Antibodies against the DNABII protein integration host factor (IHF) inhibit sinus implant biofilms.

OBJECTIVES: Chronic rhinosinusitis is a common, costly condition often treated with endoscopic sinus surgery and intraoperative placement of intranasal sinus implant materials. Whereas these materials aid in postoperative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pretreatment of sinus implant materials with antibody against an essential bacterial biofilm structural component, the DNABII family of DNA-binding proteins, as a strategy to prevent biofilm formation. METHODS: Sinus implant materials were equilibrated in immunoglobulin G (IgG)-enriched antiserum against the DNABII protein integration host factor (IHF), individually or in combination with amoxicillin-clavulanate prior to inoculation with nontypeable Haemophilus influenzae (NTHI), a predominant pathogen of chronic rhinosinusitis. After 16 hours, the bacterial burden was quantitated and compared to pretreatment with saline, IgG-enriched naive serum, or amoxicillin-clavulanate alone. RESULTS: NTHI readily formed biofilms on all three materials in vitro. However, pretreatment of each material with IgG-enriched anti-IHF resulted in a significant decrease in bacterial burden compared to controls (P ≤ 0.05). Moreover, a significant and synergistic outcome was achieved with a cocktail of anti-IHF plus amoxicillin-clavulanate (P ≤ 0.05) with complete inhibition of NTHI biofilm formation on all three materials. CONCLUSIONS: Biofilm formation was well supported in vitro on three sinus implant materials that vary in composition and resorption characteristics; however, pretreatment of each with DNABII protein targeted antibodies in combination with a previously ineffective antibiotic was highly effective to prevent the formation NTHI biofilms. These data demonstrate the potential for clinical utility of pretreatment of sinus implant and additional surgical materials with anti-DNABII antibodies. LEVEL OF EVIDENCE: NA Laryngoscope, 130:1364-1371, 2020.

A bacterial-biofilm-induced oral osteolytic infection can be successfully treated by immuno-targeting an extracellular nucleoid-associated protein.

Periodontal disease exemplifies a chronic and recurrent infection with a necessary biofilm component. Mucosal inflammation is a hallmark response of the host seen in chronic diseases, such as colitis, gingivitis, and periodontitis (and the related disorder peri-implantitis). We have taken advantage of our recently developed rat model of human peri-implantitis that recapitulates osteolysis, the requirement of biofilm formation, and the perpetuation of the bona fide disease state, to test a new therapeutic modality with two novel components. First we used hyperimmune antiserum directed against the DNABII family of proteins, now known to be a critical component of the extracellular matrix of bacterial biofilms. Second we delivered the antiserum as cargo in biodegradable microspheres to the site of the biofilm infection. We demonstrated that delivery of a single dose of anti-DNABII in poly(lactic-co-glycolic acid) (PLGA) microspheres induced significant resolution of experimental peri-implantitis, including marked reduction of inflammation. These data support the continued development of a DNABII protein-targeted therapeutic for peri-implantitis and other chronic inflammatory pathologies of the oral cavity in animals and humans.

Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.

CRISPR Immunological Memory Requires a Host Factor for Specificity.

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.

Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein.

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

Antibodies directed against integration host factor mediate biofilm clearance from Nasopore.

OBJECTIVES/HYPOTHESIS: Intranasal resorbable packing, such as Nasopore, is commonly used during sinus surgery despite a paucity of evidence that demonstrates clinical benefit. We theorized that Nasopore supports bacterial growth and biofilm formation. The DNABII family of bacterial nucleic acid binding proteins stabilizes the extracellular polymeric substance of the biofilm, thus protecting bacteria from host defenses and traditional antibiotics. We tested the hypothesis that use of anti-IHF antibodies in conjunction with antibiotics would enhance biofilm eradication from Nasopore. STUDY DESIGN: In vitro experiments. METHODS: Nontypeable Haemophilus influenzae (NTHI) biofilms were grown on Nasopore. Following 24-hour incubation, biofilms were incubated for an additional 16 hours with either medium alone, naïve rabbit serum, rabbit anti-IHF serum, amoxicillin/clavulanate, or anti-IHF serum + amoxicillin/clavulanate. Computer statistics (COMSTAT) analysis was performed on images of biofilms obtained via confocal microscopy. RESULTS: NTHI readily formed a biofilm on Nasopore. Treatment with amoxicillin/clavulanate alone mediated an increase in biomass by 92% to 6.63 µ(2) /µ(3) compared to incubation in sterile medium alone (3.46 µ(2) /µ(3)). Treatment with anti-IHF alone reduced the biomass by 77% to 1.29 µ(2) /µ(3) compared to incubation with naïve rabbit serum (5.53 µ(2) /µ(3)). Anti-IHF + amoxicillin/clavulanate reduced biomass by 88% to 0.66 µ(2) /µ(3) (P <0.02) compared to incubation with naïve rabbit serum. CONCLUSION: Antibiotics alone were ineffective in eradicating NTHI biofilms that had formed on Nasopore in vitro. Anti-IHF antibodies plus amoxicillin/clavulanate therapy synergistically reduced biofilm biomass by 88%. These data support clinical studies for the use of anti-IHF combined with antibiotics to reduce biofilm formation on intranasal packing.

Paratuberculose em ruminantes no Brasil / Paratuberculosis in ruminants in Brasil: a review

A paratuberculose ou doença de Johne é uma enterite granulomatosa causada por Mycobacterium avium subsp. paratuberculosis (Map) e comumente afeta ruminantes domésticos, no entanto, pode infectar várias espécies de mamíferos. Está presente nos cinco continentes e é considerada endêmica em algumas regiões pela Organização Internacional de Epizootias (OIE). Pertence à lista de enfermidades notificáveis, que compreende as doenças transmissíveis de importância sócio-econômica e/ou em saúde-pública, cujo controle é necessário para o comércio internacional de animais e alimentos de origem animal. A importância da doença de Johne não se restringe somente aos prejuízos econômicos causados à indústria animal, mas também na possível participação do Map na íleocolite granulomatosa que afeta seres humanos, conhecida como doença de Crohn. No Brasil, a paratuberculose já foi descrita em diversas espécies de ruminantes e em vários estados. Embora os relatos naturais da enfermidade sejam pontuais, acredita-se na possibilidade da transmissão interespecífica e na disseminação do agente através da compra e venda de animais infectados. O objetivo deste artigo foi reunir as informações disponíveis referentes aos aspectos epidemiológicos, clínico-patológicos e laboratoriais da paratuberculose em bovinos, bubalinos, caprinos e ovinos no Brasil, e salientar a necessidade de implementação de medidas de controle sanitário da enfermidade no país, o que possibilitaria a melhoria da qualidade e valorização dos produtos de origem animal no mercado internacional.

Paratuberculosis also known as Johne's disease, is a granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast bacillus that preferentially resides within host intestinal macrophages. The condition is most commonly seen in domestic ruminants, however MAP can also infect other mammalian species. Paratuberculosis shows a global distribution and is considered endemic in some regions. The World Organization for Animal Health (OIE, Office International des Epizooties), have classified paratuberculosis as a notificable disease; considered to be of socio-economic and/or public-health importance, the control of which is necessary for the international trade of animal and animal products. The importance of paratuberculosis is related primarily to economic losses in the animal industry and also because of a potential role for this bacterium in the pathogenesis of Crohn´s disease, a debilitating condition affecting the digestive tract of humans. In Brazil, paratuberculosis has been reported in a variety of ruminant species and shows a broad geographic distribution. The reported incidence of natural cases in Brazil has been limited, but it is believed that interespecific transmission of MAP and dissemination of the agent is driven by the commercialization of infected animals. The main objective of this paper was to collate the published epidemiological, clinic-pathological and diagnostic information in relation to paratuberculosis in cattle, buffaloes, goats and sheep in Brazil. Moreover, it served as a platform to emphasize the requirement to implement sanitary policies for control of MAP in the county, which may serve to improve the quality and value of animal products on international markets.

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins.

Bacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

Interaction of an IHF-like protein with the Rhizobium etli nifA promoter.

The nifA gene fulfills an essential role in the regulation of nitrogen fixation genes in Rhizobium etli. Transcription analysis of the nifA gene, assessed using promoter deletions, indicated an oxygen-independent expression, threefold higher during symbiosis as compared with free-living conditions. Electrophoretic mobility shift assays using those nifA promoter deletion fragments, which were actively transcribed, demonstrated the specific interaction with R. etli cellular protein(s) resulting in the formation of two DNA-protein complexes. An interacting protein was purified by liquid chromatography on Heparin Sepharose and Mono S columns. The purified 12 kDa R. etli protein cross-reacted with antibodies directed against Escherichia coli integration host factor (IHF). Furthermore, purified E. coli IHF was able to specifically bind to the R. etli nifA promoter region. These results point to an as yet undisclosed function of IHF in the regulation of R. etli nifA expression.

Identification of host translation inhibitory factor of Campoletis sonorensis ichnovirus on the tobacco budworm, Heliothis virescens.

Parasitization of a wasp, Campoletis sonorensis, against the larvae of Heliothis virescens depresses synthesis of specific host proteins related to growth and immunity. It has been suggested that the inhibition of host gene expression is targeted at a posttranscriptional level. This study aimed to verify the identity of host translation inhibitory factor (HTIF) derived from wasp parasitization. To identify HTIF, the proteins in the parasitized host were fractionated using different protein purification methods, and each fraction's HTIF activity was assessed. In the course of the protein purification steps, HTIF activity was highly correlated with the fractions containing VHv 1.4 protein, which has a conserved cysteine-motif and is encoded in C. sonorensis ichnovirus (CsIV). Purified VHv 1.4 protein using an immunoaffinity column exhibited a significant HTIF effect, while the heat-inactivated VHv 1.4 did not. Both recombinant VHv 1.4 and VHv 1.1 (another cys-motif protein encoded in CsIV) proteins were synthesized in Sf 9 cells through a baculovirus expression system. The purified recombinant VHv 1.4 and VHv 1.1 exhibited significant HTIF activities in a nanomolar range. However, VHv1.4 protein showed about four times higher HTIF activity than did VHv 1.1 protein. Both HTIFs acted directly on translation machinery because they inhibited a cell-free in vitro translation system using rabbit reticulocyte lysate. Both HTIFs are likely to discriminate specific target mRNAs because they inhibited translation of RNA extracts from the Tn 368 cell line, but not from Sf 9 cells. In addition, they inhibited translation of RNAs from fat body, hemocytes, and testis, but not from epidermis, gut, labial gland, and nerve tissues of H. virescens. These results indicate that both cys-motif proteins of VHv 1.4 and VHv 1.1 play a role as HTIF in C. sonorensis parasitization.