Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.

Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Access to highly specialized growth substrates and production of epithelial immunomodulatory metabolites determine survival of Haemophilus influenzae in human airway epithelial cells.

Haemophilus influenzae (Hi) infections are associated with recurring acute exacerbations of chronic respiratory diseases in children and adults including otitis media, pneumonia, chronic obstructive pulmonary disease and asthma. Here, we show that persistence and recurrence of Hi infections are closely linked to Hi metabolic properties, where preferred growth substrates are aligned to the metabolome of human airway epithelial surfaces and include lactate, pentoses, and nucleosides, but not glucose that is typically used for studies of Hi growth in vitro. Enzymatic and physiological investigations revealed that utilization of lactate, the preferred Hi carbon source, required the LldD L-lactate dehydrogenase (conservation: 98.8% of strains), but not the two redox-balancing D-lactate dehydrogenases Dld and LdhA. Utilization of preferred substrates was directly linked to Hi infection and persistence. When unable to utilize L-lactate or forced to rely on salvaged guanine, Hi showed reduced extra- and intra-cellular persistence in a murine model of lung infection and in primary normal human nasal epithelia, with up to 3000-fold attenuation observed in competitive infections. In contrast, D-lactate dehydrogenase mutants only showed a very slight reduction compared to the wild-type strain. Interestingly, acetate, the major Hi metabolic end-product, had anti-inflammatory effects on cultured human tissue cells in the presence of live but not heat-killed Hi, suggesting that metabolic endproducts also influence HI-host interactions. Our work provides significant new insights into the critical role of metabolism for Hi persistence in contact with host cells and reveals for the first time the immunomodulatory potential of Hi metabolites.

P38 MAPK pathway regulates the expression of resistin in porcine alveolar macrophages via Ets2 during Haemophilus parasuis stimulation.

Haemophilus parasuis is a widespread bacterial pathogen causing acute systemic inflammation and leading to the sudden death of piglets. Resistin, a multifunctional peptide hormone previously demonstrated to influence the inflammation in porcine, was extremely increased in H. parasuis-infected tissues. However, the mechanism of resistin expression regulation in porcine, especially during pathogen infection, remains unclear. In the present study, we explored for the first time the transcription factor and signaling pathway mediating the expression of pig resistin during H. parasuis stimulation. We found that H. parasuis induced the expression of pig resistin in a time- and dose-dependent manner via the transcription factor Ets2 in porcine alveolar macrophages during H. parasuis stimulation. Moreover, the expression of Ets2 was mediated by the activation of the p38 MAPK pathway induced by H. parasuis, thus promoting resistin production. These results revealed a novel view of the molecular mechanism of pig resistin production during acute inflammation induced by pathogenic bacteria.

Lipoprotein(a), an Opsonin, Enhances the Phagocytosis of Nontypeable Haemophilus influenzae by Macrophages.

We recently showed that both nontypeable Haemophilus influenzae (NTHi) and its surface plasminogen- (Plg-) binding proteins interact with lipoprotein(a) (Lp(a)) in a lysine-dependent manner. Because Lp(a) can be taken up by macrophages, we postulated that it serves as an opsonin to enhance phagocytosis of NTHi by macrophages. Based on colony-forming unit (CFU) counts, Lp(a) was found to increase U937 macrophage-mediated phagocytosis of NTHi49247 and NTHi49766 by 34% and 43%, respectively, after 120 min. In contrast, Lp(a) did not enhance phagocytosis of Escherichia coli BL21 or E. coli JM109, which were unable to bind to Lp(a). As with U937 macrophages, Lp(a) was capable of increasing phagocytosis of NTHi49247 by peripheral blood mononuclear cell-derived macrophages. Opsonic phagocytosis by Lp(a) was inhibited by the addition of recombinant kringle IV type 10 (rKIV10), a lysine-binding competitor; moreover, Lp(a) did not increase phagocytosis of NTHi by U937 macrophages that were pretreated with a monoclonal antibody against the scavenger receptor CD36. Taken together, our observation suggests that Lp(a) might serve as a lysine-binding opsonin to assist macrophages in rapid recognition and phagocytosis of NTHi.

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis.

In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media.

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Phloretin, an Apple Polyphenol, Inhibits Pathogen-Induced Mucin Overproduction.

SCOPE: Bacterial infection induces mucus overproduction, contributing to acute exacerbations and lung function decline in chronic respiratory diseases. A diet enriched in apples may provide protection from pulmonary disease development and progression. This study examined whether phloretin, an apple polyphenol, inhibits mucus synthesis and secretion induced by the predominant bacteria associated with chronic respiratory diseases. METHODS AND RESULTS: The expression of mucus constituent mucin 5AC (MUC5AC) in FVB/NJ mice and NCI-H292 epithelial cells is analyzed. Nontypeable Haemophilus influenzae (NTHi)-infected mice developed increased MUC5AC mRNA, which a diet containing phloretin inhibited. In NCI-H292 cells, NTHi, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa increased MUC5AC mRNA, which phloretin inhibited. Phloretin also diminished NTHi-induced MUC5AC protein secretion. NTHi-induced increased MUC5AC required toll-like receptor 4 (TLR4) and NADH oxidase 4 (NOX4) signaling and subsequent activation of the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway. Phloretin inhibited NTHi-induced TLR4/NOX4 and EGFR/MAPK signaling, thereby preventing increased MUC5AC mRNA. EGFR activation can also result from increased EGFR ligand synthesis and subsequent ligand activation by matrix metalloproteinases (MMPs). In NCI-H292 cells, NTHi increased EGFR ligand and MMP1 and MMP13 mRNA, which phloretin inhibited. CONCLUSIONS: In summary, phloretin is a promising therapeutic candidate for preventing bacterial-induced mucus overproduction.

The Nontypeable Haemophilus influenzae Major Adhesin Hia Is a Dual-Function Lectin That Binds to Human-Specific Respiratory Tract Sialic Acid Glycan Receptors.

NTHi is a human-adapted pathogen that colonizes the human respiratory tract. Strains of NTHi express multiple adhesins; however, there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1 and HMW2 (HMW1/2). Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein that has a critical role in colonization of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2-3- and 2-6-linked sialic acid glycans found in the human respiratory tract. Here, we show that the high-affinity binding domain of Hia, binding domain 1 (BD1), is responsible for binding to α2-6-sialyllactosamine (2-6 SLN) glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower-affinity binding activity for 2-3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and HMW2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of this major NTHi adhesin and putative vaccine candidate will direct and inform development of future vaccines and therapeutic strategies for this important pathogen.IMPORTANCE Host-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work, we show that the major NTHi adhesin Hia preferentially binds to Neu5Ac-α2-6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for microbes that colonize the human airway.

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.

Rate and Predictors of Bacteremia in Afebrile Community-Acquired Pneumonia.

BACKGROUND: Although blood cultures (BCs) are the "gold standard" for detecting bacteremia, the utility of BCs in patients with community-acquired pneumonia (CAP) is controversial. This study describes the proportion of patients with CAP and afebrile bacteremia and identifies the clinical characteristics predicting the necessity for BCs in patients who are afebrile. METHODS: Bacteremia rates were determined in 4,349 patients with CAP enrolled in the multinational cohort study The Competence Network of Community-Acquired Pneumonia (CAPNETZ) and stratified by presence of fever at first patient contact. Independent predictors of bacteremia in patients who were afebrile were determined using logistic regression analysis. RESULTS: Bacteremic pneumonia was present in 190 of 2,116 patients who were febrile (8.9%), 101 of 2,149 patients who were afebrile (4.7%), and one of 23 patients with hypothermia (4.3%). Bacteremia rates increased with the CURB-65 score from 3.5% in patients with CURB-65 score of 0 to 17.1% in patients with CURB-65 score of 4. Patients with afebrile bacteremia exhibited the highest 28-day mortality rate (9.9%). Positive pneumococcal urinary antigen test (adjusted OR [AOR], 4.6; 95% CI, 2.6-8.2), C-reactive protein level > 200 mg/L (AOR, 3.1; 95% CI, 1.9-5.2), and BUN level ≥ 30 mg/dL (AOR, 3.1; 95% CI, 1.9-5.3) were independent positive predictors, and antibiotic pretreatment (AOR, 0.3; 95% CI, 0.1-0.6) was an independent negative predictor of bacteremia in patients who were afebrile. CONCLUSIONS: A relevant proportion of patients with bacteremic CAP was afebrile. These patients had an increased mortality rate compared with patients with febrile bacteremia or nonbacteremic pneumonia. Therefore, the relevance of fever as an indicator for BC necessity merits reconsideration.

COX-2 mediated crosstalk between Wnt/ß-catenin and the NF-κB signaling pathway during inflammatory responses induced by Haemophilus parasuis in PK-15 and NPTr cells.

Haemophilus parasuis infection causes typical acute systemic inflammation in pigs, is characterized by fibrinous polyserositis inflammation, and results in great economic losses to the swine industry worldwide. However, the molecular details of how the host modulates the acute inflammatory response induced by H. parasuis are largely unknown. In previous studies, we found that H. parasuis high-virulence strain SH0165 infection induced the activation of both Wnt/ß-catenin and NF-κB signaling in PK-15 and NPTr cells. In this study, we found that the activation of NF-κB, a central hub in inflammatory signaling, was impeded by the Wnt/ß-catenin pathway during H. parasuis infection. In contrast, blocking NF-κB activity had no effect on the Wnt/ß-catenin pathway during H. parasuis infection. Furthermore, we found that the inhibitory effect of ß-catenin on NF-κB activity was mediated by its target gene, pig cyclooxygenase-2 (COX-2). Therefore, we demonstrated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway, which leads to decreased NF-κB activity, reducing the acute inflammatory response in pigs. Additionally, the data provide a possible perspective for understanding the anti-inflammatory role of Wnt/ß-catenin in pigs during bacterial infection.

Diagnosis and Treatment of Adults with Community-acquired Pneumonia. An Official Clinical Practice Guideline of the American Thoracic Society and Infectious Diseases Society of America.

Background: This document provides evidence-based clinical practice guidelines on the management of adult patients with community-acquired pneumonia.Methods: A multidisciplinary panel conducted pragmatic systematic reviews of the relevant research and applied Grading of Recommendations, Assessment, Development, and Evaluation methodology for clinical recommendations.Results: The panel addressed 16 specific areas for recommendations spanning questions of diagnostic testing, determination of site of care, selection of initial empiric antibiotic therapy, and subsequent management decisions. Although some recommendations remain unchanged from the 2007 guideline, the availability of results from new therapeutic trials and epidemiological investigations led to revised recommendations for empiric treatment strategies and additional management decisions.Conclusions: The panel formulated and provided the rationale for recommendations on selected diagnostic and treatment strategies for adult patients with community-acquired pneumonia.

Seasonality of antimicrobial resistance rates in respiratory bacteria: A systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance (AMR) rates may display seasonal variation. However, it is not clear whether this seasonality is influenced by the seasonal variation of infectious diseases, geographical region or differences in antibiotic prescription patterns. Therefore, we assessed the seasonality of AMR rates in respiratory bacteria. METHODS: Seven electronic databases (Embase.com, Medline Ovid, Cochrane CENTRAL, Web of Science, Core Collection, Biosis Ovid, and Google Scholar), were searched for relevant studies from inception to Jun 25th, 2019. Studies describing resistance rates of Streptococcus pneumoniae and Haemophilus influenzae were included in this review. By using random-effects meta-analysis, pooled odd ratios of seasonal AMR rates were calculated using winter as the reference group. Pooled odd ratios were obtained by antibiotic class and geographical region. RESULTS: We included 13 studies, of which 7 were meta-analyzed. Few studies were done in H. influenzae, thus this was not quantitively analyzed. AMR rates of S. pneumoniae to penicillins were lower in other seasons than in winter with pooled OR = 0.71; 95% CI = 0.65-0.77; I2 = 0.0%, and to all antibiotics with pooled OR = 0.68; 95% CI = 0.60-0.76; I2 = 14.4%. Irrespective of geographical region, the seasonality of AMR rates in S. pneumoniae remained the same. CONCLUSION: The seasonality of AMR rates could result from the seasonality of infectious diseases and its accompanied antibiotic use.

Antibiotic modulation of mucins in otitis media; should this change our approach to watchful waiting?

BACKGROUND: Gel-forming mucins (GFMs) play important roles in otitis media (OM) pathogenesis. Increased mucin expression is activated by pathogens and proinflammatory cytokines. Bacterial biofilms influence inflammation and resolution of OM and may contribute to prolonged mucin production. The influence of specific pathogens on mucin expression and development of chronic OM with effusion (OME) remains an area of significant knowledge deficit. OBJECTIVES: To assess the relationship between GFM expression, specific pathogens, middle ear mucosal (MEM) changes, biofilm formation, and antibiotic utilization. METHODS: Mixed gender chinchillas were inoculated with nontypeable Haemophilus influenzae (NTHi) strain 86028NP or Streptococcus pneumoniae (SP) strain TIGR4 via transbulla injection. Antibiotic was administered on day 3-5 post inoculation. GFM expression was measured by quantitative PCR. Biofilm formation was identified and middle ear histologic changes were measured. RESULTS: SP infection resulted in higher incidence of biofilm and ME effusion compared with NTHi infection. However, NTHi persisted in the ME longer than SP with no substantive bacterial clearance detected on day 10 compared with complete bacterial clearance on day 10 for 50-60% of the SP-infected chinchillas. Both infections increased MEM inflammatory cell infiltration and thickening. NTHi upregulated the Muc5AC, Muc5B and Muc19 expression on day 10 (p = 0.0004, 0.003, and 0.002 respectively). SP-induced GFM upregulations were trended toward significant. In both NTHi and SP infections, the degree of GFM upregulation had a direct relationship to increased MEM hypertrophy, inflammatory cell infiltration and biofilm formation. Antibiotic treatment reduced the incidence of ME effusion and biofilm, limited the MEM changes and reversed the GFM upregulation. In NTHi infection, the rate of returning to baseline level of GFMs in treated chinchillas was quicker than those without treatment. CONCLUSIONS: In an animal model of OM, GFM genes are upregulated in conjunction with MEM hypertrophy and biofilm formation. This upregulation is less robust and more quickly ameliorated to a significant degree in the NTHi infection with appropriate antibiotic therapy. These findings contribute to the understanding of pathogen specific influences on mucin expression during OM pathogenesis and provide new data which may have implications in clinical approach for OM treatment.

Suppression of Alternative Lipooligosaccharide Glycosyltransferase Activity by UDP-Galactose Epimerase Enhances Murine Lung Infection and Evasion of Serum IgM.

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE. Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization.

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.

Effects of Baicalin on piglet monocytes involving PKC-MAPK signaling pathways induced by Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (HPS) is the causative agent of Glässer's disease, characterized by arthritis, fibrinous polyserositis and meningitis, and resulting in worldwide economic losses in the swine industry. Baicalin (BA), a commonly used traditional Chinese medication, has been shown to possess a series of activities, such as anti-bacterial, anti-viral, anti-tumor, anti-oxidant and anti-inflammatory activities. However, whether BA has anti-apoptotic effects following HPS infection is unclear. Here, we investigated the anti-apoptotic effects and mechanisms of BA in HPS-induced apoptosis via the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) pathway in piglet's mononuclear phagocytes (PMNP). RESULTS: Our data demonstrated that HPS could induce reactive oxygen species (ROS) production, arrest the cell cycle and promote apoptosis via the PKC-MAPK signaling pathway in PMNP. Moreover, when BA was administered, we observed a reduction in ROS production, suppression of cleavage of caspase-3 in inducing apoptosis, and inhibition of activation of the PKC-MAPK signaling pathway for down-regulating p-JNK, p-p38, p-ERK, p-PKC-α and PKC-δ in PMNP triggered by HPS. CONCLUSIONS: Our data strongly suggest that BA can reverse the apoptosis initiated by HPS through regulating the PKC-MAPK signaling pathway, which represents a promising therapeutic agent in the treatment of HPS infection.

Azithromycin treats diffuse panbronchiolitis by targeting T cells via inhibition of mTOR pathway.

Azithromycin (AZM), that is a macrolide antibiotic, has been found to treat diffuse panbronchiolitis (DPB) effectively. However, the mechanism of action underlying the therapeutic effects remains unclear. We selected 64 patients with DPB from 305 patients who were diagnosed with DPB at the outpatient clinic in Shanghai Pulmonary Hospital from Jan 2010 to Oct 2014. The primary PBLs, CD4 + T cells, and Jurkat T cells were treated with AZM or erythromycin (EM), and the effects of AZM and EM on IL-17A and CXCL-2 production, proliferation, apoptosis and autophagy were evaluated. AZM and EM significantly inhibited IL-17A and CXCL-2 production in patients' PBLs (all P < 0.05). AZM significantly inhibited proliferation and promoted apoptosis of T cells from DPB patients. AZM can enhance autophagosome formation of T cells by suppressing S6RP phosphorylation, which is a downstream target of mTOR pathway (all P < 0.05). AZM and EM significantly decreased secreted IL-17A levels (P < 0.05) in the primary CD4 + T cells of patients with DPB. AZM may treat DPB patients by targeting cytokine production, proliferation, apoptosis and autophagy of T cell. The mechanism of therapeutic effects of AZM on DPB may be associated with a specific inhibition of mTOR pathway in the T lymphocytes.