Discovery of Small-Molecule VapC1 Nuclease Inhibitors by Virtual Screening and Scaffold Hopping from an Atomic Structure Revealing Protein-Protein Interactions with a Native VapB1 Inhibitor.

Nontypeable (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics.

Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System.

Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H.

In hot water: effects of climate change on Vibrio-human interactions.

Sea level rise and the anthropogenic warming of the world's oceans is not only an environmental tragedy, but these changes also result in a significant threat to public health. Along with coastal flooding and the encroachment of saltwater farther inland comes an increased risk of human interaction with pathogenic Vibrio species, such as Vibrio cholerae, V. vulnificus and V.

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection.

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity.

Antimicrobial Efficacy and Safety of a Novel Gas Plasma-Activated Catheter Lock Solution.

Antimicrobial lock solutions are important for prevention of microbial colonization and infection of long-term central venous catheters. We investigated the efficacy and safety of a novel antibiotic-free lock solution formed from gas plasma-activated disinfectant (PAD). Using a luminal biofilm model, viable cells of methicillin-resistant , , , and in mature biofilms were reduced by 6 to 8 orders of magnitude with a PAD lock for 60 min.

Better living through chemistry: Addressing emerging antibiotic resistance.

The increasing emergence of multidrug-resistant bacteria is recognized as a major threat to human health worldwide. While the use of small molecule antibiotics has enabled many modern medical advances, it has also facilitated the development of resistant organisms. This minireview provides an overview of current small molecule drugs approved by the US Food and Drug Administration (FDA) for use in humans, the unintended consequences of antibiotic use, and the mechanisms that underlie the development of drug resistance.

Wake me when it's over - Bacterial toxin-antitoxin proteins and induced dormancy.

Toxin-antitoxin systems are encoded by bacteria and archaea to enable an immediate response to environmental stresses, including antibiotics and the host immune response. During normal conditions, the antitoxin components prevent toxins from interfering with metabolism and arresting growth; however, toxin activation enables microbes to remain dormant through unfavorable conditions that might continue over millions of years. Intense investigations have revealed a multitude of mechanisms for both regulation and activation of toxin-antitoxin systems, which are abundant in pathogenic microorganisms.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage.

Background: Microvascular leakage of plasma proteins is a hallmark of inflammation that leads to tissue dysfunction. There are no current therapeutic strategies to reduce microvascular permeability. The purpose of this study was to identify the role of Rnd3, an atypical Rho family GTPase, in the control of endothelial barrier integrity.

The ToxAvapA toxin-antitoxin locus contributes to the survival of nontypeable Haemophilus influenzae during infection.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA) module, NTHI1912-1913, is a host inhibition of growth (higBA) homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection.

Development of a novel treatment for leukemia directed at tumor-associated mRNA splicing.

This report describes a novel approach to cancer therapy that targets genes that are preferentially alternatively spliced and expressed in leukemia. We developed CD44v6 and CD44v8 splicing constructs fused with GFP or a humanized fragment of Pseudomonas aeruginosa exotoxin A (hPE24). Transfection of K562 leukemia cells with the GFP-linked splicing constructs led to subsequent production of detectable levels of GFP.

Toxin-antitoxin loci vapBC-1 and vapXD contribute to survival and virulence in nontypeable Haemophilus influenzae.

Background: Nontypeable Haemophilus influenzae (NTHi) is a significant human pathogen responsible for respiratory tract infections and the most common cause of recurrent otitis media. Type II toxin-antitoxin (TA) systems are genetic elements that code for a stable protein toxin and a labile antitoxin that are thought to be involved in metabolic regulation of bacteria by enabling a switch to a dormant state under stress conditions. The contribution to infection persistence of the NTHi TA loci vapBC-1 and vapXD was examined in this study.

Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days.

Regulation of the vapBC-1 toxin-antitoxin locus in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA) gene pairs have been shown to facilitate entry into a reversible bacteriostatic state.

Use of the EpiAirway model for characterizing long-term host-pathogen interactions.

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa (1; 2). To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA).

A role for long chain myosin light chain kinase (MLCK-210) in microvascular hyperpermeability during severe burns.

Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunction during trauma. Previous studies suggest the involvement of myosin light chain (MLC) phosphorylation-triggered endothelial contraction in the development of microvascular hyperpermeability. Myosin light chain kinase (MLCK) plays a key role in the control of MLC-phosphorylation status; thus, it is thought to modulate barrier function through its regulation of intracellular contractile machinery.

VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease.

Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family.

Haemophilus influenzae luxS mutants form a biofilm and have increased virulence.

To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay.

Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus.

Background: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene HI0450).

Construction of a nontypeable Haemophilus influenzae-specific ectopic delivery vector.

Complementation of chromosomal mutations in trans can introduce artifacts due to the number of episomal copies of the gene in question. One solution is to study the gene expressed at a single ectopic site in cis. We have designed and constructed a vector that allows homologous recombination into a gene encoding a frame-shifted IS1016-V6 protein in the Haemophilus influenzae Rd KW20 chromosome (HI1018).

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase.

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange.

Chemically defined media for growth of Haemophilus influenzae strains.

A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are >/= 10(9) CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and beta-NAD).

Haemophilus influenzae Rd KW20 has virulence properties.

Haemophilus influenzae is a human-adapted commensal and pathogen that can cause mucosal infections such as sinusitis, otitis media and bronchitis. Certain strains also cause bacteraemia and meningitis. Clinical isolates are genetically heterogeneous and are often recalcitrant to standard genetic manipulation.

H. influenzae Consortium: integrative study of H. influenzae-human interactions.

Developments in high-throughput analysis tools coupled with integrative computational techniques have enabled biological studies to reach new levels. The ability to correlate large volumes of diverse data types into cohesive models of organism function has spawned a new systematic approach to biological investigation. The creation of a new consortium has been proposed to investigate a single organism utilizing these comprehensive approaches.