Nontypeable Haemophilus influenzae released from biofilm residence by monoclonal antibody directed against a biofilm matrix component display a vulnerable phenotype.

Bacterial biofilms contribute significantly to pathogenesis, recurrence and/or chronicity of the majority of bacterial diseases due to their notable recalcitrance to clearance. Herein, we examined kinetics of the enhanced sensitivity of nontypeable Haemophilus influenzae (NTHI) newly released (NRel) from biofilm residence by a monoclonal antibody against a bacterial DNABII protein (α-DNABII) to preferential killing by a ß-lactam antibiotic. This phenotype was detected within 5 min and lasted for ~ 6 h. Relative expression of genes selected due to their known involvement in sensitivity to a ß-lactam showed transient up-regulated expression of penicillin binding proteins by α-DNABII NTHI NRel, whereas there was limited expression of the ß-lactamase precursor. Transient down-regulated expression of mediators of oxidative stress supported similarly timed vulnerability to NADPH-oxidase sensitive intracellular killing by activated human PMNs. Further, transient up-regulated expression of the major NTHI porin aligned well with observed increased membrane permeability of α-DNABII NTHI NRel, a characteristic also shown by NRel of three additional pathogens. These data provide mechanistic insights as to the transient, yet highly vulnerable, α-DNABII NRel phenotype. This heightened understanding supports continued validation of this novel therapeutic approach designed to leverage knowledge of the α-DNABII NRel phenotype for more effective eradication of recalcitrant biofilm-related diseases.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Assessment of TLR3 and MDA5-Mediated Immune Responses Using Knockout Quail Fibroblast Cells.

Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) are double-stranded RNA (dsRNA)-recognizing receptors that mediate innate immune responses to virus infection. However, the roles played by these receptors in the pathogenesis of avian viruses are poorly understood. In this study, we generated TLR3 and MDA5 single knockout (SKO) and TLR3-MDA5 double knockout (DKO) quail fibroblast cells and examined dsRNA receptor-mediated innate immune responses in vitro. The knockout cells were then stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or influenza A virus. Endosomal stimulation of TLR3 by adding poly(I:C) in cell culture media or cytoplasmic stimulation of MDA5 by transfecting poly(I:C) resulted in significant increases of TLR3, MDA5, interferon (IFN) ß, and interleukin 8 gene expression levels in wild type (WT) cells. Endosomal poly(I:C) treatment induced a higher level expression of most of the genes tested in MDA5 SKO cells compared with WT cells, but not in TLR3 SKO and DKO cells. Cytoplasmic transfection of poly(I:C) led to significant upregulation of all four genes in WT, TLR3 SKO, and MDA5 SKO cells at 8 hr posttransfection and negligible gene expression changes in TLR3-MDA5 DKO cells. Upon infection with a strain of influenza virus with compromised IFN antagonistic capability, WT cells produced the highest amount of biologically active type I IFN followed by TLR3 SKO and MDA5 SKO cells. DKO cells did not produce detectable amounts of type I IFN. However, the IFN did not induce an antiviral state fast enough to block virus replication, even in WT cells under the experimental conditions employed. In summary, our data demonstrate that TLR3 and MDA5 are the key functional dsRNA receptors in quail and imply their coordinated roles in the induction of innate immune responses upon virus infection.

Evaluación de las respuestas inmunitarias mediadas por TLR3 y MDA5 utilizando células de fibroblastos de codorniz con genes eliminados. El receptor tipo Toll 3 (TLR3) y el gene 5 asociado a la diferenciación de melanoma (MDA5) son receptores de reconocimiento de ARN de doble cadena (dsRNA) que median las respuestas inmunitarias innatas a la infección por virus. Sin embargo, no se conocen bien las funciones que desempeñan estos receptores en la patogenia de los virus aviares. En este estudio, se generaron células de fibroblastos de codorniz con eliminación simple de los genes TLR3 y MDA5 (SKO) y eliminación doble de los genes TLR3-MDA5 (DKO) y se examinaron las respuestas inmunitarias innatas mediadas por el receptor de dsRNA in vitro. Posteriormente, las células con genes eliminados se estimularon con un ligando sintético de ARN de doble cadena poliinosínico: ácido policitidílico [poli (I: C)] o con el virus de la influenza A. La estimulación endosómica de TLR3 mediante la adición de poli(I: C) en medios de cultivo celular, o la estimulación citoplásmica de MDA5 mediante la transfección de poli(I: C), dieron como resultado aumentos significativos de los niveles de expresión de los genes para TLR3, MDA5, interferón (IFN) ß e interleucina 8 en células de tipo silvestre (WT). El tratamiento con poli(I: C) endosómico indujo un nivel de expresión más alto de la mayoría de los genes analizados en las células con eliminación simple del gene MDA5 en comparación con las células silvestres, pero no en las células con eliminación simple del gene TLR3 y con eliminación doble de genes. La transfección citoplásmica de poli(I: C) condujo a una regulación positiva significativa de los cuatro genes en las células silvestres, en las células con eliminación simple del gene TLR3 y en las células con eliminación simple del gene MDA5 a las ocho horas posteriores a la transfección y cambios insignificantes en la expresión de genes en las células con eliminación doble de los genes TLR3 y MDA5. Durante la infección con una cepa del virus de la influenza con una capacidad antagonista para IFN comprometida, las células silvestres produjeron la mayor cantidad de IFN de tipo I biológicamente activo, seguidas de las células con eliminación simple del gene TLR3 y de las células con eliminación simple del gene MDA5. Las células con eliminación doble de genes no produjeron cantidades detectables de IFN de tipo I. Sin embargo, el IFN no indujo un estado antiviral lo suficientemente rápido como para bloquear la replicación del virus, incluso en células silvestres bajo las condiciones experimentales empleadas. En resumen, los datos de este estudio demuestran que TLR3 y MDA5 son los receptores de ARN de doble cadena funcionales clave en la codorniz e implican sus funciones coordinadas en la inducción de respuestas inmunitarias innatas durante la infección por virus.