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

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

Role of c-Jun N-Terminal Protein Kinase 1/2 (JNK1/2) in Macrophage-Mediated MMP-9 Production in Response to Moraxella catarrhalis Lipooligosaccharide (LOS)

Moraxella catarrhalis is a Gram negative bacterium and a leading causative agent of otitis media (OM) in children. Recent reports have provided strong evidence for the presence of high levels of matrix metalloproteinase (MMPs) in effusion fluids from children suffering with OM, however, the precise mechanisms by which MMPs are generated are currently unknown. We hypothesized that MMPs are secreted from macrophages in the presence of M. catarrhalis lipooligosaccharide (LOS). In this report, we demonstrate that in vitro stimulation of murine macrophage RAW 264.7 cells with LOS leads to secretion of MMP-9 as determined by ELISA and zymogram assays. We have also shown that inhibition of ERK1/2 and p38 kinase completely blocked LOS induced MMP-9 production. In contrast, inhibition of JNK1/2 by the specific inhibitor SP600125 actually increased the level of expression and production of MMP-9 at both mRNA and protein levels, respectively by almost five fold. This latter result was confirmed by knocking down JNK1/2 using siRNA. Similar results have been observed in murine bone marrow derived macrophages in vitro. In contrast to and in parallel with the LOS-induced increased levels of MMP-9 in the presence of SP600125, we found a corresponding dose-dependent inhibition of TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) secretion. Results of subsequent in vitro studies provided evidence that when JNK1/2 was inhibited prior to stimulation with LOS, it significantly increased both the extent of macrophage cell migration and invasion compared to control cells or cells treated with LOS alone. The results of these studies contribute to an increased understanding of the underlying pathophysiology of OM with effusion in children

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

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

An Improved Parallel Inverse Design Method of EMU Wheel Profile from Wheel Flange Wear Viewpoint

An improved parallel inverse design method is proposed for wheel profile optimization. The dominant merit of this method is the ability to automatically search the target performance curve and obtain the optimized profile without artificial experience. With the help of vehicle system dynamic theory, an EMU model has been established in Simpack, and the dynamic performance is calculated with two profiles, i.e., optimization profile and original profile. The contact and mechanical characters are analyzed by Hertz’s theory, Kalker global algorithm, and CONTACT program. It is found that the rolling radius difference (RRD) with the optimization profile is higher than the original one, especially when the lateral displacement is greater than 3 mm. The creep force density with the optimization profile is significant with a wheelset displacement of 6∼9 mm. Compared with the original one, the distribution of contact points with the optimization profile is more uniform, and the contact position is more biased towards the root of the wheel flange. It means the optimization profile can provide higher RRD value and creep force with large lateral displacement, which is beneficial for reducing wheel flange wear. The dynamic simulation indicates that the optimization profile can help reduce the wheel flange force and wheel flange wear in a sharp curve. Meanwhile, the dynamic behaviors and wheel tread wear on a tangent track or a large curved track are also favorable with the optimization profile

Impaired Proinflammatory Response in Stringently Defined Otitis-prone Children During Viral Upper Respiratory Infections

BACKGROUND: Viral upper respiratory infections (URIs) are common and often precipitate acute otitis media (AOM), caused by bacterial otopathogens, in young children. Acute inflammatory responses initiated in the early phase of viral URI contribute to preventing the development of AOM. Stringently-defined otitis-prone (sOP) children are susceptible to recurrent AOM. METHODS: We assessed proinflammatory cytokine and chemokine levels in the nasopharynxes during viral URIs, and examined the different nasopharyngeal responses between viral URI events and the following AOM episodes in both sOP and non-otitis-prone (NOP) children. RESULTS: The sOP children exhibited significantly more AOM episodes per child (8.86-fold higher), viral URIs (P \u3c .0001), and viral URIs followed by AOMs (P \u3c .0001) than the NOP children. The sOP children had lower nasal proinflammatory levels of interleukin (IL)-6 (P = .05), IL-10 (P = .001), tumor necrosis factor (TNF)-α (P = .004), and regulated on activation, normal T-cell-expressed and -secreted (RANTES; P = .002) than NOP children during viral URIs. NOP children had higher levels of IL-6 (P = .02), IL-10 (P = .02), interferon-γ (P = .003), TNF-α (P = .006), IL-1β (P = .022), monocyte chemoattractant protein 1 (P = .028), RANTES (P = .005), IL-2 (P = .002), and IL-17 (P = .007) during viral URIs versus AOMs following the URIs, when compared to sOP children. CONCLUSIONS: We conclude that sOP children have more frequent viral URIs than NOP children, due to deficient antiviral nasopharyngeal proinflammatory cytokine and chemokine responses