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.
Maintaining genome integrity requires the accurate and complete replication of chromosomal DNA. This is of the utmost importance for embryonic stem cells (ESCs), which differentiate into cells of all lineages, including germ cells. However, endogenous and exogenous factors frequently induce stalling of replication forks in every cell cycle, which can trigger mutations and chromosomal instabilities. We show here that the oncofetal, nonhistone chromatin factor HMGA2 equips cells with a highly effective first-line defense mechanism against endonucleolytic collapse of stalled forks. This fork-stabilizing function most likely employs scaffold formation at branched DNA via multiple DNA-binding domains. Moreover, HMGA2 works independently of other human factors in two heterologous cell systems to prevent DNA strand breaks. This fork chaperone function seemingly evolved to preserve ESC genome integrity. It is hijacked by tumor (stem) cells to also guard their genomes against DNA-damaging agents widely used to treat cancer patients.
Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a “target and release” chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.
HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoidassociated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUa (PG1258) and the HUb (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUb using comparative expression profiling of the PG0121 (HUb) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUb has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake. INTRODUCTIONPorphyromonas gingivalis is a Gram-negative obligate anaerobe belonging to the family Bacteroidaceae that persists as a natural member of the human oral microbiota. A shift in the microbial community leading to outgrowth of this anaerobe is directly linked to periodontitis, a chronic inflammatory disease that leads to destruction of the tissues supporting the gums and ultimately, exfoliation of the teeth (Choil et al., 1990;Dzink et al., 1988;Grossi et al., 1994;Lamont & Jenkinson, 2000;Moore et al., 1991). This commensal can colonize, invade and multiply within gingival epithelial cells, as well as penetrate into deeper epithelial cell layers, potentially releasing the whole organism and/or virulence factors into the bloodstream (reviewed by Yilmaz, 2008). In addition to its ability to cause disease in the oral cavity, there are data indicating a role in systemic disease, including its ability to invade vascular endothelial cells (Dorn et al., 2000(Dorn et al., , 2002Jandik et al., 2008) and to cause aggregation of platelets (Pham et al., 2002). For many pathogenic bacteria, surface polysaccharides play a key role in immune modulation et al., 1996). HU typically acts as an accessory protein in virtually all types of nucleoprotein-mediated processes (reviewed by...
Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual‐modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. Upon proteolytic cleavage by Arg‐specific gingipain (RgpB), five‐fold photoacoustic enhancement and >100‐fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU mL−1 bacteria in vitro. RgpB activity was imaged in porcine jaws with low‐nanomolar sensitivity. Diagnostic efficacy was evaluated in gingival crevicular fluid samples from subjects with and without periodontal disease, wherein activation was correlated to qPCR‐based detection of P. gingivalis (Pearson's r=0.71). Finally, photoacoustic imaging of RgpB‐cleaved probe was achieved in murine brains ex vivo, with relevance and potential utility for disease models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.
Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. This probe harnesses the intramolecular dimerization of peptide-linked cyanine dyes to induce fluorescence and photoacoustic off-states. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU/mL bacteria in vitro. RgpB activity was imaged in the subgingival pocket of porcine jaws with 25 nM sensitivity. The diagnostic efficacy of the probe was evaluated in gingival crevicular fluid (GCF) samples from subjects with (n = 14) and without (n = 6) periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson’s r = 0.71). The highest activity was seen in subjects with the most severe disease. Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, demonstrating relevance and potential utility for animal models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer’s disease.
K-antigen capsule, a key virulence determinant of the oral pathogen Porphyromonas gingivalis, is synthesized by proteins encoded in a series of genes transcribed as a large polycistronic message. Previously, we identified a 77-base pair inverted repeat region with the potential to form a large stem-loop structure at the 5′ end of this locus. PG0121, one of two genes flanking the capsule operon, was found to be co-transcribed with the operon and to share high similarity to the DNA binding protein HU from Escherichia coli. A null mutation in PG0121 results in down-regulation of transcription of the capsule synthesis genes and production of capsule. Furthermore, we have also shown that PG0121 gene can complement multiple deficiencies in a strain of E. coli that is deficient for both the alpha and beta subunits of HU. Here, we examined the biochemical properties of the interaction of PG0121 to DNA with the emphasis on the kinds of nucleic acid architectures that may be encountered at the 77-bp inverted repeat. We have concluded that although some DNA binding characteristics are shared with E. coli HU, HU PG0121 also shows some distinct characteristics that set it apart from other HU-like proteins tested to date. We discuss our results in the context of how PG0121 may affect the regulation of the K-antigen capsule expression.
Evidence has been accumulating for a role of inflammation in the development of Alzheimer's disease (AD), a progressive neurodegenerative disorder causing a common form of dementia in the elderly. C1q, part of the initiation component of the classical complement pathway (CCP), is associated with beta-sheet, fibrillar amyloid plaques in AD brain. In vitro, beta-amyloid peptide in fibrillar beta-sheet conformation (fAbeta) can activate CCP via interaction of specific negatively charged amino acids of the beta-amyloid fibril with human C1q. Previous results using peptide inhibitors led to the hypothesis that a highly positively charged domain consisting of three arginine residues, such as that present in the N-terminal collagen-like region of the human C1q A chain, may be critical for the activation event. However, mouse C1q A chain lacks two of the three arginines in the corresponding C1q A chain collagen-like region. To test the hypothesis that this divergent activation domain results in a weaker C' activation and thus may contribute to the lower neuronal loss observed in transgenic mouse models of AD, a partially humanized C1q A chain knock-in mouse was generated. The mouse C1q A chain gene was modified by homologous recombination to replace 4 residues in the 13-20 amino acid region to mimic the corresponding sequence from human A chain. No significant differences in the expression of C1q were found in sera from mice homozygous for the humanized C1q A chain compared to littermate wild type mice. Two distinct C1 activation assays demonstrated that activation by fAbeta was not significantly different in the homozygous humanized C1q A chain mice. Activation of C1 by DNA, previously hypothesized to interact with this C1q A chain arginine-rich sequence was also not significantly different in the knock-in mouse. Molecular modeling based on the published crystal structure of human C1q B chain globular head and a beta-sheet model for fibrillar amyloid suggests an alternative arginine ladder in the globular head domain may provide the functional C1 activating interaction domains. The humanized C1q mouse generated here should provide a better animal model for assessing the mechanisms of C1 activation and the contribution of C1q to human health and disease.
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