A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Biofilm Formation by Nontypeable Haemophilus Influenzae and Resistance to Complement-Mediated Clearance.

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae.

An infant mouse model of influenza-driven nontypeable Haemophilus influenzae colonization and acute otitis media suitable for preclinical testing of novel therapies.

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 105 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children.

Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.

A Case Report: Ruptured Popliteal Aneurysm Caused by Haemophilus Influenzae Infection.

Mycotic aneurysms arise from infection of an arterial wall secondary to septic emboli from endocarditis. Although rare, most mycotic aneurysms involve the abdominal aorta, with Staphylococcus aureus and Salmonella spp being the most common causative organisms. We report a case of an 81-year-old woman with a ruptured mycotic popliteal aneurysm from Haemophilus influenzae infection.

Defective monocyte-derived macrophage phagocytosis is associated with exacerbation frequency in COPD.

BACKGROUND: Lower airway bacterial colonisation (LABC) in COPD patients is associated with increased exacerbation frequency and faster lung function decline. Defective macrophage phagocytosis in COPD drives inflammation, but how defective macrophage function contributes to exacerbations is not clear. This study investigated the association between macrophage phagocytosis and exacerbation frequency, LABC and clinical parameters. METHODS: Monocyte-derived macrophages (MDM) were generated from 92 stable COPD patients, and at the onset of exacerbation in 39 patients. Macrophages were exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4 h, then phagocytosis measured by fluorimetry and cytokine release by ELISA. Sputum bacterial colonisation was measured by PCR. RESULTS: Phagocytosis of H. influenzae was negatively correlated with exacerbation frequency (r = 0.440, p < 0.01), and was significantly reduced in frequent vs. infrequent exacerbators (1.9 × 103 RFU vs. 2.5 × 103 RFU, p < 0.01). There was no correlation for S. pneumoniae. There was no association between phagocytosis of either bacteria with age, lung function, smoking history or treatment with inhaled corticosteroids, or long-acting bronchodilators. Phagocytosis was not altered during an exacerbation, or in the 2 weeks post-exacerbation. In response to phagocytosis, MDM from exacerbating patients showed increased release of CXCL-8 (p < 0.001) and TNFα (p < 0.01) compared to stable state. CONCLUSION: Impaired COPD macrophage phagocytosis of H. influenzae, but not S. pneumoniae is associated with exacerbation frequency, resulting in pro-inflammatory macrophages that may contribute to disease progression. Targeting these frequent exacerbators with drugs that improve macrophage phagocytosis may prove beneficial.

Contribution of dipeptidyl peptidase 4 to non-typeable Haemophilus influenzae-induced lung inflammation in COPD.

Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1ß, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.

Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands.

Haemophilus influenzae is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive H. influenzae strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.

Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines.

PURPOSE OF REVIEW: The human upper respiratory tract is colonized with a variety of bacterial microorganisms including Haemophilus influenzae. The species H. influenzae consists of typeable and nontypeable H. influenzae (NTHi) variants. Typeable H. influenzae are subdivided into types a through f, based on the polysaccharide capsule, whereas the NTHi strains do not express a polysaccharide capsule. In this review, we highlight the current advances in the field of H. influenzae, with the focus on bacterial virulence mechanisms that facilitate bacterial colonization and disease, particularly for NTHi. RECENT FINDINGS: In the past decade, it has become apparent that NTHi has the ability to cause invasive infections. Recently, a number of adhesins have been shown to be crucial for bacterial colonization and invasion and these proteins were investigated as vaccine antigens. Although NTHi lacks a polysaccharide capsule, it expresses lipooligosaccharide that contribute to adhesion and evasion of complement-mediated killing, both contributing to bacterial virulence, which could potentially be targeted by novel antimicrobial drugs or vaccines. SUMMARY: The unraveling of H. influenzae virulence mechanisms resulted in the identification of promising targets for novel antimicrobials and vaccine antigens aiming to prevent or treat both typeable and nontypeable H. influenzae infections.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Bacterial co-infections with SARS-CoV-2.

The pandemic coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected millions of people worldwide. To date, there are no proven effective therapies for this virus. Efforts made to develop antiviral strategies for the treatment of COVID-19 are underway. Respiratory viral infections, such as influenza, predispose patients to co-infections and these lead to increased disease severity and mortality. Numerous types of antibiotics such as azithromycin have been employed for the prevention and treatment of bacterial co-infection and secondary bacterial infections in patients with a viral respiratory infection (e.g., SARS-CoV-2). Although antibiotics do not directly affect SARS-CoV-2, viral respiratory infections often result in bacterial pneumonia. It is possible that some patients die from bacterial co-infection rather than virus itself. To date, a considerable number of bacterial strains have been resistant to various antibiotics such as azithromycin, and the overuse could render those or other antibiotics even less effective. Therefore, bacterial co-infection and secondary bacterial infection are considered critical risk factors for the severity and mortality rates of COVID-19. Also, the antibiotic-resistant as a result of overusing must be considered. In this review, we will summarize the bacterial co-infection and secondary bacterial infection in some featured respiratory viral infections, especially COVID-19.

Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

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. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivoIMPORTANCE Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in cis wild-type toxin under the same conditions.

Analysis of Invasive Nontypeable Haemophilus influenzae Isolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes.

Nontypeable Haemophilus influenzae (NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genes oafA ON and lic2A OFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of an O-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

Nontypeable Haemophilus influenzae Invasive Blood Isolates Are Mainly Phosphorylcholine Negative and Show Decreased Complement-Mediated Killing That Is Associated with Lower Binding of IgM and CRP in Comparison to Colonizing Isolates from the Oropharynx.

Nontypeable Haemophilus influenzae (NTHi) bacteria express various molecules that contribute to their virulence. The presence of phosphocholine (PCho) on NTHi lipooligosaccharide increases adhesion to epithelial cells and is an advantage for the bacterium, enabling nasopharyngeal colonization, as measured in humans and animal models. However, when PCho is expressed on the lipooligosaccharide, it is also recognized by the acute-phase protein C-reactive protein (CRP) and PCho-specific antibodies, both of which are potent initiators of the classical pathway of complement activation. In this study, we show that blood isolates, which are exposed to CRP and PCho-specific antibodies in the bloodstream, have a higher survival in serum than oropharyngeal isolates, which was associated with a decreased presence of PCho. PCholow strains showed decreased IgM, CRP, and complement C3 deposition, which was associated with increased survival in human serum. Consistent with the case for the PCholow strains, removal of PCho expression by licA gene deletion decreased IgM, CRP, and complement C3 deposition, which increased survival in human serum. Complement-mediated killing of PChohigh strains was mainly dependent on binding of IgM to the bacterial surface. These data support the hypothesis that a PCholow phenotype was selected in blood during invasive disease, which increased resistance to serum killing, mainly due to lowered IgM and CRP binding to the bacterial surface.

A Cross-Reactive Protein Vaccine Combined with PCV-13 Prevents Streptococcus pneumoniae- and Haemophilus influenzae-Mediated Acute Otitis Media.

Acute otitis media is one of the most common childhood infections worldwide. Currently licensed vaccines against the common otopathogen Streptococcus pneumoniae target the bacterial capsular polysaccharide and confer no protection against nonencapsulated strains or capsular types outside vaccine coverage. Mucosal infections such as acute otitis media remain prevalent, even those caused by vaccine-covered serotypes. Here, we report that a protein-based vaccine, a fusion construct of epitopes of CbpA to pneumolysin toxoid, confers effective protection against pneumococcal acute otitis media for non-PCV-13 serotypes and enhances protection for PCV-13 serotypes when coadministered with PCV-13. Having cross-reactive epitopes, the fusion protein also induces potent antibody responses against nontypeable Haemophilus influenzae and S. pneumoniae, engendering protection against acute otitis media caused by emerging unencapsulated otopathogens. These data suggest that augmenting capsule-based vaccination with conserved, cross-reactive protein-based vaccines broadens and enhances protection against acute otitis media.

Efficacy of Aminomethyl Spectinomycins against Complex Upper Respiratory Tract Bacterial Infections.

The most frequent ailment for which antibiotics are prescribed is otitis media (ear infections), which is most commonly caused by Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae Treatment of otitis media is complicated by the fact that the bacteria in the middle ear typically form biofilms, which can be recalcitrant to antibiotic treatment. Furthermore, bacterial respiratory infections can be greatly exacerbated by viral coinfection, which is particularly evidenced by the synergy between influenza and S. pneumoniae In this study, we sought to ascertain the in vivo efficacy of aminomethyl spectinomycin lead 1950, an effective antibacterial agent both in vitro and in vivo against Streptococcus pneumoniae in the context of complex respiratory infections and acute otitis media. A single dose of 1950 significantly reduced bacterial burden in the respiratory tract for all three pathogens, even when species were present in a coinfection model. Additionally, a single dose of 1950 effectively reduced pneumococcal acute otitis media from the middle ear. The agent 1950 also proved efficacious in the context of influenza-pneumococcal super infection. These data further support the development of this family of compounds as potential therapeutic agents against the common causes of complex upper respiratory tract infections and acute otitis media.

Phasevarions of bacterial pathogens - phase-variable epigenetic regulators evolving from restriction-modification systems.

Phase-variable DNA methyltransferases control the expression of multiple genes via epigenetic mechanisms in a wide variety of bacterial species. These systems are called phasevarions, for phase-variable regulons. Phasevarions regulate genes involved in pathogenesis, host adaptation and antibiotic resistance. Many human-adapted bacterial pathogens contain phasevarions. These include leading causes of morbidity and mortality worldwide, such as non-typeable Haemophilus influenzae, Streptococcus pneumoniae and Neisseria spp. Phase-variable methyltransferases and phasevarions have also been discovered in environmental organisms and veterinary pathogens. The existence of many different examples suggests that phasevarions have evolved multiple times as a contingency strategy in the bacterial domain, controlling phenotypes that are important in adapting to environmental change. Many of the organisms that contain phasevarions have existing or emerging drug resistance. Vaccines may therefore represent the best and most cost-effective tool to prevent disease caused by these organisms. However, many phasevarions also control the expression of current and putative vaccine candidates; variable expression of antigens could lead to immune evasion, meaning that vaccines designed using these targets become ineffective. It is therefore essential to characterize phasevarions in order to determine an organism's stably expressed antigenic repertoire, and rationally design broadly effective vaccines.

Electronic cigarette vapour increases virulence and inflammatory potential of respiratory pathogens.

INTRODUCTION: Bacteria have been extensively implicated in the development of smoking related diseases, such as COPD, by either direct infection or bacteria-mediated inflammation. In response to the health risks associated with tobacco exposure, the use of electronic cigarettes (e-cigs) has increased. This study compared the effect of e-cig vapour (ECV) and cigarette smoke (CSE) on the virulence and inflammatory potential of key lung pathogens (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa). METHODS: Biofilm formation, virulence in the Galleria mellonella infection model, antibiotic susceptibility and IL-8/TNF-α production in A549 cells, were compared between bacteria exposed to ECV, CSE and non-exposed bacteria. RESULTS: Statistically significant increases in biofilm and cytokine secretion were observed following bacterial exposure to either ECV or CSE, compared to non-exposed bacteria; the effect of exposure to ECV on bacterial phenotype and virulence was comparable, and in some cases greater, than that observed following CSE exposure. Treatment of A549 cells with cell signaling pathway inhibitors prior to infection, did not suggest that alternative signaling pathways were being activated following exposure of bacteria to either ECV or CSE. CONCLUSIONS: These findings therefore suggest that ECV and CSE can induce changes in phenotype and virulence of key lung pathogens, which may increase bacterial persistence and inflammatory potential.

Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNA-laden exosomes from middle-ear epithelium in vitro. -Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.