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 NT(Hi) survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene H10450). Both vapDHi and a flanking gene, H10451, exhibit the genetic and physical characteristics of a toxin/antitoxin ( TA) locus, with VapDHi serving as the toxin moiety and H10451 as the antitoxin. We propose the name VapXHi for the H10451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections. Results: Isogenic mutants in vapDHi were attenuated for survival inside human respiratory epithelial cells (NCI-H292) and human brain microvascular endothelial cells (HBMEC), the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHi allele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHi allele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHi survival defect in HBMEC. Subunits of full-length VapDHi homodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHi or vapXHi, overexpression of the VapDHi toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHi antitoxin on a compatible plasmid. Conclusion: We conclude that vapDHi and vapXHi may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells

Toxin-Antitoxic 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. Results: Deletions in vapBC-1, vapXD and vapBC-1 vapXD significantly decreased the survival of NTHi co-cultured with primary human respiratory tissue at the air-liquid interface and in the chinchilla model of otitis media. The TA deletions did not affect the growth dynamics of the mutants in rich media, their ultra-structural morphology, or display appreciable synergy during NTHi infections. The toxin and antitoxin proteins of both pairs heterodimerized in vivo. Consistent with our previous findings regarding the VapC-1 toxin, the NTHi VapD toxin also displayed ribonuclease activity. Conclusions: We conclude that the vapBC-1 and vapXD TA loci enhance NTHi survival and virulence during infection in vitro and in vivo using a mechanism of mRNA cleavage, and that these conserved TA pairs represent new targets for the prophylaxis and therapy of otitis media and other NTHi-caused mucosal diseases

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. We deleted toxAvapA and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term in vitro co-culture with primary human respiratory tissues, which revealed that the DeltatoxAvapA mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways

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 Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans in mature biofilms were reduced by 6 to 8 orders of magnitude with a PAD lock for 60 min. Subsequent 24-h incubation of PAD-treated samples resulted in no detectable regrowth of viable bacteria or fungi. As a comparison, the use of a minocycline-EDTA-ethanol lock solution for 60 min led to regrowth of bacteria and fungi, up to 10(7) to 10(9) CFU/ml, in 24 h. The PAD lock solution had minimal impact on human umbilical vein endothelial cell viability, whereas the minocycline-EDTA-ethanol solution elicited cell death in nearly half of human endothelial cells. Additionally, PAD treatment caused little topological change to catheter materials. In conclusion, PAD represents a novel antibiotic-free, noncytotoxic lock solution that elicits rapid and broad-spectrum eradication of biofilm-laden microbes and shows promise for the prevention and treatment of intravascular catheter infections

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 potential therapeutic benefit of Rnd3 protein delivery to ameliorate microvascular leakage was also investigated. Methods and Results-Using immunofluorescence microscopy, Rnd3 was observed primarily in cytoplasmic areas around the nuclei of human umbilical vein endothelial cells. Permeability to fluorescein isothiocyanate-albumin and transendothelial electrical resistance of human umbilical vein endothelial cell monolayers served as indices of barrier function, and RhoA, Rac1, and Cdc42 activities were determined using G-LISA assays. Overexpression of Rnd3 significantly reduced the magnitude of thrombin-induced barrier dysfunction, and abolished thrombin-induced Racl inactivation. Depleting Rnd3 expression with siRNA significantly extended the time course of thrombin-induced barrier dysfunction and Racl inactivation. Time-lapse microscopy of human umbilical vein endothelial cells expressing GFP-actin showed that co-expression of mCherry-Rnd3 attenuated thrombin-induced reductions in local lamellipodia that accompany endothelial barrier dysfunction. Lastly, a novel Rnd3 protein delivery method reduced microvascular leakage in a rat model of hemorrhagic shock and resuscitation, assessed by both intravital microscopic observation of extravasation of fluorescein isothiocyanate-albumin from the mesenteric microcirculation, and direct determination of solute permeability in intact isolated venules. Conclusions-The data suggest that Rnd3 can shift the balance of RhoA and Racl signaling in endothelial cells. In addition, our findings suggest the therapeutic, anti-inflammatory potential of delivering Rnd3 to promote endothelial barrier recovery during inflammatory challenge

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. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes

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. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage

Competition binding of 50TIR and 50US by Vap proteins.

<p>Gel shift products from addition of 50US substrate into samples containing 50TIR substrate and either (A) VapBC-1 or (B) VapC-1 at a 150∶1 molar ratio of VapC-1 protein to DNA. VapB-1 and VapC-1 are at a 3∶1 molar ratio in the VapBC-1 samples, but VapC-1∶DNA molar ratios are reported since VapC-1 is the DNA binding protein and the actual amount of VapBC-1 complexes cannot be determined. The gels show the products of the following samples: 50TIR without protein (<i>lane 1</i>), protein with only 50TIR (<i>lane 2</i>), the addition of a 1∶1, 5∶1, 10∶1 or 50∶1 molar ratio of cold 50US:50TIR (<i>lanes 3–6</i>), and DNA only at 50∶1 molar ratio of 50US:50TIR (<i>lane 6</i>). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.</p

VapBC-1 specifically interacts with the <i>vapB-1</i> TIR.

<p>(A) The sequence of the 50TIR is shown with <i>arrows</i> indicating the inverted repeat regions and bases above and below the sequence indicating the position of the G to C and T to G substitutions in the TG50TIR and 2M50TIR substrates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032199#pone-0032199-t003" target="_blank">Table 3</a>). Gel shift products from a titration of VapBC-1 with (B) 50TIR or (C) TG50TIR are shown. The lanes in panels <i>B</i> and <i>C</i> contain the following samples: DNA only (<i>lane 1</i>), the addition of VapBC-1 at a VapC-1∶DNA ratio of 10, 25, 50, 100, 200, 400 or 800 molar ratio with the DNA substrate (<i>lanes 2–8</i>), and VapC-1 only at an 800∶1 protein to DNA ratio (<i>lane 9</i>). (D) A comparison of VapBC-1 binding at a 400∶1 molar ratio with each DNA substrate (<i>lanes 2, 4</i>, and <i>6</i>). <i>Lanes 1, 3</i>, and <i>5</i> contain only DNA. The identity of each band is noted at the right of each gel. Each gel represents one of three independent experiments.</p