Glen McGillivary

Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

A Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Homologue Plays a Pivotal Role in Nontypeable Haemophilus influenzae Colonization of the Chinchilla Nasopharynx via the Outer Membrane Protein P5-Homologous Adhesin

ABSTRACT In vitro studies suggest an important role for CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) in infection by multiple gram-negative bacteria. However, in vivo evidence supporting this role is lacking, largely because the bacterial adhesins involved in this host-microbe association do not bind to murine-derived CEACAM1. One of several adhesins expressed by nontypeable Haemophilus influenzae (NTHI), the outer membrane protein P5-homologous adhesin (or P5), is essential for colonization of the chinchilla nasopharynx and infection of the middle ear. Here we reveal that NTHI P5 binds to the chinchilla homologue of CEACAM1 and that rabbit anti-human carcinoembryonic antigen blocks NTHI colonization of the chinchilla nasopharynx, providing the first demonstration of a role for CEACAM receptor binding by any bacterial pathogen in vivo.

Respiratory syncytial virus-induced dysregulation of expression of a mucosal beta-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae.

Otitis media (OM) is a polymicrobial disease wherein upper respiratory tract viruses compromise host airway defences, which allows bacterial flora of the nasopharynx (NP) access to the middle ear. We have shown, in vitro, that respiratory syncytial virus (RSV), a viral co‐pathogen of OM, reduces transcript abundance of the antimicrobial peptide (AP), chinchilla beta‐defensin‐1 (cBD‐1). Here, we demonstrated that chinchillas inoculated with RSV expressed ∼40% less cBD‐1 mRNA and protein than did mock‐challenged animals. Further, concurrent RSV infection resulted in a 10–100‐fold greater recovery of non‐typeable Haemophilus influenzae (NTHI) from nasopharyngeal lavage fluids, compared with chinchillas challenged with NTHI in the absence of viral co‐infection. Additionally, when either: anti‐cBD‐1 antibody (to bind secreted AP) or recombinant cBD‐1 (to increase AP concentration at the mucosal surface) were delivered to chinchillas, we demonstrated that disruption of the availability of a single AP influenced the relative load of NTHI in the upper respiratory tract. Collectively, our data suggested that effectors of innate immunity regulate normal bacterial colonization of the NP and, further, virus‐induced altered expression of APs can result in an increased load of NTHI within the NP, which likely promotes development of OM.

The Multifunctional Host Defense Peptide SPLUNC1 Is Critical for Homeostasis of the Mammalian Upper Airway

Otitis media (OM) is a highly prevalent pediatric disease caused by normal flora of the nasopharynx that ascend the Eustachian tube and enter the middle ear. As OM is a disease of opportunity, it is critical to gain an increased understanding of immune system components that are operational in the upper airway and aid in prevention of this disease. SPLUNC1 is an antimicrobial host defense peptide that is hypothesized to contribute to the health of the airway both through bactericidal and non-bactericidal mechanisms. We used small interfering RNA (siRNA) technology to knock down expression of the chinchilla ortholog of human SPLUNC1 (cSPLUNC1) to begin to determine the role that this protein played in prevention of OM. We showed that knock down of cSPLUNC1 expression did not impact survival of nontypeable Haemophilus influenzae, a predominant causative agent of OM, in the chinchilla middle ear under the conditions tested. In contrast, expression of cSPLUNC1 was essential for maintenance of middle ear pressure and efficient mucociliary clearance, key defense mechanisms of the tubotympanum. Collectively, our data have provided the first in vivo evidence that cSPLUNC1 functions to maintain homeostasis of the upper airway and, thereby, is critical for protection of the middle ear.

Extracellular DNA within a nontypeable Haemophilus influenzae induced biofilm binds human beta defensin-3 and reduces its antimicrobial activity

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are associated with multiple chronic infections of the airway, including otitis media. Extracellular DNA (eDNA) is part of the biofilm matrix and serves as a structural component. Human β-defensin-3 (hBD-3) is a cationic antimicrobial host defense protein (AMP) critical to the protection of the middle ear. We hypothesized that anionic eDNA could interact with and bind hBD-3 and thus shield NTHI in biofilms from its antimicrobial activity. We demonstrated that recombinant hBD-3 [(r)hBD-3] bound eDNA in vitro and that eDNA in biofilms produced by NTHI in the chinchilla middle ear co-localized with the orthologue of this AMP. Incubation of physiological concentrations of (r)hBD-3 with NTHI genomic DNA abrogated the ability of this innate immune effector to prevent NTHI from forming robust biofilms in vitro. Establishment of NTHI biofilms in the presence of both DNase I and (r)hBD-3 resulted in a marked reduction in the overall height and thickness of the biofilms and rescued the antimicrobial activity of the AMP. Our results demonstrated that eDNA in NTHI biofilms sequestered hBD-3 and thus diminished the biological activity of an important effector of innate immunity. Our observations have important implications for chronicity of NTHI-induced diseases.

Respiratory syncytial virus-induced dysregulation of expression of a mucosal β-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae

Otitis media (OM) is a polymicrobial disease wherein upper respiratory tract viruses compromise host airway defences, which allows bacterial flora of the nasopharynx (NP) access to the middle ear. We have shown, in vitro, that respiratory syncytial virus (RSV), a viral co‐pathogen of OM, reduces transcript abundance of the antimicrobial peptide (AP), chinchilla beta‐defensin‐1 (cBD‐1). Here, we demonstrated that chinchillas inoculated with RSV expressed ∼40% less cBD‐1 mRNA and protein than did mock‐challenged animals. Further, concurrent RSV infection resulted in a 10–100‐fold greater recovery of non‐typeable Haemophilus influenzae (NTHI) from nasopharyngeal lavage fluids, compared with chinchillas challenged with NTHI in the absence of viral co‐infection. Additionally, when either: anti‐cBD‐1 antibody (to bind secreted AP) or recombinant cBD‐1 (to increase AP concentration at the mucosal surface) were delivered to chinchillas, we demonstrated that disruption of the availability of a single AP influenced the relative load of NTHI in the upper respiratory tract. Collectively, our data suggested that effectors of innate immunity regulate normal bacterial colonization of the NP and, further, virus‐induced altered expression of APs can result in an increased load of NTHI within the NP, which likely promotes development of OM.

Genomic analysis of the F3031 Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius by PCR-based subtractive hybridization.

ABSTRACT PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive Haemophilus influenzae biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a Thermotoga maritima hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-like proteins. The presence of DNA related to these loci is variable among BPF isolates and nontypeable H. influenzae strains, while neither of them was detected in strains of types a to f. The data indicate that BPF-causing strain F3031 harbors unique chromosomal regions, most of which appear to be acquired from unrelated microbial sources.

Replication of Respiratory Syncytial Virus Is Inhibited by the Host Defense Molecule Viperin

Respiratory syncytial virus (RSV) is an important viral pathogen of otitis media, bronchiolitis, and pneumonia. As infection of the upper airways is a precondition for the development of these diseases, understanding RSV pathogenesis and the host response induced by RSV in this niche may enable the development of novel therapeutic strategies against this virus. We have used a microarray approach and showed that expression of the gene that encodes the antiviral protein viperin was significantly upregulated in the chinchilla nasopharynx up to 1 week after RSV challenge. Overexpression of human viperin in vitro diminished the ability of RSV to infect HeLa or A549 cells. Furthermore, transduction of the chinchilla airways with a recombinant adeno-associated virus vector that encodes viperin resulted in reduced titers of RSV in the nasopharyngeal lavage fluid. Collectively, these data indicated that viperin plays a significant role in the innate immune defense against RSV.

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans afeABCD siderophore-independent iron acquisition system

ABSTRACT The ActinobacillusactinomycetemcomitansafeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichiacoli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.

Abrogation of nontypeable Haemophilus influenzae Protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media

The pneumococcal polysaccharide conjugate vaccine which includes a nonacylated protein D carrier from Haemophilus influenzae has been recently licensed for use in many countries. While this vaccine is protective against nontypeable Haemophilus influenzae (NTHI)-induced acute otitis media (OM), the mechanism underlying this protective efficacy is not yet fully understood. Protein D/glycerophosphodiester phosphodiesterase (PD/GlpQ) is an outer membrane lipoprotein expressed by NTHI that has been ascribed several functions, including host cell adherence and phosphorylcholine (PCho) acquisition. We found that a pd/glpQ NTHI mutant exhibited reduced adherence to airway epithelial cells, diminished phosphorylcholine (PCho) decoration of biofilms, and compromised fitness during experimental acute OM compared to the parent strain. We also found that exposure of NTHI to antibodies directed against the vaccine formulation recapitulated the PCho decoration and NTHI adherence phenotypes exhibited by PD/GlpQ-deficient NTHI, providing at least two likely mechanisms by which the pneumococcal polysaccharide-PD/GlpQ conjugate vaccine induces protection from NTHI-induced OM.