharacterization of CPAF (chlamydialprotease/proteasome-likeactivityfactor), its role in chlamydial infection and pathogenesis remains unclear. We now report that aChlamydia trachomatisstrain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficientC. trachomatisstrain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promotingC. trachomatissurvival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.
Burkholderia pseudomallei Rapidly Infects the Brain Stem and Spinal Cord via the Trigeminal Nerve after Intranasal Inoculation
Authors: St. John James A. , Walkden Heidi , Nazareth Lynn , Beagley Kenneth W. , Ulett Glen C. , Batzloff Michael R. , Beacham Ifor R. , Ekberg Jenny A. K. 
Published: July 2016

ABSTRACT

Infection with
Burkholderia pseudomallei
causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that
B. pseudomallei
can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice,
B. pseudomallei
caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers.
B. pseudomallei
rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 μm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of
B. pseudomallei
that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that
B. pseudomallei
can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity.
Regulation of Ribosomal Protein Operons
rplM-rpsI
,
rpmB-rpmG
, and
rplU-rpmA
at the Transcriptional and Translational Levels
Authors: Aseev Leonid V. , Koledinskaya Ludmila S. , Boni Irina V. 
Published: July 2016

ABSTRACT

It is widely assumed that in the best-characterized model bacterium
Escherichia coli
, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several
E. coli
r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the
in vivo
regulation of three r-protein operons,
rplM-rpsI
,
rpmB-rpmG
, and
rplU-rpmA
. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal
lacZ
gene, we show here that at the translation level only one of these operons,
rplM-rpsI
, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, while
rpmB-rpmG
and
rplU-rpmA
are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of
E. coli
r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of
rplM-rpsI
expression
in vivo
, acting at a target within the
rplM
translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.

IMPORTANCE
It is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.
The Last
r
Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin
Authors: Chen Yi , Young Ry 
Published: July 2016

ABSTRACT

The latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4
r
genes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another
r
locus,
rIII
, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in both
Escherichia coli
B strains and
E. coli
K-12 strains. In T4Δ
rIII
infections, LIN was briefly established but was unstable. The overexpression of a cloned
rIII
gene alone impeded T-mediated lysis temporarily. However, coexpression of
rIII
and
rI
resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm.

IMPORTANCE
Lysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene
rIII
encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.
Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators
Authors: Nock Adam M. , Wargo Matthew J. 
Published: July 2016

ABSTRACT

Burkholderia thailandensis
is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant,
Pseudomonas aeruginosa
. Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar to
P. aeruginosa
. Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in
B. thailandensis
. In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of the
B. thailandensis
choline catabolic pathway (
gbdR1
,
gbdR2
, and
souR
). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of
gbdR1
(
BTH_II1869
) and
gbdR2
(
BTH_II0968
). We also identified a functional ortholog for
P. aeruginosa
souR
, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to
P. aeruginosa
GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the
B. thailandensis
SouR ortholog (
BTH_II0994
) is required for catabolism of choline and its metabolites as carbon sources, whereas in
P. aeruginosa
, SouR function can by bypassed by GbdR. The strategy employed by
B. thailandensis
represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection.

IMPORTANCE
Many proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential regulation of these components can help us understand both the evolution of these systems and the potential roles these pathways have in the biology of each bacterium. Here, we describe the transcriptome response of
Burkholderia thailandensis
to the eukaryote-enriched molecule choline, identify the regulatory pathway governing choline catabolism, and compare the pathway to that previously described for
Pseudomonas aeruginosa
. These data support a multitiered regulatory network in
B. thailandensis
, with conserved orthologs in the select agents
Burkholderia pseudomallei
and
Burkholderia mallei
, as well as the opportunistic lung pathogens in the
Burkholderia cepacia
clade.
Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis
Authors: Bennallack Philip R. , Bewley Kathryn D. , Burlingame Mark A. , Robison Richard A. , Miller Susan M. , Griffitts Joel S. 
Published: July 2016

ABSTRACT

Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in
Bacillus subtilis
. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2.

IMPORTANCE
The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism
B. subtilis
was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.
Single-Stranded DNA Uptake during Gonococcal Transformation
Authors: Hepp Christof , Gangel Heike , Henseler Katja , Günther Niklas , Maier Berenike 
Published: July 2016

ABSTRACT

Neisseria gonorrhoeae
is naturally competent for transformation. The first step of the transformation process is the uptake of DNA from the environment into the cell. This transport step is driven by a powerful molecular machine. Here, we addressed the question whether this machine imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at similar rates. The fluorescence signal associated with the uptake of short DNA fragments labeled with a single fluorescent marker molecule was quantified. We found that ssDNA with a double-stranded DNA uptake sequence (DUS) was taken up with a similar efficiency as dsDNA. Imported ssDNA was degraded rapidly, and the thermonuclease Nuc was required for degradation. In a
nuc
deletion background, dsDNA and ssDNA with a double-stranded DUS were imported and used as the substrates for transformation, whereas the import and transformation efficiencies of ssDNA with single-stranded DUS were below the detection limits. We conclude that the DNA uptake machine requires a double-stranded DUS for efficient DNA recognition and transports ssDNA and dsDNA with comparable efficiencies.

IMPORTANCE
Bacterial transformation enables bacteria to exchange genetic information. It can speed up adaptive evolution and enhances the potential of DNA repair. The transport of DNA through the outer membrane is the first step of transformation in Gram-negative species. It is driven by a powerful molecular machine whose mechanism remains elusive. Here, we show for
Neisseria gonorrhoeae
that the machine transports single- and double-stranded DNA at comparable rates, provided that the species-specific DNA uptake sequence is double stranded. Moreover, we found that single-stranded DNA taken up into the periplasm is rapidly degraded by the thermonuclease Nuc. We conclude that the secondary structure of transforming DNA is important for the recognition of self DNA but not for the process of transport through the outer membrane.
Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics
Authors: Page Stephani C. , Immormino Robert M. , Miller Thane H. , Bourret Robert B. 
Published: July 2016

ABSTRACT

Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to environmental stimuli. TCSs mediate responses through phosphotransfer from a conserved histidine on a sensor kinase to a conserved aspartate on the receiver domain of a response regulator. Typically, signal termination occurs through dephosphorylation of the receiver domain, which can catalyze its own dephosphorylation. Despite strong structural conservation between receiver domains, reported autodephosphorylation rate constants (
k
dephos
) span a millionfold range. Variable receiver domain active-site residues D + 2 and T + 2 (two amino acids C terminal to conserved phosphorylation site and Thr/Ser, respectively) influence
k
dephos
values, but the extent and mechanism of influence are unclear. We used sequence analysis of a large database of naturally occurring receiver domains to design mutant receiver domains for experimental analysis of autodephosphorylation kinetics. When combined with previous analyses,
k
dephos
values were obtained for CheY variants that contained D + 2/T + 2 pairs found in 54% of receiver domain sequences. Tested pairs of amino acids at D + 2/T + 2 generally had similar effects on
k
dephos
in CheY, PhoB
N
, or Spo0F. Acid or amide residues at D + 2/T + 2 enhanced
k
dephos
. CheY variants altered at D + 2/T + 2 exhibited rate constants for autophosphorylation with phosphoramidates and autodephosphorylation that were inversely correlated, suggesting that D + 2/T + 2 residues interact with aspects of the ground or transition states that differ between the two reactions.
k
dephos
of CheY variants altered at D + 2/T + 2 correlated significantly with
k
dephos
of wild-type receiver domains containing the same D + 2/T + 2 pair. Additionally, particular D + 2/T + 2 pairs were enriched in different response regulator subfamilies, suggesting functional significance.

IMPORTANCE
One protein family, defined by a conserved domain, can include hundreds of thousands of known members. Characterizing conserved residues within a conserved domain can identify functions shared by all family members. However, a general strategy to assess features that differ between members of a family is lacking. Fully exploring the impact of just two variable positions within a conserved domain could require assessment of 400 (i.e., 20 × 20) variants. Instead, we created and analyzed a nonredundant database of receiver domain sequences. Five percent of D + 2/T + 2 pairs were sufficient to represent 50% of receiver domain sequences. Using protein sequence analysis to prioritize mutant choice made it experimentally feasible to extensively probe the influence of positions D + 2 and T + 2 on receiver domain autodephosphorylation kinetics.
JC Polyomavirus Infection of Primary Human Renal Epithelial Cells Is Controlled by a Type I IFN-Induced Response
Authors: Assetta Benedetta , De Cecco Marco , O’Hara Bethany , Atwood Walter J. 
Published: July 2016

ABSTRACT
The JC and BK human polyomaviruses (JCPyV and BKPyV, respectively) establish lifelong persistent infections in the kidney. In immunosuppressed individuals, JCPyV causes progressive multifocal leukoencephalopathy (PML), a fatal neurodegenerative disease, and BKPyV causes polyomavirus-associated nephropathy (PVN). In this study, we compared JCPyV and BKPyV infections in primary human renal proximal tubule epithelial (HRPTE) cells. JCPyV established a persistent infection, but BKPyV killed the cells in 15 days. To identify the cellular factors responsible for controlling JCPyV infection and promoting viral persistence, we profiled the transcriptomes of JCPyV- and BKPyV-infected cells at several time points postinfection. We found that infection with both viruses induced interferon production but that interferon-stimulated genes (ISGs) were only activated in the JCPyV-infected cells. Phosphorylated STAT1 and IRF9, which are responsible for inducing ISGs, translocated to the nucleus of JCPyV-infected cells but did not in BKPyV-infected cells. In BKPyV-infected cells, two critical suppressors of cytokine signaling, SOCS3 and SOCS1, were induced. Infection with BKPyV but not JCPyV caused reorganization of PML bodies that are associated with inactivating antiviral responses. Blockade of the interferon receptor and neutralization of soluble interferon alpha (IFN-α) and IFN-β partially alleviated the block to JCPyV infection, leading to enhanced infectivity. Our results show that a type I IFN response contributes to the establishment of persistent infection by JCPyV in HRPTE cells.

IMPORTANCE
The human polyomaviruses JCPyV and BKPyV both establish lifelong persistent infection in the kidneys. In immunosuppressed patients, BKPyV causes significant pathology in the kidney, but JCPyV is only rarely associated with disease in this organ. The reasons behind this striking difference in kidney pathology are unknown. In this study, we show that infection of primary human renal tubule epithelial cells with JCPyV and BKPyV results in divergent innate immune responses that control JCPyV but fail to control BKPyV. This is the first study that directly compares JCPyV and BKPyV infection
in vitro
in the same cell type they naturally infect, and the significant differences that have been uncovered could in part explain the distinct disease outcomes.